Par Daniel

Isui Aguilar

Dirigée Par Guy

Tran Van Nhieu
email: guy.tran-van-nhieu@college-de-france.fr

Agathe Subtil

Fabrizia Stavru

Cesar Valencia-Gallardo

Charles Bou-Nader

Daniel-Isui Aguilar-Salvador

Marc Fontecave

Tina Izard

Guy Tran Van Nhieu

Hamed Khakzad

Charles Bou- Nader

Christian Malosse

Borges Diogo

Lima

Chakir Bello

Benjamin Cocom-Chan

Nicole Quenech'du

Bilal Mazhar

Delphine Javelaud

Jacques Fattaccioli

Alain Mauviel

Atef Asnacios

Julia Chamot-Rooke

Lars Malmström

Daniel Isui Aguilar

Characterization of the role of IpaA on the cytoskeletal anchorage of Shigella flexneri

Keywords: Shigella, Type III secretion, invasion, IpaA, vinculin, talin Article Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion Shigella, Type III secretion, invasion, IpaA, vinculin

Shigella invades cells of the intestinal epithelium by injecting effector proteins through a Type III Secretion System (T3SS). The type III effector IpaA binds to talin and vinculin through three Vinculin Binding Sites (VBSs), promoting cytoskeletal anchorage to promote bacterial internalization. As opposed to other invasive bacteria, Shigella does not show constitutive cell binding activity but triggers transient adhesion through T3SS activation. IpaA VBS3 was found to be structurally analogous to talin H5 helix, which, together with H1-H4, forms the R1 talin bundle. Functional analysis identified IpaA VBS3 is necessary for talin recruitment to Shigella entry foci and formation of nascent adhesions and filopodia. IpaA VBS3 also stimulates filopodial capture of bacteria and stabilizes cell adhesions in invaded cells. On the other hand, structural models of IpaA VBSs/Vinculin interactions indicate a novel mode of vinculin activation, involving conformational changes in vinculin head domain that leads to trimerization and stabilization of focal adhesions. IpaA was also observed to be injected by "kissing-and-running" bacteria, facilitating additional bacterial binding and invasion events. This IpaA-dependent priming was dependent on IpaA VBS3 but not on IpaA VBS1-2. Injected IpaA was observed to form clusters apposed to the cell apical side and to induce integrin clustering. Overall, the results indicate that IpaA triggers general changes in cell adhesive properties and promotes a cooperative mechanism of Shigella invasion.

Titre :

Caractérisation du rôle de la protéine IpaA sur l'ancrage cytosquelettique de Shigella flexneri Résumé : Shigella envahit les cellules de l'épithélium intestinal en injectant des protéines effectrices via un système de sécrétion de type III (T3SS). L'effecteur de type III IpaA se lie à la taline et à la vinculine par l'intermédiaire de trois sites de liaison à la vinculine (VBS), favorisant ainsi l'ancrage cytosquelettique ainsi que l'internalisation des bactéries. Contrairement à d'autres bactéries invasives, Shigella ne montre pas d'activité de liaison cellulaire constitutive mais déclenche une adhésion transitoire par l'activation de T3SS. IpaA VBS3 s'est avéré être structurellement analogue à l'hélice de la taline H5, qui, avec H1-H4, forme le faisceau de taline R1. L'analyse fonctionnelle identifiée IpaA VBS3 est nécessaire au recrutement de la taline dans les foyers d'entrée de Shigella et à la formation d'adhérences naissantes et de filopodes. IpaA VBS3 stimule également la capture filopodiale des bactéries et stabilise les adhérences cellulaires dans les cellules envahies. D'autre part, les modèles structurels des interactions IpaA VBS / Vinculin indiquent un nouveau mode d'activation de la vinculine, impliquant des changements conformationnels dans le domaine de la tête de la vinculine, conduisant à la trimérisation et à la stabilisation des adhérences focales. IpaA a également été observé comme étant injecté par une bactérie "s'embrassant et courant", facilitant des événements supplémentaires de liaison bactérienne et d'invasion. Cet amorçage dépendant d'IpaA dépendait d'IpaA VBS3 mais pas d'IpaA VBS1-2. Une fois injecté, IpaA, forme des grappes apposées sur le côté apical de la cellule pour induire une grappe d'intégrines. Globalement, les résultats indiquent que l'IpaA provoque des modifications générales des propriétés adhésives des cellules et favorise un mécanisme coopératif d'invasion de Shigella.

Mots clefs :

Shigella, Sécrétion Type III, invasion, IpaA, vinculine, taline where the old spiny lobster still waves his pincers the fish eaten by God the snake that gulped a rabbit the rabbit that always teased the coyote the coyote that swallowed a wasps' nest the honey that wells from my breasts your navel I am and you will not see me die You may think everyone has gone away but you will not see me die There will be a seed hidden in the scrub by the path that to this land will return and will seed the future and will feed our souls and our stories will be reborn and you will not see me die because we will stay strong because we will always be alive because our song will be eternal because we will be us and you and our children's children and the earth's quaking that will shake the sea and we will be many hearts anchored to the essence of the binnizá and you will not see me die you will not see me die TABLE 1. Specifically, the symptoms caused by Shigella spp. reflect the pathogen ability to invade and multiply inside the colonic mucosa, triggering an intense inflammatory reaction, which eventually leads to destruction of infected tissue and is rarely followed by a systemic infection. The first symptoms are usually fever, headache, malaise, anorexia and vomiting, followed by watery diarrhea. In healthy individuals the symptoms recede in a few days. However, in some individuals, infection can lead to excretion of stools containing blood and mucus with abdominal cramps and tenesmus and in young infants can also lead to necrotising enterocolitis. In rare cases it can lead to meningitis, osteomyelitis, arthritis, splenic abscess, rectal prolapse, intestinal obstruction, toxic megacolon and perforation [START_REF] Kotloff | Shigellosis[END_REF].

List of Tables

Humans are the only known natural host for Shigella, which is predominantly transmitted by faecal-oral contact favored by inadequate sanitation and hygiene. In rare cases, transmission has also been correlated with houseflies presence [START_REF] Farag | Housefly Population Density Correlates with Shigellosis among Children in Mirzapur, Bangladesh: A Time Series Analysis[END_REF]. In a study carried out in volunteers from a correctional facility, it was determined that around 100 colony-forming units suffice to produce a symptomatic infection (DuPont et al. 1989). Case reports of asymptomatic long-term carriage exist, but the prevalence at the population level is unknown [START_REF] Levine | Long-Term Shigella-Carrier State[END_REF]

Epidemiology

It is estimated that Shigella causes 269 million cases yearly worldwide, leading to ~210 thousand deaths. The global distribution of Shigella is strongly correlated with income levels, being the lower-income regions, such as sub-saharan Africa and south Asia (Figure 1), where incidence and mortality rates are higher [START_REF] Khalil | Morbidity and Mortality due to Shigella and Enterotoxigenic Escherichia Coli Diarrhoea: The Global Burden of Disease Study 1990-2016[END_REF].

Figure 1. Shigella diarrhea mortality rate per 100 000 people in 2016 (Modified from [START_REF] Khalil | Morbidity and Mortality due to Shigella and Enterotoxigenic Escherichia Coli Diarrhoea: The Global Burden of Disease Study 1990-2016[END_REF].

Figure 1. Shigella diarrhea mortality rate

In particular, infants under five years are highly vulnerable. In this population the incidence is estimated in 116 episodes per 1000 child-years. Elderly people are also at high risk of mortality. Shigella is the most common cause of diarrhea in adults older than 70 years, leading to 74 thousand deaths [START_REF] Khalil | Morbidity and Mortality due to Shigella and Enterotoxigenic Escherichia Coli Diarrhoea: The Global Burden of Disease Study 1990-2016[END_REF]. In highincome countries, Shigella is associated with travellers who visit regions where the incidence is high. It is estimated that 2-9% of the cases of traveller's diarrhea is due to these bacterial species [START_REF] Shah | Global Etiology of Travelers' Diarrhea: Systematic Review from 1973 to the Present[END_REF]. Other relevant and recent outbreaks in Europe have occurred among refugees from the Middle East living under poor sanitation conditions, where antibiotic-resistant strains of Shigella have been detected [START_REF] Georgakopoulou | Resistant Shigella Strains in Refugees, August-October 2015, Greece[END_REF].

Shigella spp.

Shigella discovery is attributed to Kiyoshi Shiga in 1898, who cultured the bacillus from a patient during an epidemic in Japan [START_REF] Shiga | Studien über Die Epidemische Dysenterie in Japan, Unter Besonderer Berücksichtigung Des Bacillus Dysenteriae[END_REF]. Originally named Bacillus dysenteriae, the strain was subsequently called Shigella dysenteriae serotype 1.

Discovered soon after E. coli, Shigella was placed in a different genus although it can also be considered as an E. coli sub-species. Absence of flagellum-based motility and lack of lactose fermentation were the main diagnostic criteria for its identification.

A different strain of Shigella was isolated by Simon Flexner, which is now known as Shigella flexneri. Yet another group was observed by Boyd, who undertook a project that resulted in several thousands cultures from different cases with different antiserum agglutination properties. Based on this collection he proposed a classification that forms the backbone of the current classification system [START_REF] Boyd | The Antigenic Structure of the Mannitol-Fermenting Group of Dysentery Bacilli[END_REF], further improved by Ewing [START_REF] Ewing | SHIGELLA NOMENCLATURE[END_REF].

Shigella classification

Shigella is a Gram negative, non-motile, non-pigmented and facultatively anaerobic bacteria. It belongs to the Enterobacteraceae family and it has a morphology of straight rods of 1-3 x 0.7-1 μM [START_REF] Strockbine | Shigella." Bergey's Manual of Systematics of Archaea and Bacteria[END_REF].

The existing classification of the Shigella genus has mainly historical reasons, and it has been kept as a separate genus from E. coli mainly due to the seriousness of the disease. There are 4 validated species within the Shigella genus, and within each species several serotypes have been classified: S. dysenteriae (with 15 serotypes), S. boydii (19 serotypes), S. flexneri (14 serotypes and subserotypes) and S. sonnei [START_REF] Strockbine | Shigella." Bergey's Manual of Systematics of Archaea and Bacteria[END_REF]. S. dysenteriae and S. flexneri are the main cause of bacillary dysentery epidemics.

The division of each species into serotypes is based on the lipopolysaccharide O antigen. S. flexneri serotypes share a common basic lipopolysaccharide structure and differ by the addition of glucosyl and/or O-acetyl residues [START_REF] Simmons | Structure and Biology of Shigella Flexneri O Antigens[END_REF]. S. boydii and S. dysenteriae have well differentiated serotypes from other Shigella species, although they are highly related to E. coli O antigen (P. R. [START_REF] Edwards | Edwards and Ewing's Identification of Enterobacteriaceae[END_REF]. S. sonnei has only one serotype, which reflects its clonal origin and low variation level [START_REF] Karaolis | Sequence Variation in Shigella Sonnei (Sonnei), a Pathogenic Clone of Escherichia Coli, over Four Continents and 41 Years[END_REF]

Evolution of Shigella

Despite being categorized as a separate genus, current evidence indicates that Shigella species are biotypes or clones of Escherichia coli. Early studies using Multi Locus Enzyme Electrophoresis to compare 1600 E. coli strains with 123 strains of the four species of Shigella clustered into E. coli groups, rather than clustering in a separate group [START_REF] Ochman | Enzyme Polymorphism and Genetic Population Structure in Escherichia Coli and Shigella[END_REF]. Phylogenetic analysis of the 16S ribosomal DNA also placed Shigella species and E. coli in the same phylogenetic group [START_REF] Cilia | Sequence Heterogeneities among 16S Ribosomal RNA Sequences, and Their Effect on Phylogenetic Analyses at the Species Level[END_REF]. Analysis of polymorphisms in housekeeping genes of Shigella and E.coli also suggests that different Shigella species are the result of convergent evolution rather than a single speciation event, with up to 8 separate divergence events happening within a time frame of 35,000 to 270,000 years from the present [START_REF] Pupo | Multiple Independent Origins of Shigella Clones of Escherichia Coli and Convergent Evolution of Many of Their Characteristics[END_REF], although more recent analysis indicate that the number of divergence event could be as low as four [START_REF] Sahl | Defining the Phylogenomics of Shigella Species: A Pathway to Diagnostics[END_REF]. Convergent evolution is further stressed by the fact that phenotypic characteristics such as lack of motility and lactose fermentation have different genetic basis in different strains across the genus [START_REF] Pupo | Multiple Independent Origins of Shigella Clones of Escherichia Coli and Convergent Evolution of Many of Their Characteristics[END_REF][START_REF] Al Mamun | Cloning and Characterization of the Region III Flagellar Operons of the Four Shigella Subgroups: Genetic Defects That Cause Loss of Flagella of Shigella Boydii and Shigella Sonnei[END_REF][START_REF] Ito | Possible Mechanisms Underlying the Slow Lactose Fermentation Phenotype in Shigella Spp[END_REF]. Also, the high diversity of the O antigen come from different mechanisms of acquisition; S. flexneri variations come mostly from phage-encoded enzymes [START_REF] Simmons | Structure and Biology of Shigella Flexneri O Antigens[END_REF], while several other unique antigens are identical to other E. coli strains (P. R. [START_REF] Edwards | Edwards and Ewing's Identification of Enterobacteriaceae[END_REF]. S. sonnei O antigen synthesis gene is encoded on a plasmid, presumably acquired by lateral transfer [START_REF] Kopecko | Genetic and Physical Evidence for Plasmid Control of Shigella Sonnei Form I Cell Surface Antigen[END_REF]. Furthermore, a whole-genome-alignmentphylogeny classifies Shigella serotypes in 5 distinct clades, differing from their current classification and reflecting its multiple origin (Figure 2). S. sonnei also encodes a Type VI Secretion System that contributes to killing of other microbiota competitors like E. coli, which can prevent pathogenic infections [START_REF] Anderson | Shigella Sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy[END_REF]. This advantage may be relevant for its increasing prevalence in developed countries, where S. sonnei is the most common Shigella serotype [START_REF] Kotloff | Shigellosis[END_REF]. One of the most relevant evolutionary events in the Shigella species is the appearance of a large virulence plasmid pINV, which will be further discussed in more detail. In addition to this event, other plasmids with different functions have also been acquired by different species, including the plasmid pHS-2, which control the O antigen chain-lengtht and the plasmids pDPT1 and pSSE3, which encode the production of colicins [START_REF] Calcuttawala | Activity Spectrum of Colicins Produced by Shigella Sonnei and Genetic Mechanism of Colicin Resistance in Conspecific S. Sonnei Strains and Escherichia Coli[END_REF]. Most recently, acquisition of plasmids encoding antibiotic resistance have been found, including resistance to sulfonamide, streptomycin and tetracycline [START_REF] Holt | Shigella Sonnei Genome Sequencing and Phylogenetic Analysis Indicate Recent Global Dissemination from Europe[END_REF], fluoroquinolones [START_REF] Gu | Comparison of the Prevalence and Changing Resistance to Nalidixic Acid and Ciprofloxacin of Shigella between Europe-America and Asia-Africa from 1998 to 2009[END_REF]) and azithromycin [START_REF] Baker | Intercontinental Dissemination of Azithromycin-Resistant Shigellosis through Sexual Transmission: A Cross-Sectional Study[END_REF].

At the genomic level, Shigella spp. has incorporated pathogenesis-associated genomic regions, or Pathogenicity Islands (PAI), which enhance its virulence and adaptation to its niche. Three PAIs have been identified in S. flexneri, with the role of some encoded proteins identified. LPS modification enzymes [START_REF] Allison | Serotype-Converting Bacteriophages and O-Antigen Modification in Shigella Flexneri[END_REF] allow the evasion of the humoral immune response, which targets mainly the O antigen; enterotoxins ShET1 and ShET2 play a role in the watery diarrhea [START_REF] Fasano | Shigella Enterotoxin 1: An Enterotoxin of Shigella Flexneri 2a Active in Rabbit Small Intestine in Vivo and in Vitro[END_REF] Proteases [START_REF] Rajakumar | Use of a Novel Approach, Termed Island Probing, Identifies the Shigella Flexneri She Pathogenicity Island Which Encodes a Homolog of the Immunoglobulin A Protease-like Family of Proteins[END_REF] play different roles, Pic plays a role in the degradation of the extracellular environment in the gut and in the host [START_REF] Henderson | Characterization of Pic, a Secreted Protease of Shigella Flexneri and Enteroaggregative Escherichia Coli[END_REF][START_REF] Al-Hasani | The Immunogenic SigA Enterotoxin of Shigella Flexneri 2a Binds to HEp-2 Cells and Induces Fodrin Redistribution in Intoxicated Epithelial Cells[END_REF]; iron acquisition genes in the aerobactin operon allow Shigella to counteract the iron-deficient environment [START_REF] Moss | The selC-Associated SHI-2 Pathogenicity Island of Shigella Flexneri[END_REF][START_REF] Nassif | Evaluation with an iuc::Tn10 Mutant of the Role of Aerobactin Production in the Virulence of Shigella Flexneri[END_REF]; colicin resistance genes may play a role in its survival in the intestinal lumen [START_REF] Vokes | The Aerobactin Iron Transport System Genes in Shigella Flexneri Are Present within a Pathogenicity Island[END_REF]. S. dysenteriae type 1 also produces the Shiga toxin, which inhibits host protein synthesis. This leads to fluid accumulation, paralysis and cell death. Because the kidney highly expresses the toxin receptor Gb3, this toxin can also lead to hemorrhagic uremic syndrome [START_REF] Johannes | Shiga Toxins -from Cell Biology to Biomedical Applications[END_REF].

Shigella virulence plasmid

The Shigella large virulence plasmid varies in size, being as large as 220 kb (Figure 3). This plasmid is sufficient to confer invasion in eukaryotic cells by otherwise noninvasive E. coli strains (Sansonetti et al. 1983). This plasmid contains around 280 open reading frames (ORFs), of which half are associated with insertion sequence elements suggesting a highly polyphyletic origin. For example, Yersinia low-calciumresponse plasmid harbors a sequence homologous to a region in the ipa-mxi-spa gene cluster. Other ORFs homologous to genes of M. tuberculosis, S. typhimurium, M. bovis, E. coli, and V. cholerae have also been reported [START_REF] Venkatesan | Complete DNA Sequence and Analysis of the Large Virulence Plasmid of Shigella Flexneri[END_REF].

Importantly, the virulence plasmid harbors a 32 kb gene cluster that encodes the Mxi-Spa Type III Secretion System (Figure 3), which structure and role in invasion will be further described. In the same cluster, there are also a series of effector proteins, termed "Invasion Plasmid Antigens", or Ipas, that mediate bacterial invasion and manipulate the immune response. The transcriptional regulation of this region is controlled by temperature, osmolarity and pH, with optimal conditions being 37°C, physiological saline concentration and pH 7.4 [START_REF] Maurelli | Temperature-Dependent Expression of Virulence Genes in Shigella Species[END_REF][START_REF] Porter | A Role for H-NS in the Thermo-Osmotic Regulation of Virulence Gene Expression in Shigella Flexneri[END_REF][START_REF] Nakayama | Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella Sonnei virF Gene[END_REF]. The virF regulatory gene is normally silenced by the chromosomally encoded H-NS protein until a shift in temperature to 37°C releases the transcriptional silencing by adjusting the DNA structure at the virfF promoter [START_REF] Maurelli | Identification of a Chromosomal Gene Controlling Temperature-Regulated Expression of Shigella Virulence[END_REF][START_REF] Stoebel | Anti-Silencing: Overcoming H-NS-Mediated Repression of Transcription in Gram-Negative Enteric Bacteria[END_REF].

VirF promotes the transcription of VirB, which antagonizes H-NS repression.

Specifically, VirB activates the mxi and spa genes that encode the T3SS, chaperon and protein effector genes and the regulator gene mxiE [START_REF] Schroeder | Molecular Pathogenesis of Shigella Spp.: Controlling Host Cell Signaling, Invasion, and Death by Type III Secretion[END_REF]. O antigen biosynthesis cluster (blue); genes encoding proteins with other known functions (green). ipa, invasion plasmid antigen gene; icsP, also known as sopA; T3SS, type III secretion system. Modified from [START_REF] The | The Genomic Signatures of Shigella Evolution, Adaptation and Geographical Spread[END_REF].

Overview of Shigella invasion

Before reaching the intestinal epithelium, Shigella needs to overcome physiological barriers throughout the digestive tract. Shigela spp. are resistant to acidic environments, presumably through glutamate-dependent acid-resistance pathway which allows them to survive the passage through the stomach [START_REF] Jennison | The Acid-Resistance Pathways of Shigella Flexneri 2457T[END_REF]. Afterwards, its primary sites of infection are the terminal ileum, colon and rectum. Another barrier Shigella has to overcome is the mucin layer that covers the intestinal epithelium. This layer consists of a heterogeneous meshwork of proteins including the large glycoproteins mucins (>2 x 10 6 Da). All four species of Shigella produce mucinase and neuraminidase [START_REF] Haider | Production of Mucinase and Neuraminidase and Binding of Shigella to Intestinal Mucin[END_REF], which may allow them to degrade this barrier and get access to the intestinal epithelial cells.

Anatomy of the intestinal epithelium

The intestinal epithelium is a singe-cell layer that extends across the 4 regions of the intestine (jejunum, ileum, caecum and colon). Its structure differs between regions.

The small intestine is rich in villi, protrusions that extend into the lumen and maximize the contact area and therefore the nutrient adsorption. In opposition, villi are absent in the colon. The epithelium also forms invaginations named "crypts of Lieberkün". [START_REF] Crosnier | Organizing Cell Renewal in the Intestine: Stem Cells, Signals and Combinatorial Control[END_REF]).

Enterocytes are polarized and form a highly tight barrier through tight junctions, adherens junctions and desmosomes. This barrier is permeable to ions, nutrients and water, while restricting the passage of proteins, lipids and microorganisms from the lumen to the lamina propria (Takuya Suzuki 2013). In addition to epithelial cells, there is also an abundant population of immune cells interspaced with epithelial cells. Tuft cells detect the presence of helminths [START_REF] Gerbe | Intestinal Epithelial Tuft Cells Initiate Type 2 Mucosal Immunity to Helminth Parasites[END_REF]) and M cells perform uptake and presentation of luminal antigens to immune cells [START_REF] Ohno | Intestinal M Cells[END_REF].

M cells are associated to subepithelial lymphoid aggregates, termed gut-associated lymphoid tissue, which prime adaptive immune cell responses in the intestine by presenting antigens to dendritic cells. The best characterized of these aggregates are the Peyer's patches in the small intestine [START_REF] Cornes | Number, Size, and Distribution of Peyer's Patches in the Human Small Intestine: Part I The Development of Peyer's Patches[END_REF], which consists of B cell lymphoid follicles and T cell areas. Also, in the large intestine, there are structures termed cecal patches in the ileocaecal valve and colonic patches in the colon and rectum. Both patches resemble in function and structure to Peyer patches, albeit smaller in size [START_REF] Owen | Ultrastructural and Cytoarchitectural Features of Lymphoreticular Organs in the Colon and Rectum of Adult BALB/c Mice[END_REF].

In the lamina propria, there are also CD4+ and CD8+ T cells, with an effector memory phenotype. There is also a large population of IgA and IgM producing B cells and macrophages. These antibodies are shed into the lumen of the intestine and prevent bacterial adhesion to the epithelium. Eosinophils and mast cells are also present along the intestinal tract and contribute to the regulation of the inflammatory response (for a review see [START_REF] Mowat | Regional Specialization within the Intestinal Immune System[END_REF]).

Shigella invasion routes of the intestinal epithelium

The initial routes of invasion are not entirely clear and will be further discussed in detail. Early observation of biopsies from Shigella-infected patients suggested the gut-associated lymphoid tissue as one of the primary sites targeted [START_REF] Mathan | Morphology of Rectal Mucosa of Patients with Shigellosis[END_REF]). In the rabbit ileal loop model, M cells were reported as the initial route of entry [START_REF] Wassef | Role of M Cells in Initial Antigen Uptake and in Ulcer Formation in the Rabbit Intestinal Loop Model of Shigellosis[END_REF]. However, a guinea pig model that recapitulates several clinical features of the human infection shows that epithelial cells are invaded as early as 2-6 hours post-administration [START_REF] Shim | New Animal Model of Shigellosis in the Guinea Pig: Its Usefulness for Protective Efficacy Studies[END_REF].

Using a guinea pig intra-rectal inoculation model, it was observed that enterocytes at the colonic crypts mouths were targeted at the onset of colonization and bacteria spread by cell-to-cell transmission into the crypts along the infection [START_REF] Arena | Bioimage Analysis ofShigellainfection Reveals Targeting of Colonic Crypts[END_REF]. In the rabbit ileal loop model, there is evidence that Shigella traverses the cell monolayer through M cells [START_REF] Sansonetti | Infection of Rabbit Peyer's Patches by Shigella Flexneri: Effect of Adhesive or Invasive Bacterial Phenotypes on Follicle-Associated Epithelium[END_REF], a step that would allow it to infect macrophages in the M cell pouch. According to this model, after inducing the pyroptotic death of macrophages, Shigella would invade epithelial cells from the basal side. Pyroptosis also leads to the release of pro-inflammatory cytokines IL-1β and IL-18, and invasion of epithelial release of IL-8. This causes a strong inflammatory response that recruits polymorphonuclear leukocytes and destabilizes the intestinal epithelium, hence facilitating invasion from lumenal bacteria.

The Shigella Type III Secretion System

The Shigella T3SS structure

A key element for the pathogenesis of Shigella is its T3SS. This apparatus functions as a needle, translocating effector proteins from the bacterial cytoplasm to the host cell cytosol (Figure 5). Assembly of fully functional T3SS apparatus is regulated by temperature but the injection of T3 effectors is dependent on host membrane contact [START_REF] Veenendaal | The Type III Secretion System Needle Tip Complex Mediates Host Cell Sensing and Translocon Insertion[END_REF]).

The T3SS is generally divided in three parts: the basal body, the needle and the tip complex. The basal body anchors the apparatus to the bacterial membranes (Figure 5). MxiD and MxiM form a ring in the outer membrane. On the cytoplasmic side, a Cring formed by the proteins Spa33, MxiK and MxiN mediates the recognition, sorting and secretion of effectors, where the ATPase activity of Spa47 is involved (B. Hu et al. 2015;[START_REF] Burgess | Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation[END_REF][START_REF] Morita-Ishihara | Shigella Spa33 Is an Essential C-Ring Component of Type III Secretion Machinery[END_REF]). Spa32 interacts with Spa40 and controls the length of the needle [START_REF] Botteaux | The 33 Carboxyl-Terminal Residues of Spa40 Orchestrate the Multi-Step Assembly Process of the Type III Secretion Needle Complex in Shigella Flexneri[END_REF][START_REF] Tamano | Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length[END_REF]. Other proteins are also present, but their roles remain unclear.

The needle is composed of the major MxiH and the minor MxiI subunits. MxiH assembles in a helical fashion and constitutes the needle rod with a 2-3 nm inner diameter [START_REF] Blocker | Structure and Composition of the Shigella Flexneri 'Needle Complex', a Part of Its Type III Secreton[END_REF], while MxiI is proposed to be involved, together with MxiC, in the secretion activation upon contact of the host membrane [START_REF] Hajjami | The Inner-Rod Component of Shigella Flexneri Type 3 Secretion System, MxiI, Is Involved in the Transmission of the Secretion Activation Signal by Its Interaction with MxiC[END_REF][START_REF] Cherradi | Interplay between Predicted Inner-Rod and Gatekeeper in Controlling Substrate Specificity of the Type III Secretion System[END_REF].

Before contact with the host membrane the needle tip complex composed of IpaD and perhaps IpaB remains in a closed conformation [START_REF] Veenendaal | The Type III Secretion System Needle Tip Complex Mediates Host Cell Sensing and Translocon Insertion[END_REF][START_REF] Cheung | Three-Dimensional Electron Microscopy Reconstruction and Cysteine-Mediated Crosslinking Provide a Model of the Type III Secretion System Needle Tip Complex[END_REF]. IpaB and IpaC are translocated upon contact with host cells, likely by interacting with membrane enriched in cholesterol and sphingolipidscontaining membranes (Goot et al. 2004). IpaB and IpaC form the translocon allowing the translocation of effector proteins.

Shigella T3SS effector proteins

At 37°C the transcriptional activator VirF is expressed and activates virB expression.

VirB is also a transcriptional activator that activates the ipa (Invasion Plasmid Antigens) operon and the mxi (Membrane Expression of Ipa) -spa (Surface Presentation of Ipa) operon. Expression of these operons triggers bacterial entry mediated by the T3SS [START_REF] Du | The Type III Secretion System Apparatus Determines the Intracellular Niche of Bacterial Pathogens[END_REF], along with other effector genes. Injection of effector proteins is temporarily divided into two waves. After initial host cell contact, the injection of the first wave of effectors happens within minutes (Enninga et al. 2005).

Shigella T3SS uses chaperons to prevent degradation and to target effector proteins to the secretion apparatus. The IpgC chaperone also regulates the expression of the second wave of T3S effectors. Prior to activation of T3 secretion, the IpgC chaperone associates with IpaB and IpaC and prevents their premature interaction in the bacterial cytosol [START_REF] Parsot | The Various and Varying Roles of Specific Chaperones in Type III Secretion Systems[END_REF][START_REF] Pilonieta | The Chaperone IpgC Copurifies with the Virulence Regulator MxiE[END_REF]. After activation of T3 secretion, IpaB and IpaC are secreted, releasing IpgC, which may then interact with the MxiE transcriptional activator. This allows the formation of the complex IpgC-MxiE that promotes triggering the transcription of genes harboring a MxiE box, (Figure 6) corresponding to genes of the second wave of effectors [START_REF] Kane | MxiE Regulates Intracellular Expression of Factors Secreted by the Shigella Flexneri 2a Type III Secretion System[END_REF].

Genes belonging to the second wave of effectors are mainly involved in high-jacking host signaling, apoptosis inhibition and regulation of long-term effects on the host immune response (For review, see [START_REF] Mattock | How Do the Virulence Factors of Shigella Work Together to Cause Disease?[END_REF]. The incorporation of actin monomers is asymmetrical. The barbed end (or plus end)

of actin filaments incorporates ATP-actin monomers, while the pointed end (or minus end) is more prone to be disassembled (Figure 7A). Simultaneous incorporation of monomers at one end and disassembly on the other gives rise to treadmilling.

Actin sequestering proteins

In the intracellular milieu, actin binds to profilin. This complex represents the main source of monomers for polymerization. Profilin catalyzes the ADP/ATP exchange in actin monomers, while inhibiting the spontaneous filament nucleation property of actin monomers [START_REF] Dominguez | Actin Structure and Function[END_REF]. Profilin-actin also binds to other proteins involved in actin polymerization, such as formins and Ena/VASP proteins [START_REF] Kovar | Control of the Assembly of ATP-and ADP-Actin by Formins and Profilin[END_REF]) (Figure 7).

Thymosin-β4 is a peptide of 43 residues. In its N-terminus it has short helix that binds the actin barbed end and the rest of the helix, forms an extended region that binds the front surface of actin and a second helix that caps actin pointed end [START_REF] Xue | Structural Basis of Thymosin-β4/profilin Exchange Leading to Actin Filament Polymerization[END_REF]. This results in the blockage of engagement of actin monomers into polymerization. However, profilin and thymosin-β4 are constantly exchanging actin monomers, allowing elongation of actin filaments [START_REF] Pantaloni | How Profilin Promotes Actin Filament Assembly in the Presence of Thymosin Beta 4[END_REF].

Severing proteins

Depolymerization in cells is enhanced by proteins of the ADF/Cofilin family, which includes Actin Depolymerization Factor (ADF), cofilin-1, cofilin-2 and twinfilin. This family of proteins have a higher affinity for ADP-actin than for ATP-actin, and therefore bind to ADP-actin fiber stretches and promote the severing of these filaments (Barbara W. [START_REF] Bernstein | ADF/Cofilin: A Functional Node in Cell Biology[END_REF].

Gelsolin proteins also sever actin filaments and cap their barbed ends. Most of its family members' activity is regulated by calcium binding. Mutants of this protein have defects in cellular motility and decreased blood clotting. Modified from [START_REF] Svitkina | The Actin Cytoskeleton and Actin-Based Motility[END_REF]).

Actin nucleators

Actin nucleators initiate the de novo polymerization of actin filaments. [START_REF] Pollard | Regulation of Actin Filament Assembly by Arp2/3 Complex and Formins[END_REF]. Given the Arp2/3 structure, the daughter filament elongates at an angle of 70° from the mother filament [START_REF] Amann | Direct Real-Time Observation of Actin Filament Branching Mediated by Arp2/3 Complex Using Total Internal Reflection Fluorescence Microscopy[END_REF]. At a large scale, polymerization of new branched filaments upon existing filaments give rise to branched actin networks, which are enriched in lamellipodia (Figure 7B).

In opposition to Arp2/3, formins nucleate and elongate unbranched actin filaments in filopodia, stress fibers and actin cables. They are characterized by the presence of the Formin Homology Domains 1 and 2, which mediate their interaction with profilin and their nucleation activity, respectively. Their ATP-dependent activity results in a highly processive actin polymerization [START_REF] Romero | Formin Is a Processive Motor That Requires Profilin to Accelerate Actin Assembly and Associated ATP Hydrolysis[END_REF]). In a similar fashion, Ena/VASP proteins also promote the nucleation and elongation of actin filaments (Figure 7C), and works in neurons show that they are particularly important for filopodial formation [START_REF] Lebrand | Critical Role of Ena/VASP Proteins for Filopodia Formation in Neurons and in Function Downstream of Netrin-1[END_REF]).

Capping proteins

Capping protein and CapZ bind to the barbed end of actin filaments, blocking both the addition and loss of actin subunits [START_REF] Isenberg | An Actin-Binding Protein from Acanthamoeba Regulates Actin Filament Polymerization and Interactions[END_REF][START_REF] Xu | Effect of Capping Protein, CapZ, on the Length of Actin Filaments and Mechanical Properties of Actin Filament Networks[END_REF], therefore preventing the assembly/disassembly of filaments. In conjunction with the branching activity of Arp2/3, a network of short and highly branched actin filaments can be formed (Figure 7B) (M. [START_REF] Edwards | Capping Protein Regulators Fine-Tune Actin Assembly Dynamics[END_REF].

Cross-linking proteins

Individual actin filaments can be bundled together through the action of actin crosslinkers, such as α-actinin isoforms 1 and 4 or fimbrin. These proteins contain Actin-Binding Domains (ABD) that connects two separate actin fibers. α-actinin has also the ability to associate with other cytoskeletal, signaling and membrane molecules [START_REF] Sjöblom | α-Actinin Structure and Regulation[END_REF].

The organization into bundles by filament bundling facilitates the mechanical contraction exerted by myosins molecular motors. In particular, Myosin II transforms the chemical energy released by ATP hydrolysis into mechanical work that results in pulling of actin filaments (Figure 8). The pulling activity is the result of the myosin II polymerization into bipolar filaments, with the motor domains walking in opposing cross-linked actin filaments (Figure 8) [START_REF] Sweeney | Motor Proteins[END_REF]. 9).

Although not proven for all αβ pairs, some integrins can transition from a "bent closed" or inactive form, to an extended closed and finally to an extended open. This transition is associated with an increase in the affinity of the integrin for its substrate. process termed "inside-out activation", as opposed to "outside-in signaling"

implicating the activation of integrins through the binding of their extracellular ligand.

Talin recruitment to the membrane is presumably achieved through its interaction with Rap1-GTP-Interacting-Adaptor-Molecule (RIAM), which links membrane associated Rap1 with cytoplasmic talin [START_REF] Lee | RIAM Activates Integrins by Linking Talin to Ras GTPase Membrane-Targeting Sequences[END_REF] and concentrates it at the tip of actin growing filaments in lamellipodial and filopodial protrusions [START_REF] Lagarrigue | A RIAM/lamellipodin-talin-integrin Complex Forms the Tip of Sticky Fingers That Guide Cell Migration[END_REF].

Integrins display catch-slip bonds with RGD-ligands [START_REF] Kong | Demonstration of Catch Bonds between an Integrin and Its Ligand[END_REF], ICAM (W.

Chen, Lou, and Zhu 2010) and VCAM [START_REF] Choi | Dynamic Control of β1 Integrin Adhesion by the plexinD1-sema3E Axis[END_REF]. In an ideal bond between molecules, the lifetime of the interaction would not depend on force. However, bonds usually behave under non-ideal regimes. Under a slip regime, the bond between two molecules will decrease its lifetime as force is applied. Counter-intuitively, catch-slip bonds undergo first a catch regime where application of force will increase the lifetime of the bond, therefore strengthening the interaction. This catch regime is followed by a slip regime until detachment [START_REF] Rakshit | Biomechanics of Cell Adhesion: How Force Regulates the Lifetime of Adhesive Bonds at the Single Molecule Level[END_REF].

In order for the force to be effectively transmitted between the actomyosin cytoskeleton and the extracellular environment, integrin molecules need to be connected to the actin cytoskeleton. This task is performed by linker proteins such as talin, tensin, filamin and α-actinin, which bind simultaneously to integrins and F-actin.

This force-enhanced process leads to the maturation of adhesion structures through the recruitment of additional scaffolding and signaling molecules.

Adhesion formation and maturation

Filopodia are finger-like structures that protrude out of the cells' edge. Its extension is driven by actin polymerization mediated by formins and regulated by Rho GTPases, capping proteins and Ena/VASP [START_REF] Gupton | Filopodia: The Fingers That Do the Walking[END_REF][START_REF] Lagarrigue | A RIAM/lamellipodin-talin-integrin Complex Forms the Tip of Sticky Fingers That Guide Cell Migration[END_REF][START_REF] Albuschies | The Role of Filopodia in the Recognition of Nanotopographies[END_REF]. Deeper into the cell edge, short-lived adhesions (~60 seconds turnover) can be found in the lamellipodium, termed "nascent adhesions".

Larger adhesions, known as "focal complexes" with a persistence of several minutes can be found further back from the leading edge. These focal complexes can mature into large focal adhesions (3-10 μm long) located at the extremes of stress fibers, with a persistence time of 30-60 min [START_REF] Zimerman | Early Molecular Events in the Assembly of the Focal Adhesion-Stress Fiber Complex during Fibroblast Spreading[END_REF].

There is evidence that adhesions at the shaft of the filopodia and nascent adhesions mature into focal adhesions upon the advancement of lamellipodia (Figure 10) [START_REF] Wong | Fibroblasts Probe Substrate Rigidity with Filopodia Extensions before Occupying an Area[END_REF]. Nascent adhesion formation is independent of myosin II and contain integrins, talin, kindlin, vinculin, α-actinin, paxilin and FAK. These adhesions can either disassemble or further mature into focal adhesions in a process largely driven by tension and crosslinking exerted by myosin II.

Talin and Vinculin molecular clutch

The molecular clutch hypothesis postulates that focal adhesions mediate the interactions between the retrograde-moving actin and ligand-bounded cytoskeleton.

This interaction regulates the protrusive activity of the actin cytoskeleton. In adherent migrating cells, the cortical actin cytoskeleton is organized into two structurally and functionally distinct regions: the lamellipodium and the lamellum. In the lamellipodium Arp2/3-driven actin polymerization generates pushing force. However, when the molecular clutch is not engaged (i.e. when actin fibers are not tethered to the extracellular matrix), actin movement result in treadmilling behaviour towards the interior of the cell due to the resistance from the plasma membrane, generating rearward movement. When the clutch is engaged, on the other hand, forces required to counteract membrane resistance are transmitted to the matrix and protrusions at the cell edge are generated (Figure 11). Interaction between actin cytoskeleton and the extracellular matrix involves transmembrane proteins and linker molecules. In focal adhesions, the linker integrins bind to the extracellular matrix. In addition, the role of talin as an integrin-actin linker molecule is remarkable, because it possesses structural characteristics of a molecular mechanosensor regulated by force. One characteristic is its ability to scaffold vinculin in a force-dependent manner (Figure 12). Talin is organized in two domains, the N-terminal FERM domain can bind phospholipids and integrin, responsible for the inside-out signaling activity through integrins [START_REF] Anthis | The Structure of an Integrin/talin Complex Reveals the Basis of inside-out Signal Transduction[END_REF]).

The C-terminal domain contains 13 helical bundles (designated R1-R13) that contain two actin binding sites on the rod domain and one in the FERM domain at least 11 putative Vinculin Binding sites (VBSs), each one constituted by a single amphipathic α-helix (Gingras et al. 2005). However, all of the VBSs helices are buried within α- Talin bundles unfold in a hierarchical manner, depending on the force required for their unfolding. It has been shown that all bundles, al least in vitro, are suceptible to be unfolded by stretching forces ranging from 5 to 30 pN [START_REF] Haining | All Subdomains of the Talin Rod Are Mechanically Vulnerable and May Contribute To Cellular Mechanosensing[END_REF]Yao et al. 2015). In its inactive form, vinculin has a globular structure consisting of two domains: a head domain and a tail domain connected by an unstructured linker (Figure 13). Both head and tail domains consist of α-helix bundles, the head is organized in four subdomains (named VD1-VD4), while the tail domain contains a single bundle (termed Vt) [START_REF] Borgon | Crystal Structure of Human Vinculin[END_REF]). The vinculin intra-molecular interaction occurs between the Vt and head subdomains VD1 and VD4 (Izard et al. 2004;[START_REF] Cohen | Two Distinct Head-Tail Interfaces Cooperate to Suppress Activation of Vinculin by Talin[END_REF]. When these interactions are disrupted, the actin binding site on Vt is free to interact with F-actin under a catch-bond regime (D. L. [START_REF] Huang | Vinculin Forms a Directionally Asymmetric Catch Bond with F-Actin[END_REF]. Reported VBSs from talin and α-actinin bind to the VD1 domain (Izard et al. 2004). However, whether talin VBSs exposure alone is sufficient for vinculin activation in vivo is a matter of debate. There are observations that phosphorylation on vinculin tyrosine residues 100 and 1065 by Src [START_REF] Auernheimer | Vinculin Phosphorylation at Residues Y100 and Y1065 Is Required for Cellular Force Transmission[END_REF] and Factin binding (H. Chen, Choudhury, and Craig 2006) facilitates vinculin activation.

Also, PI(4, 5)P2 is required for vinculin sequestration at FAs and contributes to their stabilization [START_REF] Chinthalapudi | Lipid Binding Promotes Oligomerization and Focal Adhesion Activity of Vinculin[END_REF]. interaction with VD1 disrupts heat-tail interaction and leads to interaction with F-actin (purple). Modified from [START_REF] Bakolitsa | Structural Basis for Vinculin Activation at Sites of Cell Adhesion[END_REF]Park et al. 2014).

Force generation and maturation

Initial nascent adhesion formation is dependent on actin polymerization, in which Rac1 activation and Arp2/3 have been implicated [START_REF] Wu | Arp2/3 Is Critical for Lamellipodia and Response to Extracellular Matrix Cues but Is Dispensable for Chemotaxis[END_REF].

This is in contrast to nascent adhesions that mature into focal adhesions in a process that requires stress fibre assembly and myosin II activity [START_REF] Oakes | Tension Is Required but Not Sufficient for Focal Adhesion Maturation without a Stress Fiber Template[END_REF]. Myosin II activity is sustained by Rho through the action of Rho associated protein kinase (ROCK). As adhesion structures mature, its composition changes and generates a complex 3D organization (Figure 14). Signalling proteins like FAK and Paxilin localize next to the integrin cytoplasmic tails, together with the Talin head. However, talin rod protrudes inwards, colocalizing with vinculin, which is initially recruited to the plasma membrane, but relocalizes as the adhesion matures and talin VBS are unveiled by force. Talin tail, which binds to F-actin colocalizes with proteins like zyxin, VASP and α-actinin [START_REF] Case | Integration of Actin Dynamics and Cell Adhesion by a Three-Dimensional, Mechanosensitive Molecular Clutch[END_REF]. Gram-negative bacteria, as described in Table 2. Bacteria also express adhesion proteins at their surface that promote a tight binding to host cells that are relevant for attachment and invasion. Theses proteins commonly target host surface molecules or the extracellular matrix, as can be seen in table 3.

Y. pseudotuberculosis Invasin β1-integrin

In addition to classically described adhesin-receptor interactions, some bacteria have evolved more sophisticated forms of targeting their niche, which in some cases include the manipulation of the cell intracellular machinery, notably the cell cytoskeleton.

E. coli FimH catch-bond adhesion

Uropathogenic E. coli, a bacterial species that colonizes the urinary tract, expresses a variant of the type 1 fimbriae whose affinity for its receptor is enhanced under high flow velocity conditions (high shear-stress) [START_REF] Thomas | Bacterial Adhesion to Target Cells Enhanced by Shear Force[END_REF].

Type I fimbriae are assembled via the chaperone/usher pathway, which consists of: a chaperon protein which interacts with the pilus and usher proteins including an usher protein, which forms the translocation pore in the outer membrane. Around 1000 copies of the subunit FimA forms the fimbriae's rod, while a single copy of the FimF,

FimG and FimH subunits constitute the adhesive tip. FimH, located at the fimbriae distal part, is highly variable, which reflects different specificities for different targets [START_REF] Mortezaei | Structure and Function of Enterotoxigenic Escherichia Coli Fimbriae from Differing Assembly Pathways[END_REF]. FimH from the uropathogenic E.coli strain J96 contains three aminoacid substitutions relative to the gene encoded by the intestinal E. coli strain F18 that confer its catch-bond binding properties for the mannose receptor [START_REF] Thomas | Bacterial Adhesion to Target Cells Enhanced by Shear Force[END_REF]. Conformational changes between the pilin domain and the lectin domain mediate this process. The physical separation of these domains induced by force removes the allosteric inhibition exerted by the pilin domain upon the binding of the lectin domain for its target (Figure 15), increasing the affinity of the lectin for the mannose resiude [START_REF] Sauer | Catch-Bond Mechanism of the Bacterial Adhesin FimH[END_REF]. Listeria entry in non-phagocytic cells is mediated by two surface-expressed proteins:

Internalin A (InlA) and Internalin B (InlB), which bind to their eukaryotic receptors E-cadherin [START_REF] Gaillard | Entry of L. Monocytogenes into Cells Is Mediated by Internalin, a Repeat Protein Reminiscent of Surface Antigens from Gram-Positive Cocci[END_REF][START_REF] Mengaud | E-Cadherin Is the Receptor for Internalin, a Surface Protein Required for Entry of L. Monocytogenes into Epithelial Cells[END_REF]) and c-Met [START_REF] Pentecost | Listeria Monocytogenes Internalin B Activates Junctional Endocytosis to Accelerate Intestinal Invasion[END_REF], respectively.

E-cadherin is part of adherens junctions that mediate cell-to-cell adhesions in the intestinal epithelium. This transmembrane protein mediates homotypic interactions between cells and are connected to the actomyosin cytoskeleton mainly through αcatenin, β-catenin and p120-catenin. Both cadherin-cadherin interactions and the cadherin-catenin complex (E-cadherin/α-catenin/β-catenin) interaction with F-actin display a catch-bond behaviour [START_REF] Buckley | Cell Adhesion. The Minimal Cadherin-Catenin Complex Binds to Actin Filaments under Force[END_REF][START_REF] Rakshit | Ideal, Catch, and Slip Bonds in Cadherin Adhesion[END_REF]. p120

phosphorylation state is also able to allosterically regulate the affinity of homophilic cadherin interactions [START_REF] Shashikanth | Allosteric Regulation of E-Cadherin Adhesion[END_REF].

Targeting E-cadherin by InlA is sufficient to promote internalization [START_REF] Lecuit | Internalin of Listeria Monocytogenes with an Intact Leucine-Rich Repeat Region Is Sufficient to Promote Internalization[END_REF]. InlA affinity for E-cadherin, however, is low, and is thought to be compensated by the high amount of molecules throughout the bacterial surface, and might allow it to dissociate from E-cadherin once internalized [START_REF] Wollert | Thermodynamically Reengineering the Listerial Invasion Complex InlA/E-Cadherin[END_REF].

Initial interaction of InlA with E-cadherin requires its presence in lipid rafts and leads to the recruitment of α-catenin, β-catenin and p120-catenin [START_REF] Lecuit | A Role for Alpha -and Beta -Catenins in Bacterial Uptake[END_REF].

Listeria internalization involves the recruitment of Src kinase [START_REF] Sousa | Src, Cortactin and Arp2/3 Complex Are Required for E-Cadherin-Mediated Internalization of Listeria into Cells[END_REF]),

which phosphorylates E-cadherin and triggers its ubiquitination by Hakai, thus marking E-cadherin molecules for endocytosis [START_REF] Bonazzi | Successive Post-Translational Modifications of E-Cadherin Are Required for InlA-Mediated Internalization of Listeria Monocytogenes[END_REF]. Src kinase, together with Rac1 promote the recruitment of cortactin, an activator of Arp2/3. This cascade leads to a local and transient actin polymerization event that is essential for internalization. Molecular motor myosin VIIa and vezatin are also recruited by αcatenin to bacterial entry sites and might play a role in the generation of force for Listeria internalization [START_REF] Sousa | Unconventional Myosin VIIa and Vezatin, Two Proteins Crucial for Listeria Entry into Epithelial Cells[END_REF]).

Attaching and Effacing Lesion Pathogens

Pathogens in this category include Enteropathogenic E. coli (EPEC), Enterohemorrhagic E.coli (EHEC) and C. rodentium. These bacterial pathogens remain extracellular and remodel the enterocyte cell surface by effacing the microvilli and create a novel structure termed "pedestal" underneath them (Figure 16).

The pedestal formation depends on a T3SS, which inserts the bacterial Tir effector in the host membrane. In EPEC, Tir serves as a receptor for the bacterial surface protein Intimin [START_REF] Kenny | Enteropathogenic E. Coli (EPEC) Transfers Its Receptor for Intimate Adherence into Mammalian Cells[END_REF]. Tir triggers Intimin clustering and phosphorylation by host kinases of the families Abl/Arg, Src, and Tec families [START_REF] Kenny | Phosphorylation of Tyrosine 474 of the Enteropathogenic Escherichia Coli (EPEC) Tir Receptor Molecule Is Essential for Actin Nucleating Activity and Is Preceded by Additional Host Modifications[END_REF]. This phosphorylation results in the binding of the adaptor proteins Nck1 and 2 through their SH domains [START_REF] Gruenheid | Enteropathogenic E. Coli Tir Binds Nck to Initiate Actin Pedestal Formation in Host Cells[END_REF]. This leads to the further recruitment of N-WASP and ultimately to the activation of the AcRP2/3 complex [START_REF] Kalman | Enteropathogenic E. Coli Acts through WASP and Arp2/3 Complex to Form Actin Pedestals[END_REF], resulting in actin polymerization. In EHEC, Arp2/3 is activated upon the multimerization induced by the translocated bacterial protein EspFU [START_REF] Campellone | EspFU Is a Translocated EHEC Effector That Interacts with Tir and N-WASP and Promotes Nck-Independent Actin Assembly[END_REF].

Salmonella "triggered" internalization

Salmonella enterica species encompass a group of bacterial enteropathogenic strains that cause diseases in animals and humans. S. Typhi and a few other non-Typhoidal serotypes can cause serious systemic disease in humans. All strains share the ability to invade intestinal epithelial cells. S. Typhi can also invade the spleen, liver and gallbladder [START_REF] Dougan | Salmonella Enterica Serovar Typhi and the Pathogenesis of Typhoid Fever[END_REF]. Invasion of Salmonella is associated with the generation of membrane ruffles around its entry site, generated by the action of injected effector proteins, by a so-called "trigger mechanism".

Salmonella expresses a T3SS in its Pathogenicity Island 1 (SPI-1) that translocates protein effectors that manipulate the host cytoskeleton. The SopB, SopE and SopE2 effectors promote Salmonella uptake by host cells and a triple mutant prevents cytoskeletal remodeling and entry [START_REF] Zhou | A Salmonella Inositol Polyphosphatase Acts in Conjunction with Other Bacterial Effectors to Promote Host Cell Actin Cytoskeleton Rearrangements and Bacterial Internalization[END_REF]. SopB activity as a phosphatidylinositol 4, 5 bisphosphate phosphatase leads to ruffle formation. Its activity causes the accumulation of PI3P, PI(3,4)P2 and PI(3,4,5)P3 [START_REF] Mallo | SopB Promotes Phosphatidylinositol 3-Phosphate Formation on Salmonella Vacuoles by Recruiting Rab5 and Vps34[END_REF] which, among other effects lead to the activation of RhoG [START_REF] Patel | Differential Activation and Function of Rho GTPases during Salmonella-Host Cell Interactions[END_REF].

The SopE2 and SopE effectors are GEF mimics. The former activates Cdc42 [START_REF] Stender | Identification of SopE2 from Salmonella Typhimurium, a Conserved Guanine Nucleotide Exchange Factor for Cdc42 of the Host Cell[END_REF] and the latter activates both Cdc42 and Rac1 [START_REF] Hardt | S. Typhimurium Encodes an Activator of Rho GTPases That Induces Membrane Ruffling and Nuclear Responses in Host Cells[END_REF][START_REF] Friebel | SopE and SopE2 from Salmonella Typhimurium Activate Different Sets of RhoGTPases of the Host Cell[END_REF]. RhoG and Rac1 activation contributes to ruffle formation and bacteria uptake, while Cdc42 activation is important for downstream signal transduction [START_REF] Patel | Differential Activation and Function of Rho GTPases during Salmonella-Host Cell Interactions[END_REF]. Actin is also directly targeted by Salmonella through SipA and SipC. SipA prevents actin depolymerization by binding to and stabilizing actin filaments [START_REF] Mcghie | Control of Actin Turnover by a Salmonella Invasion Protein[END_REF]) SipC, which is also part of the T3SS translocon, nucleates and bundles actin filaments [START_REF] Hayward | Direct Nucleation and Bundling of Actin by the SipC Protein of Invasive Salmonella[END_REF].

Shigella Invasion and Cytoskeleton remodelling

At initial stages of the invasion process, binding of bile salts to IpaD favors exposure of IpaB at the tip complex [START_REF] Epler | Ultrastructural Analysis of IpaD at the Tip of the Nascent MxiH Type III Secretion Apparatus of Shigella Flexneri[END_REF]. IpaB binds to the extracellular portion of the host receptor CD44 (Skoudy et al. 2000;Lafont et al. 2002). Shigella interacts with filopodia through the tip complex proteins IpaB and IpaD and is pulled towards the cell body (Figure 17A) (Romero et al. 2011). This interaction is mediated by a limited number of T3SS apparatus in the bacterial surface, which can withstand stall forces exerted by retracting filopodia in the ranges of tens of picoNewtons. (Romero et al. 2012). Bacterial invasion is achieved by the interplay of proteins from the first wave (Figure 16B). In addition to its role as part of the translocon, IpaC also possesses the ability to induce actin polymerization through Src [START_REF] Mounier | The IpaC Carboxyterminal Effector Domain Mediates Src-Dependent Actin Polymerization during Shigella Invasion of Epithelial Cells[END_REF]) and Rac [START_REF] Van Nhieu | IpaC Induces Actin Polymerization and Filopodia Formation during Shigella Entry into Epithelial Cells[END_REF]). IpgB1 and IpgB2 are GEFs that target and activate Rac1 and RhoA, respectively (Z. [START_REF] Huang | Structural Insights into Host GTPase Isoform Selection by a Family of Bacterial GEF Mimics[END_REF], contributing to the formation of Arp2/3dependent membrane ruffles. IpgD has a phosphatidylinositol 4, 5 bisphosphate 4phosphatase activity, this decreases the tethering force between the plasma membrane and the actin cytoskeleton [START_REF] Niebuhr | Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri Effector IpgD Reorganizes Host Cell Morphology[END_REF]. IpgD also inhibits calcium signaling leading to the preservation of cell adhesion structures (Sun et al. 2017). Furthermore, its activity leads to a positive feedback loop that promotes ARF6 activation, leading to a Rac1-dependent actin remodelling (Garza-Mayers et al.

2015)

. IpaA role will be further discussed in detail.

Once Shigella is internalized inside a vacuole, it escapes into the host cytosol. The Shigella T3 apparatus activity has been shown to trigger vacuolar lysis [START_REF] Du | The Type III Secretion System Apparatus Determines the Intracellular Niche of Bacterial Pathogens[END_REF]), IpgD-mediated interaction of Rab-11 containing vacuoles and macropinosomes with the bacterial vacuole might also affect the stability of the vacuole [START_REF] Mellouk | Shigella Subverts the Host Recycling Compartment to Rupture Its Vacuole[END_REF][START_REF] Weiner | Macropinosomes Are Key Players in Early Shigella Invasion and Vacuolar Escape in Epithelial Cells[END_REF].

Shigella does not express a flagellum and is non-motile in the extracellular environment. However, once free in the cytosol, Shigella exhibits intracellular motility.

The above-mentioned outer membrane protein IcsA is expressed at one pole of the bacteria and binds N-WASP, which activates Arp2/3, leading to the formation of an actin comet tail [START_REF] Egile | Activation of the CDC42 Effector N-WASP by the Shigella Flexneri IcsA Protein Promotes Actin Nucleation by Arp2/3 Complex and Bacterial Actin-Based Motility[END_REF]). This activity is further enhanced by the T3SSdependent recruitment of the N-WASP activator, TOCA-1 [START_REF] Egile | Activation of the CDC42 Effector N-WASP by the Shigella Flexneri IcsA Protein Promotes Actin Nucleation by Arp2/3 Complex and Bacterial Actin-Based Motility[END_REF][START_REF] Leung | Bacterial Actin Assembly Requires Toca-1 to Relieve N-Wasp Autoinhibition[END_REF]. During cell-to-cell spreading, the type 3 effectors OspE1 and OspE2 interact with PDLIM to activate protein kinase C involved in cell spreading [START_REF] Defilippi | Dissection of Pathways Implicated in Integrin-Mediated Actin Cytoskeleton Assembly. Involvement of Protein Kinase C, Rho GTPase, and Tyrosine Phosphorylation[END_REF][START_REF] Yi | Systematic Analysis of Bacterial Effector-Postsynaptic Density 95/disc Large/zonula Occludens-1 (PDZ) Domain Interactions Demonstrates Shigella OspE Protein Promotes Protein Kinase C Activation via PDLIM Proteins[END_REF]. Actin polymerization by formin Dia1, which is recruited around intracellular Shigella by IpgB2, drives intercellular spread. [START_REF] Alto | Identification of a Bacterial Type III Effector Family with G Protein Mimicry Functions[END_REF][START_REF] Heindl | Requirement for Formin-Induced Actin Polymerization during Spread of Shigella Flexneri[END_REF]. Myosin X has also been observed to be involved in the formation of Shigella-containing protrusions into neighbouring cells [START_REF] Bishai | Myosin-X Facilitates Shigella-Induced Membrane Protrusions and Cell-to-Cell Spread[END_REF].

IpaA

IpaA is a 633 aminoacid multi-domain protein [START_REF] Venkatesan | Nucleotide Sequence of Invasion Plasmid Antigen Gene ipaA from Shigella Flexneri 5[END_REF]. A fragment of IpaA consisting of its first 500 aminoacids activates RhoA and competes with the talin binding to the integrin β1 subunit. This fragment inhibits 1 integrin anchoring to stress fibers and, together with the stimulation of actomyosin contraction, results in their disassembly (Demali, Jue, and Burridge 2006).

Early studies identified IpaA-dependent vinculin recruitment in bacterial entry foci. Of note, ipaA mutants display a 10-fold decrease in invasion ability (Tran Van Nhieu, Ben-Ze'ev, and Sansonetti 1997), indicating a crucial role for IpaA during Shigella invasion. IpaA association with vinculin N-terminal head domain was also shown to activate vinculin binding to F-actin (Bourdet-Sicard et al. 1999)

IpaA Vinculin Binding Sites

The region responsible in IpaA for vinculin binding and F-actin capping was mapped to the C-terminal domain (Ramarao et al. 2007), where three vinculin binding sites were identified (Figure 18A) (Park, Valencia-Gallardo, et al. 2011) . These VBSs consist of amphipathic α-helices similar to the talin VBSs and were shown to be responsible for the previously observed vinculin recruitment to bacterial foci (Figure 17B) (Izard, Tran Van Nhieu, and Bois 2006). IpaA VBS1 dissociation constant (Kd)

for vinculin is in the picomolar range, and is at least 10-fold lower than that reported for α-actinin VBS (P. R. J. Bois et al. 2005). VBS1 interaction with vinculin occurs through the first helical bundle of vinculin D1 domain and promotes the helical-bundle conversion associated with vinculin activation. IpaA VBS2 was shown to be able to interact with the second bundle of D1 in an additive manner (Tran [START_REF] Van Nhieu | Vinculin Binding in Its Closed Conformation by a Helix Addition Mechanism[END_REF], thereby stabilizing IpaA VBS1-2-vinculin interaction, with Kd in the femtomolar range (Izard, Tran Van Nhieu, and Bois 2006). IpaA VBS 1-2 interaction with vinculin leads to its activation and is necessary for efficient bacterial entry and cell-cell spreading.

A third VBS (VBS3) was also identified. As for IpaA VBS1, the IpaA VBS3 peptide binds to and activates vinculin (Park, Valencia-Gallardo, et al. 2011). In addition to this, it was recently shown to promote filopodial capture by targeting a partially- The main goal of my thesis consisted in describing the effects of IpaA VBSs on host cell and during Shigella invasion. We therefore performed microscopy analysis of mammalian cells' adhesion structures transfected with different IpaA VBSs constructions in order to characterize its impact in the cell's cytoskeleton, with emphasis on the focal adhesion proteins vinculin and talin. We also implemented a microfluidics-based adhesion assay in order to characterize the functional impact of IpaA VBSs on the host's adhesion properties.

Together with biochemical evidence, invasion assays and structural modelling, we propose that IpaA VBS3 binds to a semi stretched conformer of talin, which stabilizes filopodial tip adhesions. In addition, IpaA VBS1-3 induces conformational changes in vinculin that lead to the unfolding of the vinculin head domain. This yet unreported mode of vinculin activation likely correspond to a mode occuring during mechanotransduction, that Shigella bypasses through the action of IpaA. The targeting of adhesion proteins to bacterial foci would stimulate the capture of additional bacteria and modify the susceptibility of the host membrane to prime it for bacterial adhesion and anchoring. This process would be further enhanced by the injection of protein effectors by kiss-and-run bacteria.

3 Article 1

Overview

Talin was identified as a binding partner of IpaA by a two-hybrid approach. This was done by using an IpaA fragment (containing the residues 1-565 and lacking IpaA VBS1-2) as a bait against a human placental cDNA library. Further biochemical characterization identified IpaA VBS3 as the binding partner of talin H1-H4 helices, mimicking talin H5 helix. IpaA VBS3 was found to be structurally analogous to talin H5 helix, which, together with H1-H4, forms the R1 talin bundle. Functional analysis identified IpaA VBS3 is necessary for talin recruitment to Shigella entry foci. When expressed in human cells it also stimulates the formation of nascent adhesions and promotes filopodia. We found that IpaA VBS3 stimulates filopodial capture of bacteria and stabilizes cell adhesions in invaded cells. My contribution to this article was the conceiving and realization of experiments, generation of IpaA VBS3 and talin constructs, microscopy analysis of adhesion structures and filopodia and in bacterial adhesion assays.

In Brief

Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion

Cesar Valencia-Gallardo, 1,2,3,4 Charles Bou-Nader, 5,6 Daniel-Isui Aguilar-Salvador, 1,2,3,4 Nathalie Carayol, 1,2,3,4 Nicole Quenech'Du, 1,2,3,4 Ludovic Pecqueur, 5,6 HaJeung Park, 7 Marc Fontecave, 5,6 Tina Izard, 8 and Guy Tran Van Nhieu 1,2,3,4,9, *

SUMMARY

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5 A ˚resolution crystal structure of IpaA VBS3 in complex with the talin H1-H4 helices shows a tightly folded a-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1-H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46.

Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella.

These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions.

INTRODUCTION

The cytoskeletal linkers talin and vinculin play important and distinct roles in integrin-mediated cell adhesion [START_REF] Atherton | Mechanosensitive components of integrin adhesions: Role of vinculin[END_REF][START_REF] Klapholz | Talin-the master of integrin adhesions[END_REF]. Although vinculin strengthens adhesion and cytoskeletal anchorage, talin is critically required for the formation and maturation of adhesion structures [START_REF] Lagarrigue | The Rap1-RIAM-talin axis of integrin activation and blood cell function[END_REF]Yan et al., 2015). Talin consists of an amino-terminal FERM domain connected to a large rod domain containing vinculin binding sites (VBSs) buried in helical bundles [START_REF] Calderwood | Talins and kindlins: partners in integrin-mediated adhesion[END_REF][START_REF] Lagarrigue | The Rap1-RIAM-talin axis of integrin activation and blood cell function[END_REF]Yan et al., 2015). Upon activation, talin bridges the cytoplasmic tail of the integrin b1 subunit with actin filaments (Yan et al., 2015). Stretching of the talin molecule due to actomyosin-dependent contractility leads to the unveiling of VBSs buried in helical bundles along the talin rod domain (Yan et al., 2015). Upon exposure, talin VBSs activate vinculin that further tethers actin filaments to adjust adhesion strength in response to substrate stiffness (Gingras et al., 2005;Yan et al., 2015). In vitro measurements on single molecules indicate that unveiling of VBSs in various talin bundles occurs with a defined hierarchy, suggesting different talin activation states.

Bacterial pathogens have evolved remarkable strategies to invade host cells [START_REF] Dunn | Uncivil engineers: Chlamydia, Salmonella and Shigella alter cytoskeleton architecture to invade epithelial cells[END_REF]Pizarro-Cerda ét al., 2012). Shigella, the causative agent of bacillary dysentery, invades intestinal epithelial cells after being captured by filopodia using a type III secretion system (T3SS) that injects bacterial effectors into host cells [START_REF] Dunn | Uncivil engineers: Chlamydia, Salmonella and Shigella alter cytoskeleton architecture to invade epithelial cells[END_REF][START_REF] Gala ´n | Bacterial type III secretion systems: specialized nanomachines for protein delivery into target cells[END_REF]Valencia-Gallardo et al., 2015). The Shigella IpaA type III effector contains three exposed VBSs located within its 145 C-terminal residues that act in concert to promote bacterial invasion (Izard et al., 2006;Park et al., 2011;[START_REF] Nhieu | Vinculin binding in its closed conformation by a helix addition mechanism[END_REF]. Here, we show that in addition to vinculin, IpaA VBS3 also directly targets talin. IpaA VBS3 forms a folded globular structure with the H1-H4 helices in the talin R1 bundle, which differs from the stretched bundle structure expected during full talin activation. Our results suggest that binding of IpaA VBS3 to a semistretched talin conformer stabilizes filopodial adhesions and stimulates bacterial capture.

RESULTS

Talin Is Recruited by IpaA and Is Required for Shigella Invasion IpaA recruits vinculin at Shigella invasion sites to trigger the formation of a transient adhesion required for bacterial internalization by epithelial cells (Tran [START_REF] Van Nhieu | Modulation of Bacterial Entry into Epithelial Cells by Association between Vinculin and the Shigella IpaA Invasin[END_REF]. Talin is also recruited at Shigella entry sites, but its role requires clarification. When analyzed using immunofluorescence microscopy, talin was detected in coat structures around internalized bacteria as early as 10 min incubation at 37 C (Figures S1A and S1B,arrowhead). Talin recruitment and coalescing around internalized bacteria occurred concomitantly with the depolymerization of actin in membrane ruffles (Figures 1A, 1B, S1A, and S1B). Cell treatment with anti-talin small interfering RNA (siRNA) did not impair actin polymerization at Shigella invasion sites, but actin coat structures surrounding invading bacteria seldom formed, reminiscent of foci induced by an ipaA mutant (Figures 1A and1B). Consistently, bacterial invasion was reduced by 4-fold in anti-talin siRNA-treated cells compared with control cells (Figure 1C). A Shigella ipaA mutant strain failed to form talin coats, suggesting a role for IpaA in talin recruitment at invasion sites (Figures 1D,1E, and S1C-S1E).

To investigate the role of IpaA VBSs in the formation of talin coat structures, cells were challenged with ipaA mutant strains complemented with IpaA derivatives. Talin coat structures were not detected for the ipaA mutant complemented with vector alone or IpaA deleted for all its VBSs (Figures 1D and1E). Complementation of the ipaA mutant with full-length IpaA, IpaA DVBS1-2, or IpaA DVBS3 restored the formation of foci with talin coat structures (Figure 1E). However, a significant decrease in the percentage of foci forming talin coat structures was observed for the ipaA mutant strains expressing IpaA DVBS1-2 or IpaA DVBS3 compared with full-length IpaA, with 19% ± 0.7% and 25% ± 5% relative to 52% ± 6.7%, respectively (Figure 1E). These results indicate that talin recruitment could occur via IpaA VBS3 or via IpaA VBS1-2, possibly through vinculin-talin interactions.

These results indicate that talin is required for Shigella invasion and is recruited at entry sites in an IpaA-dependent manner to form actin coats surrounding invading bacteria.

IpaA VBS3 Binds to Talin

To confirm and further investigate how IpaA VBS3 recruited talin, we performed a yeast two-hybrid screen using an established human placental cDNA library corresponding to a total of 82.02 million prey clones and a bait containing IpaA1-565 containing the VBS3 but devoid of the VBS1-2 sites (559-633). This screen identified 150 clones representing 16 different genes. Among these, talin was identified with very high confidence in 95 prey clones, with clones corresponding to different open reading frames in talin (Table S1). As expected, vinculin was also identified as a prey (Table S1). To test whether IpaA VBS3 could directly bind to talin, we performed native gel shift assays using synthetic peptides (STAR Methods). As observed for vinculin, IpaA VBS3 did not bind to talin R1 (talin H1-H5) but formed a complex with talin H1-H4, suggesting that the targeting of active talin in which the inhibitory H5 helix is removed by mechanical stretching [START_REF] Papagrigoriou | Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle[END_REF] (Figures 2A and S4B). In contrast, even at high molar ratios, IpaA VBS1 and IpaA VBS2 did not form any detectable complex with talin H1-H4. Isothermal titration calorimetry (ITC) measurements indicated that IpaA VBS3 bound to talin H1-H4 with high affinity (K D = 174 ± 19 nM), whereas IpaA VBS2 did not show any detectable interaction with talin H1-H4 (Figure 2B; Table 1). IpaA VBS1 interacted with talin H1-H4 with much lower affinity than IpaA VBS3 (K D = 15.2 ± 1.1 mM; Figure 2B; Table 1). When native gel shift assays using IpaA 483-633 containing all three VBS1-3 (A483) were performed, a clear migration shift corresponding to an A483-talin H1-H4 complex was observed with depletion of free talin H1-H4 (Figure 2C, arrowhead). In contrast, when IpaA 524-634 containing only IpaA VBS1-2 (A524) was incubated with talin H1-H4, no migration shift could be detected (Figure 2C,A524).

Size-exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) analysis indicated that A483 and A524 were monomers in solution, as indicated by the molecular mass determination (Figure 2D; Table S2). Of note, H1-H4 behaved as a globular dimer in SEC-MALS analysis (Figure 2D; Table S2), consistent with the crystal structure of talin rod 482-655 [START_REF] Papagrigoriou | Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle[END_REF]. When mixed prior to analysis, only single species corresponding to the talin H1-H4 dimer or A524 monomer were recovered, consistent with the native gels and ITC results (Figure 2D; Table S2). In contrast, a compact globular complex corresponding to one talin H1-H4 dimer bound to a single A483 molecule was observed (Figure 2D, talin H1-H4 + A483; Table S2). These results are in agreement with the ITC measurements and suggest that A483 strongly interacts with the talin H1-H4 via IpaA VBS3. These results indicate that in addition to vinculin, IpaA VBS3 also binds to talin with high affinity, a property not shared with other IpaA VBSs.

IpaA VBS3 Mimics Talin H5 to Form an a-Helix Bundle with Talin H1-H4 We solved the crystal structure of the IpaA VBS3-talin H1-H4 complex at 2.5 A ˚resolution (Table S3). The complex was purified using a strategy similar to that used for the IpaA VBS3-vD1 complex (Park et al., 2011). The asymmetric unit contains six molecules of talin H1-H4 organized into dimers as observed in SEC-MALS (Figure S2). Each talin H1-H4 molecule shows a clear additional density corresponding to the IpaA VBS3 peptide in vicinity of mainly the H2 and H4 helices (Figure S2B). All talin H1-H4 chains in the asymmetric unit were very similar with pairwise RMSDs in the range 0.28-0.64 A ˚over 140 Ca. The interface area between IpaA VBS3 and talin H1-H4 is 849 ± 26 A ˚2 and involves mainly hydrophobic residues located in talin a helices H2, H3, and H4 (Figures 3A and3B). In addition, electrostatic interactions, either direct, between K498 and E621, or indirect with R489 pointing within a negatively charged region, further strengthen IpaA VBS3 binding and positioning in the talin H2-H4 groove (Figures 3A and3B). Structural comparison of IpaA VBS3 in its talin-bound and vinculin-bound state (PDB: 3rf3; Figure 3B) indicates that the same IpaA VBS3 hydrophobic residues are involved in binding to talin H1-H4 and vD1. The IpaA VBS3-vinculin vD1 interaction surface area, however, is wider (1,150 A ˚2) than that of IpaA VBS3-talin H1-H4 and comprises IpaA residues F493, K497, and E490, which are not involved in talin H1-H4 recognition (Figure 3B). Strikingly, IpaA K498 specifically interacts with talin E621 and does not establish interaction with vD1 (Figure 3B). Structural comparison with talin H1-H5 (Figures 3C-3E) reveals a remarkable superposition of VBS3 with H5. I492, A495, V499, L506, and I507 of IpaA VBS3 involved in hydrophobic interaction with talin H1-H4 are homologous to L637, A640, V644, L651, and L652 in the talin a-helix H5, nesting in the identical hydrophobic groove (Figures 3C-3E). Furthermore, a strong similarity in polarity is observed between talin H5 and IpaA VBS3, with IpaA R489 corresponding to talin R634 engaged in the same polar interaction with the talin H2-H3 loop.

Contact Residues in IpaA VBS3 Determining Binding Specificity for Talin Synthetic peptides were generated to confirm the role of IpaA polar and hydrophobic residues in talin H1-H4 binding. Specifically, we tested the effects of charge at R489 and K498, and The affinity of synthetic peptides corresponding to talin VBS6, VBS9, VBS33, VBS36, VBS46, and VBS50 for talin H1-H4. Talin VBSs could be divided into four classes according to their affinity for talin H1-H4: whereas VBS33 and VBS36 did not show detectable binding, VBS6, VBS50, and VBS9 showed weak or moderate affinity for talin H1-H4. Talin VBS46, however, interacted with talin H1-H4 with an affinity comparable with that of IpaA VBS3, with an estimated K D of 60 ± 0.8 (SD) nM. Although consistently displaying high affinity for vD1, IpaA and talin VBSs bound to talin H1-H4 with a broad range of K D values. N, stoichiometry; K D , affinity constant; DH (cal/mol), enthalpy; ÀTDS (cal/mol), entropic contribution; DG (cal/mol), free enthalpy; NA, not applicable; n.d., not determined. IpaA VBS3 and talin H46 binding to talin H1-H4 is exothermic (DG < 0) and driven mainly by enthalpy (DH < 0 and DH < ÀTDS). Talin H1-H4 binding to IpaA peptides VBS1 and VBS3 show an important enthalpic and minor entropic contribution. Talin H1-H4 binding to talin VBSs shows an important enthalpic contribution and an unfavorable entropic contribution to DG. The estimated K D value of other talin VBSs for vinculin D1 (K D vD1) was inferred from previous studies (Izard et al., 2006;[START_REF] Izard | Structural basis for amplifying vinculin activation by talin[END_REF]Park et al., 2011;[START_REF] Yogesha | Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin[END_REF].

924 Cell Reports 26, 921-932, January 22, 2019 substitution of A495 rationalized by the structural alignment with IpaA and talin VBSs (Figure S3). ITC analysis indicated that the K498A substitution did not significantly decrease the talin-binding affinity of IpaA VBS3, possibly because of the complementary charge contribution by adjacent K497 (Table 1). The single charge inversion K498E, however, led to a 1.7-fold increase in the determined K D (K D = 300 ± 2.8 nM) for talin H1-H4. A similar increase was observed for the charge suppressing double mutations R489A K498A with a K D of 328 ± 1.9 nM. Consistent with a critical role for the hydrophobic interactions, the A495K substitution led to a drastic defect in IpaA VBS3 binding to talin, with a 17-fold increase in K D (K D = 2.93 ± 0.3 mM) (Table 1). These results were further confirmed in native gel shift experiments showing that as opposed to IpaA VBS3, IpaA VBS3 498E and IpaA VBS3 A495K did not induce any detectable shift of talin H1-H4 (Figure S4). In contrast, shifts were still observed in the presence of vD1, indicating that the mutations did not prevent complex formation with vinculin, although higher peptide concentrations were required for the A495K substitution consistent with a partial effect of this substitution on vinculin binding (Figure S4B).

Talin Binding by IpaA VBS3 Stimulates the Formation of Nascent Adhesions

Our evidence suggests that IpaA VBS3 targets an active conformer of talin containing a four-helix folded R1 bundle that differs from the ''opened'' configuration reported to interact with vinculin (del [START_REF] Rio | Stretching Single Talin Rod Molecules Activates Vinculin Binding[END_REF][START_REF] Papagrigoriou | Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle[END_REF].

To test the targeting of a specific talin conformer by IpaA VBS3, we took advantage of its high affinities for vinculin and talin and generated a GFP fusion as a probe to analyze its localization following cell transfection. As shown in Figure S5A, GFP-IpaA VBS3 labeled focal adhesions (FAs) of cells plated onto fibronectin-coated glass coverslips, showing a strict co-localization with talin and vinculin. As expected, siRNA-mediated depletion experiments showed that GFP-IpaA VBS3 labeling of adhesion structures was vinculin and talin dependent (Figure S5). When cells were plated on 15 kPa stiffness substrate, GFP-IpaA VBS3 labeled vinculin-and talin-containing adhesions at the cell periphery, including at the base of filopodia as well as at the edge of lamellae. Adhesion structures located toward the cell interior, however, were less enriched in IpaA VBS3, consistent with labeling of nascent adhesions (Figures S5E andS5F) [START_REF] Beningo | Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts[END_REF][START_REF] Van Hoorn | The nanoscale architecture of force-bearing focal adhesions[END_REF].

In replating experiments, GFP-IpaA VBS3 labeled talinand vinculin-containing peripheral adhesions, consistent with nascent adhesions, as well as prominent adhesions (Figures 4A-4C). As expected, control cells transfected with GFP alone showed a diffuse cytoplasmic labeling and virtually no recruitment in adhesion structures (Figures 4B and4C). When cells were transfected with the mutated IpaA VBS3 variants, 6-and 7.8-fold reductions in the number of GFP-IpaA VBS3-positive adhesions per cell were observed for IpaA VBS3 A495K and K498E, respectively, compared with parental IpaA-VBS3 (Fig- ures 4A-4C). In addition, the size of adhesions was significantly reduced in GFP-IpaA VBS3 A495K and K498E transfected cells compared with GFP-IpaA VBS3 transfectants (Figures 4D and4E). Of note, while showing reduced stimulatory effects on adhesion formation, the talin-binding deficient variants A495K and K498E showed an increase in the size of FAs compared with GFP-transfected control cells, with a median ± median absolute deviation (MAD) of 1.53 ± 0.51 and 1.21 ± 0.39, respectively (Figures 4D and 4E), likely due to their residual vinculin-binding ability.

IpaA VBS3 Regulates Filopodial Extension through Its Talin-but Not Vinculin-Binding Activity

In replating experiments, talin was also enriched in adhesions in the filopodial shaft distal moiety, while vinculin preferentially localized at the filopodial shaft proximal moiety and base (Figure 5A), with median distances ± MAD of 2.5 ± 1.8 and 1.7 ± 1.3 mm, respectively (Figure 5B; STAR Methods). In addition, GFP-IpaA VBS3 was associated with distal filopodial talin adhesions, whereas association with vinculin was observed in more proximal filopodial adhesions, with a median distance of 2.8 ± 1.5 mm compared with 1.4 ± 0.9 mm, respectively (Figures 5C andS6). VBS3-and talin-labeled adhesions were located more distally than talin-and vinculin-labeled adhesions in filopodia, suggesting the formation of distinct complexes (Figure 5C). Consistent with different talin conformers, the canonical talin-binding domain of vinculin vD1 labeled filopodial clusters that were more proximal to the cell body than those labeled with VBS3, with median distances to the cell body of 2.5 ± 1.8 and 3.1 ± 2.2 mm, respectively (Figure 5D). As expected, GFP alone did not label structures in filopodia (Figures 5E andS6).

To test the role of talin binding in the targeting of filopodial distal adhesions by IpaA VBS3, we analyzed the localization of the GFP-IpaA VBS3 A495K and K498E mutants. As opposed to GFP-IpaA VBS3, the mutated VBS3 variants impaired for talin binding showed a 2.7-fold decrease labeling of large filopodial adhesions (Figures 5A and5E). Binding of vD1 to activated talin was shown to inhibit its refolding, thereby stabilizing FAs [START_REF] Atherton | Vinculin controls talin engagement with the actomyosin machinery[END_REF][START_REF] Yao | Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation[END_REF]. We therefore expected binding (B) Molecular detail of the interaction between IpaA VBS3 with talin H1-H4 and vD1. Top: IpaA VBS3 is shown as a cartoon with its relevant interacting residues. Bottom: talin H1-H4 and vinculin D1 are illustrated by their vacuum electrostatic potential generated with APBS [START_REF] Baker | Electrostatistics of nanosystems: application to microtubules and the ribosome[END_REF]. Of note, the amino-terminal residues Thr 489 to Thr 487 of IpaA VBS3 form an additional helical turn when bound to talin H1-H4 as opposed to the vD1 complex, suggesting that this different fold further tunes recognition specificity. (C) Superimposition of talin H1-H4:IpaA VBS3 and talin H1-H5 structures. Talin H1-H4 bound to IpaA VBS3 (green) and to talin H5 (pink) are shown as a cartoon in blue and magenta, respectively. (D) Helical wheel analysis of IpaA VBS3 (top) and talin H5 (bottom) indicates that both are amphipathic a helices with conserved residues in their hydrophobic interphase. The hydrophobic residues, polar residues, and positively and negatively charged residues are colored yellow, green, blue, and red, respectively. (E) Comparison of talin H1-H4 binding to IpaA VBS3 and talin H5. Talin H1-H4 is shown in white cylinder while the electrostatic surface of IpaA VBS3 and H5 are represented as calculated with APBS.

of IpaA VBS3 to favor filopodial extension by stabilizing filopodial adhesions. Consistently, quantification indicated that filopodia in GFP-IpaA VBS3 transfectants were longer than those observed in cells transfected with GFP-talin with median lengths ± MAD of 4.1 ± 1.9 and 3.4 ± 1.9 mm, respectively (Figure 5F). Remarkably, cells transfected with GFP-IpaA VBS3 A495K and K498E showed filopodia that were significantly shorter than cells transfected with GFP-IpaA VBS3, with median lengths of 2.6 ± 0.8 and 2.9 ± 1 mm, respectively, and comparable with the filopodial length in cells transfected with vD1 (Figure 5F; median 2.5 ± 1.8 mm). In control experiments, the filopodial length in cells co-transfected with GFP and mCherry-talin or vinculin-mCherry were comparable with those of GFP-talin alone or GFP-VBS3 K498E, with median lengths of 3.0 ± 1.1 and 2.9 ± 0.1 mm, respectively (Figure 5F).

Together, these results suggest that through talin binding, IpaA VBS3 stabilizes filopodial adhesions, thereby favoring their extension. The role of IpaA VBS3 in the regulation of both filopodial and nascent adhesions further supports the link reported between these adhesion structures [START_REF] Hoffmann | Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth[END_REF]Jacquemet et al., 2015;[START_REF] Partridge | Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading[END_REF].

Talin Binding by IpaA VBS3 Stimulates Filopodial Capture and Shigella-Induced Cell Adhesion Formation Shigella invasion of epithelial cells is preceded by its capture by filopodia (Romero et al., 2011). We next analyzed the effects of GFP-IpaA VBS3 transfection in bacterial capture (Romero et al., 2011). As shown in Figure 6A, GFP-IpaA VBS3 and talin structures could be observed at the tip of 84.6% of filopodiacapturing bacteria (Figure 6A, arrowhead; 13 filopodia, n = 3), but only in 12.8% of filopodia devoid of bacteria (304 filopodia, n = 3). To test the relevance of talin binding by IpaA VBS3 during filopodial capture, cells were challenged with ipaA mutant strains complemented with various IpaA constructs. As shown in Figure 6B, deletion of all IpaA VBSs resulted in a 5-fold reduction in the number of bacterial capture compared with full-length IpaA. Although deletion of IpaA VBS1-2 containing VBS3 did not affect filopodial capture, deletion of IpaA VBS3 reduced filopodial capture by 2.4-fold (Figure 6B). The A495K or K498E mutations that impaired talin binding reduced filopodial capture to similar extent than IpaA DVBS3 (Figure 6B).

In addition to forming pseudo-adhesion structures in membrane ruffles during invasion, Shigella stabilizes FAs to prevent Cell Reports 26, 921-932, January 22, 2019 927

early detachment of infected cells (Sun et al., 2017). To investigate the role of IpaA VBS3 in this process, cells were challenged with bacteria for 30 min, and talin-containing adhesions were analyzed using immunofluorescence microscopy (STAR Methods). As shown in Figures 6C and6D, while showing less FAs than uninfected cells, cells infected with the ipaA mutant complemented with full-length IpaA remained spread and showed large adhesion structures. In contrast, cells challenged with ipaA mutant complemented with vector alone showed a significant decrease in size and number of adhesions (Figures 6C and6D). Complementation with IpaA DVBS1-2 containing VBS3 restored adhesion structures, although these latter were smaller than those observed for full-length IpaA (Figures 6C and 6D sions per cell but a clear shift in the distribution toward cells showing smaller adhesions (Figures 6C-6F). Together, these results indicate that talin binding by IpaA VBS3 plays an important role in bacterial capture by filopodia and stabilizes cell adhesions during Shigella infection.

DISCUSSION

Our work provides a mechanistic basis for how IpaA VBS3 acts as a dual talin-vinculin binder, a property not shared by IpaA VBS1 or IpaA VBS2. IpaA residues V499-L503-L507 are involved in interactions with talin or vinculin, suggesting that these constitute a common hydrophobic core essential for binding but not conferring specificity. Two other hydrophobic residues, IpaA I492-A495, however, appear to be specific for talin interaction. Also, electrostatic interactions involving residues IpaA R489 and K498 are important for talin specificity. Specifically, the lysine residue at position 498 in IpaA VBS3 is critical for specific and high-affinity binding to talin. The sequence alignment of IpaA and talin VBSs and ITC-based affinity measurements highlight that among the VBSs analyzed, only talin H46 share properties similar to IpaA VBS3 in binding to talin H1-H4 (Table 1; Figure S3). These findings indicate functional diversity between VBSs, with IpaA VBS3 and talin H46 acting as dual vinculinand talin-binding sites. Although this was not investigated in the present study, IpaA VBS3 may also bind to talin 2, a talin 1-related protein that presents conserved structural features in the R1 bundle (Figure S7). Our structural and cell labeling data suggest that IpaA VBS3 targets an active talin conformer presenting a folded R1 bundle in filopodial distal adhesions that differs from fully active talin. Stretching experiments on purified talin bundles failed to detect intermediate steps during R1 unfolding [START_REF] Yao | Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation[END_REF](Yao et al., , 2016)). Current magnetic tweezer resolution limits, however, do not enable detection of displacement of a single helix, such as that of H5 expected to occur during IpaA VBS3 binding to R1. Recent single-molecule atomic force microscopy combined with steered molecular dynamics (SMDs), however, supported the existence of 3-helix intermediates during unfolding of the talin R9 and R11 fivehelix bundles (Mykuliak et al., 2018). SMDs also predict that stronger hydrogen and salt bridge interactions between the talin H1 and H4 helices result in a torque leading to dissociation of talin H5 from H4 under applied force [START_REF] Hyto ¨nen | How force might activate talin's vinculin binding sites: SMD reveals a structural mechanism[END_REF][START_REF] Lee | Force-induced activation of talin and its possible role in focal adhesion mechanotransduction[END_REF], consistent with a talin conformer with a partially unfolded R1 bundle. Because of their structure, the talin R2 and R3 bundles are proposed to unfold first in response to stretching forces, leading to RIAM dissociation and vinculin binding (Goult et al., 2013b), while R1 and R10 unfold at intermediate forces (del Rio et al., 2009;[START_REF] Wang | Defining single molecular forces required to activate integrin and notch signaling[END_REF][START_REF] Yao | Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation[END_REF]Yao et al., , 2016)). Talin VBSs' exposure in cells, however, may be altered by ligands stabilizing talin bundles or in the talin auto-inhibited state (Goult et al., 2013a).

Early studies had pointed to a role for talin in controlling filopodial elongation and retraction [START_REF] Sydor | Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone[END_REF]. In line with this, the functional analysis of mutated IpaA VBS3 indicated that talin binding is specifically required for filopodial adhesions and bacterial capture. Consistently, IpaA A495K, and K498E mutations that decrease talin-binding activity prevented the localization of IpaA VBS3 in filopodial shaft adhesions and its effects on filopodia elongation. We propose that in crawling filopodia, IpaA VBS3 favors filopodial protrusion by stabilizing talin-mediated cytoskeletal anchorage at filopodial shaft adhesions. Talin contains a C-terminal F-actin-binding domain involved in talin dimer formation [START_REF] Gingras | The structure of the C-terminal actin-binding domain of talin[END_REF]. It is possible that talin-talin interactions are favored during initial activation steps. Interactions between talin H1-H4 and IpaA VBS3 or talin H46 may stabilize and/or contribute to the formation of higher order talin oligomers devoid of vinculin in filopodial tip and distal adhesions, acting as a molecular clutch adapted to the low force range exerted by filopodia. Adhesions involving this talin-based clutch would serve as anchoring points favoring filopodial extension through actin polymerization at the tip of filopodia.

Unlike talin VBSs, IpaA VBSs are not buried within bundles and may bind to available ligands in an opportunistic manner. Following capture by swirling filopodia, IpaA VBS3 may bind to talin R1 to stabilize the filopodial tip adhesion. Because pathogens often mimic cellular processes, a similar role may be performed by talin H46 in the R10 bundle during filopodial sensing. Upon filopodial retraction IpaA VBS3 may cooperate with IpaA VBS1-2 to trigger vinculin activation and reinforce cytoskeletal tethering. IpaA could possibly also bridge activated vinculin through its VBS1, 2 and talin through its VBS3, thereby contributing in the cross-linking of cytoskeletal network at Shigella invasion sites. As force increases, further stretching of talin will lead to the dissociation of VBS3-talin recipient bundle interaction and subsequent VBS-vinculin interaction, in line with the enrichment of vinculin at proximal filopodial shaft and basal adhesions.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

d KEY RESOURCES

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Guy Tran van Nhieu (guy.tran-van-nhieu@college-de-france.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell lines

HeLa cells (ATCC CCL-2) were incubated in RPMI (Roswell Park Memorial Institute) medium containing 5% FCS (fetal calf serum, GIBCOâ) in an incubator with 5% CO 2 . Mouse Embryo Fibroblast (MEF) cells were incubated in DMEM (11965092, GIBCO) medium containing 10% FCS (fetal calf serum, GIBCOâ) in an incubator with 5% CO 2 .

Bacterial strains

The wild-type Shigella flexneri, isogenic mutants, and complemented ipaA mutant strains, as well as wild-type Shigella expressing the AfaE adhesin were previously described (Park et al., 2011). Bacterial strains were cultured in trypticase soy broth (TCS) medium at 37 C. When specified, antibiotics were added at the following concentrations: carbenicillin 100 mg/ml, kanamycin 20 mg/ml.

METHOD DETAILS

Antibodies

The anti-Shigella serotype V LPS rabbit antibody was described previously (Tran [START_REF] Van Nhieu | Modulation of Bacterial Entry into Epithelial Cells by Association between Vinculin and the Shigella IpaA Invasin[END_REF]]. The mouse monoclonal antitalin clone 8d4 antibody was from Sigma-Aldrich. Alexa 405 coupled anti-rabbit and Alexa 488 coupled anti-mouse IgGs were from Jackson Immunoresearch. Alexa 647-coupled phalloidin was from Invitrogen.

Generation of expression constructs

Full-length human vinculin-mCherry (residues 1-1066) was generated by polymerase chain reaction using 5 0 -CTGTCGACTGATGC CAGTGTTTCATACG-3 0 / 5 0 -CTCCCGGGTCTGGTACCAGGGAGTCTTTC-3 0 primers and cloned into the pmCherry-N1 (Clontech) vector using the SalI -SmaI sites. Human talin-mCherry was from Addgene. Human talin-GFP was a gift from K. Yamada. The A483 construct was PCR amplified using 5 0 -GGCGAATTCCCGGAGACACATATTTAACACG À3 0 / 5 0 -GCCGTCGACTTAATCCT TATTGATATTCT À3 0 primers and cloned into the EcoRI -SalI sites of pGEX-4T-2 (GE Lifesciences). The GFP-A483 plasmid was generated by PCR amplification using the 5 0 -GCGATATCATGGCCAGCAAAGG-3 0 and 5 0 -GCGCGGCCGCTTAATCCTTATTGA TATTC-3 0 primers and cloned into a pcDNA3.1 NT-GFP Topo (Invitrogen). The GFP-IpaAVBS3 plasmid was generated by PCR amplification using the 5 0 -ACCCGGGGATTAAGCGGCC-3 0 and 5 0 -ACCCGGGATCCTGATTTAGTTCC-3 0 primers and the GFP-A483 plasmid as matrix. The amplicon was digested with SmaI and ligated using a T4 DNA Ligase. Mutagenesis to generate the GFP-IpaA VBS3 A495K and K498E variants was performed using the following pairs of primers 5 0 -CACGGGAAACGATATTTGAAAAAT CAAAAAAAGTAACAAACTC-3 0 ; 5 0 -GAGTTTGTTACTTTTTTTGATTTTTCAAATATCGTTTCCCGTG-3 0 , and 5 0 -CGATATTTGAAGCTT CAAAAGAAGTAACAACAAACTCCCTAA-3 0 and 5 0 -TTAGGGAGTTTGTTGTTACTTCTTTTGAAGCTTCAAATATCG-3 0 , respectively, using the Quickchange II site-directed mutagenesis procedure (Stratagene). All other enzymes were from New England Biolabs. Plasmids containing talin VBS1 (H1-H4) residues 482-636 and GST-IpaA 524-633, as well as peptides IpaA-VBS1 (611-633) and VBS2 (565-586) were previously described (Izard et al., 2006;[START_REF] Papagrigoriou | Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle[END_REF]Ramarao et al., 2007;[START_REF] Van Nhieu | Vinculin binding in its closed conformation by a helix addition mechanism[END_REF] (N-QVVLINAVKDVAKALGDLISATKAA-C, 2077-2102) were synthesized at the Scripps Institute proteomics facility (Jupiter Florida, USA). For crystallographic studies, the human talin H1-H4 domain (residues 481-636) preceded by an internal ribosome-binding site and a start codon was PCR-amplified and cloned into the EcoRI/SacI sites of pET28a-IpaA-VBS3 (Park et al., 2011) using the following pairs of primers 5-CGAATTCAGGAGGACAGCTATGCACCGAGGACACATG-3 and 5-CGAGCTCGTTACTGACGGGGCT CAGCACTG-3 to generate the bicistronic expression vector pET28a-IpaA-VBS3/VTBS-talinH1-H4. Plasmids were transformed into the E. coli BL21 (DE3) strain (Invitrogen).

Yeast double hybrid analysis

The yeast two-hybrid analysis was performed using IpaA1-565 as bait to screen a human placental RP1 library, according to standard procedures and the Y2H protocole (Hybrigenics services).

Protein purification

The talin H1-H4 derivative was purified by affinity chromatography in 25 mM Tris-HCl pH 7.4, 0.5 M NaCl and 30 mM imidazole using a HisTrap HP 5ml affinity column (GE Healthcare). For crystallographic studies, the binary complex of IpaA-VBS3 / talinH1-H4 was purified by passage over a HisTrap HP 5 mL affinity column (GE Healthcare) in 25 mM Tris-HCl pH 7.4, 0.5 M NaCl and 30 mM imidazole. The elution fractions were pooled and concentrated with a 3 kDa cut-off ultrafiltration unit (Amicon), and the buffer was exchanged to phosphate-buffered saline (PBS) and digested by thrombin digestion. The complex was loaded onto a Superdex 200 size chromatography column and fractions containing IpaA-VBS3 / talinH1-H4 complex concentrated up to 30 mg/ml. IpaA derivatives were purified by affinity chromatography in PBS (Phosphate Buffer Saline) using a GSTrap HP affinity column (GE Healthcare), cleaved with thrombin to remove the GST moiety, followed by size exclusion chromatography (HiLoad S200, Ge Healthcare). For crystallographic studies, talin H1-H4$IpaA-VBS3 binary complex was purified as described (Park et al., 2011). Protein concentration was determined using the BCA assay (Thermo Scientific). Samples were dialyzed in binding buffer and stored at À80 C at concentrations ranging from 1 to 10 mg/ ml.

Native-PAGE analysis

Talin H1-H4 and IpaA peptides were incubated in binding buffer (25 mM Tris-HCl PH 7.0, 100 mM NaCl and 1 mM b-Mercaptoethanol) for 1hr at 4 C. After incubation, samples were resuspended in 2 x Native loading buffer (62,5mM Tris pH 6,8 containing 25% Glycerol) and separated by Tris-Glycine Native-PAGE electrophoresis [START_REF] Scha ¨gger | Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis[END_REF]. Gels were stained using standard colloidal Coomassie stain.

SEC-MALS

The purified proteins IpaA483-633, IpaA524-633 and talin H1-H4 were used at 20 mM equimolar concentrations and incubated at 4 C for one hour in binding buffer (25 mM Tris-HCl PH 7.0, 100 mM NaCl and 1 mM b-Mercaptoethanol). 200 mls of the protein mixtures were analyzed by size-exclusion chromatography (SEC) on a Superdex 200 10/300 GL (GE Healthcare) using a Shimadzu Prominence HPLC. Multi-angle laser light scattering (MALS) was measured with a MiniDAWN TREOS equipped with a quasi-elastic light scattering module and a refractometer Optilab T-rEX (Wyatt Technology). Protein concentration was determined using a specific refractive index (dn/dc) of 0.183 at 658 nm.

Crystallization, structure determination, and crystallographic refinement Crystals of the IpaA-VBS3 / talin H1-H4 complex were obtained by mixing 1 ml of protein complex at 7 mg/ml in buffer 25 mM Tris pH 7.4 and 150 mM NaCl with 1 ml of 0.2 mM ammonium sulfate, 0.1 mM sodium acetate pH 4.6 and 30% PEG 4000 by vapor diffusion at 292 K. Crystals were cryoprotected with a solution consisting of the reservoir supplemented with 20% v/v ethylene glycol and flash frozen. X-ray diffraction data were collected at 100 K on a single crystal at PROXIMA-1 beamline at the SOLEIL synchrotron (Saint-Aubin, France). Data were indexed and processed using XDS [START_REF] Kabsch | XDS[END_REF] and corrected for anisotropy with the STARANISO server (staraniso.globalphasing.org). Structure solution was obtained by molecular replacement with Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF] using as search template a monomer of talin-H1-H4 derived from the pdb entry 1sj8 lacking residues 515 to 538. A clear solution was obtained (TFZ = 28.5; LLG = 2105.5) Refinement was done with autoBUSTER [START_REF] Bricogne | BUSTER version 2.9[END_REF] using NCS and alternating with manual building in Coot [START_REF] Emsley | Features and development of Coot[END_REF]. One TLS group per chain was used at the end of the refinement. Final structure is deposited on ProteinDataBank (PDB ID: 5NL1).

Isothermal titration calorimetry

Protein interaction was analyzed by microcalorimetry using an ITC200 calorimeter (MicroCal) at 25 C. 200 mls of 20-100 mM of talin H1-H4 protein in binding buffer (25 mM Tris-HCl pH 7.0, 100 mM NaCl and 1 mM b-mercaptoethanol) were added to the cell and binding was measured in the presence of different concentrations of IpaA / talin VBSs peptides. In other experiments, A483 or A524 were added to the cell and binding of talin H1-H4 (100 mM) was measured. Typically 20-40 injections of 2 mls of ligand were made with intervals of 320 s between each addition, with a reference power of 12 mcal / sec. Data was analyzed using the MicroCal software provided by manufacturer.

e4 Cell Reports 26, 921-932.e1-e6, January 22, 2019 siRNA transfection HeLa cells were seeded at a density of 10 5 cells in wells containing a 22 3 22 mm coverslip in a 6-well plate. The following day, cells were transfected with of anti-talin 1 siRNA (Stealth Select RNAi, catalog no. 1299003, Invitrogen, oligo 804; sequence: 5 0 -CCA AGAACGGAAACCUGCCAGAGUU-3 0 ) or anti-vinculin siRNA (Stealth Select RNAi, catalog no. 1299001, Invitrogen, oligo VCLH55111259) duplex at the indicated concentrations and time periods.

Cell challenge with Shigella strains

HeLa cells seeded at 2 3 10 5 cells in coverslip-containing 33 mm-diameter wells the day before, were transfected with the indicated constructs using the JetPeiâ transfection reagent. After 16 hours, cells were challenged with Shigella strains coated with poly-Llysine, as follows. Bacteria grown to an OD 600 nm of 0.6 -0.8 were washed three-times by successive centrifugation at 13 Kg for 30 s and resuspension in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 , 5 mM glucose, and 25 mM HEPES, pH = 7.3). Samples were resuspended in EM buffer containing 50 mg/ml poly-L-lysine and incubated for 15 min at 21 C, washed three times in EM bufer and resuspended in the same buffer at a final OD of OD 600 nm = 0.2. Cell samples were washed three times in EM buffer and challenged with 1 mL of the bacterial suspension and incubated at 37 C. Samples were fixed with PBS containing 3.7% PFA after 15 min incubation for the analysis of filopodial capture. Alternatively, for the analysis of FAs in infected cells, a similar procedure was used except that cells were seeded at a density of 400 000 cells / well and challenged after 16H for 60 min with poly-Llysine coated bacteria prior to fixation with a 1:1 ethanol-acetone solution for 5 min at À20 C. Samples were processed for immunofluoresence microscopy.

Immunofluorescence microscopy analysis

For GFP-IpaA VBS3, GFP and talin / vinculin co-localization experiments MEF cells were seeded at a density of 1 3 10 4 cells in a m-Dish 35 mm 15 kPa stiffness chamber (Cat #81391, ibidi) coated with 10 mg / ml of Fibronectin (Calbiochem). Cells were transfected with plasmids encoding human talin-mCherry, vinculin-mCherry, GFP-IpaA VBS3 and GFP using the Lipofectamine 2000 transfection reagent (Life Technologies). For replating experiments HeLa cells co-transfected with vinculin-mCherry and talin-GFP, were resuspended by trypsinization and replated on glass coverslips. After 20 minutes the samples were fixed, processed for immunofluorescence microscopy, and mounted on slides using Dako mounting medium (Dako, Agilent Technologies), as described (Romero et al., 2012).

Samples were analyzed using an Eclipse Ti microscope (Nikon) equipped with a 100x objective, a CSU-X1 spinning disk confocal head (Yokogawa), and a Coolsnap HQ2 camera (Roper Scientific Instruments), controlled by the Metamorph 7.7 software. The percent of internalized bacteria was scored as one if the number of internalized bacteria per foci was at least the 50% of the total number of bacteria in the foci or zero if below, in three independent experiments (n control = 27, n siRNATln = 23). For control and anti-talin siRNA transfected cells, the percent of internalized bacteria was compared using a Chi-square test (R Statistical Software).

Image processing and analysis

Analysis was performed in Icy Bioimaging Analysis software (de Chaumont et al., 2012). Scans of GFP-IpaA VBS3, Vinculin-mCherry and Talin-mCherry were performed as follows. Defined ROIs in median projections of basal in-focus planes were segmented in concentric areas from the edge of the cell. Average values of intensities were normalized to the maximum and minimum average intensities of the ROI. For talin-GFP and vinculin-mCherry filopodial distribution, saturated sum projections of images from 20 minutes-replated cells were used for Concentric Area Scan analysis. For the quantification of the number of FAs, a semi-automated protocol was developed using Icy software (de Chaumont et al., 2012). Spinning-disk fluorescent microscopy planes were used to detect GFP-IpaA VBS3 structures using HK means thresholding and overlaid binary masks obtained from the threshold projections of F-actin labeled images (Max-entropy method). FAs were detected as spots positive for both GFP-IpaA VBS3 and actin structures using Wavelet Spot Detector. FAs were detected as spots positive for both GFP-IpaA VBS3 above background intensity levels. Filopodia length and number were determined manually from actin-labeled projections. IpaA VBS3, talin, vinculin and vD1 filopodial clusters were identified using Wavelet Spot Detector in Saturated sum projections. Co-localizing clusters were determined by overlaying the separate detections. Distance to cell body was determined using ROI Inclusion analysis plugin.

QUANTIFICATION AND STATISTICAL ANALYSIS

Bacterial internalization and talin coat-structure formation were analyzed a contingency table using in a Pearson Chi-square test (R Statistical Software). A Post hoc pairwise Chi-square test (NCStats package, R Statistical Software) with FDR p value correction was further used to compare the distribution between the various strains. n > 35 foci, N = 3. The correlation of the intensity of talin and vinculin labeling was analyzed using a Wilcoxon rank sum test (R Statistical software).

For all statistical tests a D'Agostino-Pearson normality test was performed in order to decide if performing a parametric or a nonparametric test. Applied statistical test, number of datapoints (n) and number of experimental replicates (N) are indicated in the figure legends; as well as mean ± SEM, for normally-distributed data, and median ± MAD, otherwise. Integrin-mediated cell adhesion critically depends on the cytoskeletal linkers talin and vinculin 4-6 . During mechanotransduction, talin acts as a mechanosensor by exposing its VBSs as a function of the stretching force generated by acto-myosin and dependent on substrate stiffness 7 .

Article 2

Overview

Talin exposed VBSs recruit and activate vinculin, reinforcing anchorage to the actin cytoskeleton in response to mechanical load 8, 9 . Vinculin contains three repetitions (D1-D3) of a conserved domain consisting of two bundles of four helices, and a fourth D4 domain containing only one helical bundle connected to a proline-rich unstructured region and the carboxyterminal F-actin binding domain 10 .

Under its inactive folded form, intramolecular interactions between the vinculin head and tail domains prevent ligand binding.

The Shigella type III invasion effector IpaA contains 3 VBSs located in its carboxyterminal moiety 11-13 . IpaA VBS1, as for all VBSs described to date to promote vinculin activation, interacts with the first helical bundle of the D1 domain, promoting major conformational changes that disrupt the D1-tail intramolecular interactions and free the vinculin F-actin binding region 11 (Fig. 1a). IpaA VBS2, in contrast, interacts with the second helical bundle of D1 12 (Fig. 1a) and its association with IpaA VBS1 results in a very high affinity and stable IpaA VBS1-2:D1 complex ,with an estimated KD in the femtoM range 12 . While functional evidence indicates that IpaA VBS3 cooperates with IpaA VBS1-2 to stimulate bacterial invasion, the isolated peptide acts as IpaA VBS1 in promoting vinculin activation through interaction with the vinculin D1 first helical bundle and also forms a folded bundle with the talin H1-H4 helices 13, 14 .

Here, we studied the effects of the IpaA subdomains containing VBS1-2 (A524) or VBS1-3 (A483) on vinculin activation (Fig. 1b). We performed SEC-MALS (Size Exclusion Chromatography-Multi-Angle Light Scattering) to analyze complexes formed upon incubation of A483 with HV1-834 containing the D1-D4 domains, corresponding to full-length human vinculin (HV) devoid of the carboxyterminal F-actin binding domain (Fig. 1b). In addition to the 1:1 D1D4: A483 complex, larger complexes were observed corresponding to a 2:1 heterocomplex and a 3:0 D1-D4 homo-trimer (Fig. 1c). Similar 2:1 and 3:0 complexes were observed when A483 was incubated with HV1-484 containing only the vinculin D1 and D2 domains (D1D2) (Fig. 1d), indicating that vinculin homo-trimerization occurs exclusively through these vinculin head sub-domains. By contrast, when A524 was incubated with D1D2, 1:1 and 2:1 D1D2:A524 complexes were detected, but not the D1D2 homo-trimer suggesting a role for IpaA VBS3 in vinculin trimerization (Fig. 1d). Consistently, higher order D1D2 homo-complexes devoid of A483 and D1D2:A524 hetero-complexes were visualized by native PAGE (Fig. 1e and Suppl. Figs. 3c,d). These results suggest that binding of IpaA VBSs to vinculin triggers conformational changes in vinculin leading to the formation of an IpaA VBS3-dependent vinculin homo-trimer.

To further investigate initial interactions responsible for vinculin trimer formation, w e performed binding assays with HV derivatives immobilized onto a solid phase to restrict conformational changes.

These assays indicated that A483 and A524 bound to HV with a similar affinity, as estimated by their EC50 (95% confidence interval) of 6.1 (4.2-9.0) and 3.7 (1.7-8.1) nM, respectively (Suppl. Fig. 1a).

Strikingly, a large difference was observed in the binding plateau, indicating that HV presented more binding sites for A483 than for A524 (Suppl. Fig. 1a). Also, D1D2 presented more binding sites for HV1-258 containing the D1 domain only, suggesting the presence of additional sites on the D2 domain (Suppl. Fig. 1b). Consistently, BN-PAGE showed the formation of 1:1, as well as a 1:2 D1D2:A483 complex, observed with increasing A483 molar ratio (Suppl. Fig. 1c). In contrast, single 1:1 complexes were observed for D1:A483, D1:A524 or D1D2-A524 (Suppl. Figs. 1c-h), indicating that IpaA VBS3 was required to reveal additional sites on the D2 domain. Of note, D1D2 homo-trimers observed in the SEC-MALS and native gel analysis (Figs. 1d,e and Ext Data Figs. 3c,d) were not detected in BN-PAGE, suggesting that Coomassie brilliant blue interfered with the formation of higher order D1D2 complexes. Together, these results suggested that the formation of vinculin trimers triggered by A483 required the IpaA VBS3 dependent exposure of binding sites on D2. These findings were unexpected, since vinculin activating ligands have been described to bind to a single site on the D1 domain of vinculin.

To map interactions of A524 and A483 with D1D2, complexes were cross-linked, subjected to proteolysis and analyzed using Liquid Chromatography coupled to Mass Spectrometry (LC-MS) (Methods). Intermolecular links were identified from the characterization of cross-linked peptides (Suppl. Tables 123; Methods) and along with identified intramolecular links, used to produce structural models (Methods). The A524:D1 complex showed links consistent with a "canonical" conformer expected from established structures 11, 12, 15 (Suppl. Fig. 2). Similar links were identified for the A524:D1D2 complex, with a majority of links observed with the D1 domain (Fig. 2a). For both complexes, the structure shows interactions between IpaA VBS1 and VBS2 with the D1 first and second bundles, respectively, leading to helical bundle reorganization of D1 associated with vinculin activation (Figs. 2c,d; Suppl. Fig. 2 and Suppl. Tables 1,2). For the A483:D1D2 complex, structural modeling shows 2 major conformers accounting for the majority of links. In a first "closed" conformer, IpaA VBS1 and VBS2 interact with the D1 bundles in a similar manner as for A524, where the relative positioning of D1 and D2 is globally conserved compared to apo D1D2 in the A524-D1D2 complex (compare Figs. 2d and2e). In this "closed" conformer, IpaA VBS3 interacts with an interface formed by the H5 (residues 128-149) and H8 (residues 222-250) helices in the second bundle of D1 and the H13 (residues 373-397) helix in the second bundle of the D2 domain (Figs. 2e andf). The second "open" conformer, however, shows a major re-orientation of D1 and D2 subdomains with their major axis forming an angle value of ca 82° compared to the 25° observed in the native vinculin structure or the first conformer (Fig. 2g), with IpaA VBS3 docking sidewise through extensive interaction with the H5 (residues 128-149) and H8 (residues 222-250) helices of the vinculin D1 domain. Because this latter conformer leads to major changes in bundle exposure in D1 and D2, we posit that it is involved in the formation of higher order D1D2 complexes and trimer induced by A483.

To test this, we engineered a structural clamp by substituting residue Q68 in the first D1 bundle and A396 in the second D2 bundle for cysteine residues, expected to prevent the formation of the open conformer upon disulfide bridge formation (Suppl. Figs. 3a,b). Consistently, cysteine clamped D1D2-Q68C A396C did not prevent the exposure of additional sites on D2 or 1:1 complex formation induced by A524 or A483, but prevented the formation of higher order complexes (Fig. 1 e and Suppl. Figs. 3ce). We coined "supra-activation" the mode of vinculin activation induced by A483 involving major conformational changes in the vinculin head, to distinguish it from the canonical activation associated with the dissociation of vinculin head-tail domains.

We then characterized the effects of A524 and A483 expression by performing immunofluorescence staining of vinculin-containing adhesion structures. As shown in Figs 3a-c, C2.7 cells transfected with GFP-A524 formed more numerous and larger peripheral adhesions as well as actin-rich ruffles compared to control cells (Figs. 3a-d). Strikingly, GFP-A483 transfected cells formed even larger and more numerous adhesions than GFP-A524 transfected cells, but significantly less actin ruffles than GFP-A524 transfectants (Figs. 3a-d). These observations were confirmed by live TIRF microscopy showing the extreme stability of adhesions in GFP-A483 transfectants, with a median duration of at least 84 min, while GFP-A524 and control cells showed adhesions with a comparable median duration of less than 25 min (Fig. 3e; Suppl. movie 1). GFP-A524 and GFP-A483 transfectants showed slightly slower median instant rates of adhesion assembly than control cells (Suppl. Fig. 4; Suppl. movie 1) but significant 1.6-fold and 2-fold decrease in median instant rates of disassembly relative to control cells, respectively (Suppl. Fig. 4). The stability of GFP-A483-induced FAs was independent of acto-myosin contraction. Indeed, GFP-A483-induced FAs resisted the action of the Rho-kinase inhibitor Y27632 relaxing actin-myosin, with a five-and four-times slower median rate of disassembly of adhesions relative to control cells and GFP-A524 transfectants, respectively (Figs. 3f-i; Suppl. movie 2). Furthermore, large adhesions formed in GFP-A483 transfectants following addition of the inhibitor (Figs. 3f,h), a process that was not observed for the other samples, including cells transfected with GFP-A524 or GFP fused to the vinculin D1 domain (vD1) reported to delay talin refolding following stretching 16-18 (Figs. 3g-i; Suppl. movie 2). In line with the stabilization of mature FAs through vinculin supra-activation, GFP-A483 but not GFP-vD1 also delayed the Y27632-induced removal of the late adhesion marker VASP (Suppl. Fig. 5; Suppl. movie 3). Consistent with A483 bypassing mechanotransduction, GFP-A483 expression induced 5-and 1.6-fold higher yield of short term (≤ 10 min) cell adhesion to fibronectin substrates compared to control GFP and GFP-A524 transfectants, respectively (Suppl. Fig. 6a). By contrast, little difference in adhesion yield was detected at 15 min suggesting that canonical activation of vinculin also resulted in its supra-activation during mechanotranduction (Suppl. Fig. 6a). In line with this, MEF vinculin-null cells transfected with the clamped vinculin version formed fewer and smaller FAs than cells transfected with wild-type vinculin but more than mock-transfected cells (Suppl. Figs. 6b-d).

To confirm and extend these findings, cell adhesion strength was assessed measuring their detachment upon application of a controlled shear stress in a microfluidic chamber. When 1205Lu melanoma cells were allowed to adhere to fibronectin-coated surfaces for more than 25 min, little difference in resistance to shear stress could be detected among samples (Suppl. Fig. 7a). In contrast, when shear stress was applied less than 20 min following cell incubation, GFP-A483-transfected cells showed significantly higher resistance to shear stress up to 22.2 dynes.cm -2 than GFP-A524-or GFPtransfected cells, with 1.7 ± 0.2 -and 0.9 ± 0.14-fold enrichment ± SD of adherent cells for GFP-A483 and GFP-A524-transfected cells versus control GFP-transfected cells, respectively (Fig. 4b; Suppl. movie 4). In addition, similar to melanoma cells depleted for by siRNA treatment, cells transfected with the clamped vinculin version showed a decreased ability to adhere in comparison to WT vinculin-transfected cells (Suppl. Figs. 7a,b). These results are in full agreement with effects observed on adhesion structures and suggest that A483 interaction with vinculin leads to the bypass of mechanotransducing steps to promote strong adhesion.

Vinculin is paradoxically described as a prognostic marker favoring the migration of cancer cells or as a tumor suppressor stimulating cell anchorage 19-21 . These contradictory findings reflect its complex and poorly understood regulation, as well as different roles in 2D or 3D systems 22, 23 . Also, an increase of the total pool of vinculin may not correlate with increased vinculin activation. We took advantage of the unique property of A483 to study the effects of vinculin supra-activation on the motility and invasion of melanoma cells. In time-lapse microscopy experiments in 2D-chambers, GFP-A524 inhibited melanocyte motility compared to control cells, with a rate of Root Median Square Displacement (rMSD) of 3.16 and 15.6 m.min, respectively (Figs. 4c,d). An even stronger inhibition was observed for GFP-A483-transfected cells (rMSD = 2.3 m.min) (Figs. 4c,d). Transmigration of melanocytes in 3D-matrigels was similarly inhibited by A524 and A483 (Fig. 4e).

Bacteria invading through a triggering mode rely on a discrete number of T3SS-dependent contacts for which cytoskeletal tethering is likely critical for invasion 2 . As opposed to physiological substrates, bacteria cannot sustain the range of counter-forces associated with integrin-mediated adhesion to the substrate. The Shigella type III effector IpaA provides an elegant solution to this problem by promoting strong adhesion without requirement for mechanotransduction. Through the joint action of its VBSs, IpaA induces major conformational changes of the vinculin head subdomains. Understanding how these major vinculin conformational changes regulate the composition and properties of cell adhesions will bring important insights into cell adhesion processes and will be the focus of future investigations.

METHODS

Generation of constructs

Human vinculin constructs were generated by polymerase chain reaction using the forward primer 5' GCGCATATGCCAGTGTTTCATACG-3' and reverse primer 5'-CGTCGACTCACCAGGCATCTTCATCGGC-3' for D1 (residues 1-258) or 5'-CGTCGACTCAGTGTACAGCTGCTTTG-3' for D2 (residues 1-492) using a plasmid containing full-length octahistidine-tagged human vinculin (residues 1-1,066), as template 10 , and cloned into the NdeI-SalI sites of pet15b (Novagen). The IpaA constructs GFP-A524 and GFP-A483 were generated by polymerase chain reaction (PCR) and cloning into pcDNA3.1 NT-GFP Topo TA (Invitrogen) using the 5'-TCAAAGGACATTACAAAATCC-3' and 5'-GCGATATCATGGCCAGCAAAGG-3' forward primers, respectively, and the 5'-GCGCGGCCGCTTAATCCTTATTGATATTC-3' reverse primer. The GST-A483A construct was generated by PCR using 5'-GGCGAATTCCCGGAGACACATATTTAACACG-3' forward and 5'-GCCGTCGACTTAATCCTTATTGATATTCT-3' reverse primers and cloning into the EcoRI-SalI ofpGEX-4T-2 (GE Lifesciences). pGST-A524 was previously described 24 . The pGFP-vD1 plasmid was generated by polymerase chain reaction using the forward primer 5'-ACCCGGGATCCCGCC-3' and reverse primer 5'-ACCCGGGACCAGGCA-3', and cloned into peGFP. The pmCherry-human vinculin (HV) and pmCherry-VASP plasmids were from Addgene. Stealth siRNA anti-human vinculin was from Invitrogen (reference number 1299001).

Protein purification

BL21 (DE3) chemically competent E. coli (Life Technologies) was transformed with the expression constructs. D1 and D1D2 were purified essentially as described 11, 25 . For the IpaA derivatives, bacteria grown until OD600nm = 1.0 were induced with 0.5 mM IPTG and incubated for another 3 hrs. Bacteria were pelleted and washed in binding buffer 25 mM Tris PH 7.4, 100 mM NaCl and 1 mM betamercaptoethanol, containing Complete TM protease inhibitor. Bacterial pellets were resuspended in 1/50th of the original culture volume and lyzed using a cell disruptor (One shot model, Constant System Inc.). Proteins were purified by affinity chromatography using a GSTrap HP affinity column (GE Healthcare) and size exclusion chromatography (HiLoad S200, Ge Healthcare). Samples were stored aliquoted at -80°C at concentrations ranging from 1 to 10 mg/ml.

Protein complex formation analysis

Proteins were incubated at a concentration of 30 M in binding buffer for 60 min at 4°C. Samples were analyzed by SEC-MALS (Wyatt Technology Europe) using a 24 ml Superdex 200 Increase 10/300 GL filtration column and a MiniDAWN TREOS equipped with a quasi-elastic light scattering module and a refractometer Optilab T-rEX (Wyatt Technology). Data were analyzed using the ASTRA 6.1.7.17 software (Wyatt Technology Europe). Protein complex formation was visualized by PAGE under non-denaturing conditions using à 7.5% polycrylamide gel, followed by Coomassie blue staining. BN-PAGE buffer (250 mM -aminicaprionic acid and 25mM Bis-Tris PH 7,0) at 4°C for one hour. The protein mixtures were separated in a one-dimension native BN-PAGE electrophoresis as described 26 . For vinculin-IpaA protein ratio assay, vinculin-IpaA bands containing the complexes separated by BN-PAGE were cut, sliced and boiled in 2 x Laemmli SDS buffer followed by SDS-PAGE. The second dimension SDS-PAGE gels were stained (colloidal Coomassie staining) and the density of the bands was determined using Image J. The normalized vinculin:IpaA ratio of the complexes was compared using a nonparametric Kruskal-Wallis rank sum test (R statistical software).

Solid-phase binding assay

For crosslinking vinculin-IpaA complex, bands containing the complexes were cut, sliced and electroeluted in native conditions (15 mM Bis-Tris pH 7.0 and 50 mM Tricine) inside a closed dialysis membrane (SpectraPor). The soluble complexes were recovered and their buffer exchanged twice into an amine-free cross-link buffer in 25 mM HEPES pH 7.0 containing 100 mM NaCl using 10MWCO ZEBA desalting columns (Thermo Scientific). The fractions containing the complexes were incubated for 1 hr at 4°C with 10 mM N-hydroxysulfosuccinimide and 5 mM EDC (Sigma-Aldrich) following the manufacturer's recommendations. The cross-linking reaction was stopped by adding 50 mM Tris pH 7.4

and incubating for 20 minutes. Samples were denaturated in 2x SDS Laemmli buffer for 5 min at 95°C and complexes were eluted from gel slices following SDS-PAGE.

Liquid Chromatography Mass spectrometry (LC-MS)

Complexes obtained after the cross-linking step were loaded onto a 4-20% polyacrylamide gradient gels and Coomassie stained. The bands containing the complexes were cut and submitted to tryptic digestion 27 . The experiments were performed in duplicates for the 3 complexes D1:A524, D1D2:A524 and D1D2:A483. Peptides obtained after tryptic digestion were analyzed on a Q Exactive Plus instrument (Thermo Fisher Scientific, Bremen) coupled with an EASY nLC 1 000 chromatography system (Thermo Fisher Scientific, Bremen). Sample was loaded on an in-house packed 50 cm nano-HPLC column (75 μm inner diameter) with C18 resin (1.9 μm particles, 100 Å pore size, Reprosil-Pur Basic C18-HD resin, Dr.

Maisch GmbH, Ammerbuch-Entringen, Germany) and equilibrated in 98 % solvent A (H2O, 0.1 % FA) and 2 % solvent B (ACN, 0.1 % FA). A 120 minute-gradient of solvent B at 250 nL.min-1 flow rate was applied to separate peptides. The instrument method for the Q Exactive Plus was set up in DDA mode (Data Dependent Acquisition). After a survey scan in the Orbitrap (resolution 70 000), the 10 most intense precursor ions were selected for HCD fragmentation with a normalized collision energy set up to 28.

Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1 and >7 were excluded. Dynamic exclusion was enabled for 35 or 45 seconds respectively.

Data analysis

The identification of cross-linked peptides from LC-MS data was performed using SIM-XL v. 1.3 28 , with the following search parameters: EDC as cross-linker, a tolerance of 20 ppm for precursor and fragment ions, trypsin fully specific digestion with up to three missed cleavages. Carbamidomethylation of cysteines was considered as a fixed modification. All initial identification of cross-linked peptides required a primary score of SIM-XL greater than 2.5 for inter-links and 2.0 for intra-links or loop-links. As single incorrect cross-link identification might lead to a different model, a manual post-validation of the search engine results at the MS2 level was thus performed. A 2D-map showing the protein-protein interaction was generated as an output (Figs. 2a,b). Only peptides present in the 2 replicates are gathered in Supplementary Tables 1-3 and were used for the modeling.

Modeling

We used the constraints obtained from the cross-linking MS data (Suppl. Tables 123) to guide the protein structure modeling using the TX-MS protocol as described by Hauri, Khakzad et al. 29 . In short, TX-MS uses the Rosetta comparative modeling protocol (RosettaCM) 30 , and the flexible backbone docking protocol (RosettaDock) 31 to generate models and evaluate how well each model explains the MS constraints using a novel scoring function. Here, a total of 100,000 models was generated, of which the highest-scoring model is displayed in (Fig2e), supported by a total of 100 inter and intra-molecular crosslinks.

Cell lines

C2.7 myoblasts 32 and MEF vinculin null cells 8 were routinely grown in DMEM 1 g / L glucose containing 10 % FCS in a 37°C incubator containing 10 % CO2. 1205Lu melanoma cells 33 were grown in RPMI + Glutamax medium (RPMI1640) supplemented with 10 % fetal calf serum (FCS) and non-essential aminoacids in a 37°C incubator with 5 % CO2.

Immunofluorescence

TIRF (Total Internal Reflection Microscopy) analysis

C2.7 cells were transfected with pmCherry-HV or pmCherry-VASP and the indicated plasmids as described above. Samples were mounted onto a TIRF microscopy chamber on a stage of an Eclipse Ti inverted microscope (Nikon) equipped with an Apo TIRF 100 x N.A. 1.49 oil objective heated at 37°C. TIRF analysis was performed using the Roper ILAS module and an Evolve EM-CCD camera (Roper Scientific Instruments). When mentioned, Y-27632 was used at 100 μM. Image acquisition was performed every 12.5 seconds for 30 to 90 minutes.

Live cell tracking

1205Lu melanoma cells were transfected with IpaA constructs or GFP alone (control) and transferred in microscopy chamber on a 37°C 5%-CO2 stage in RPMI1640 medium containing 25 mM HEPES. For cell tracking, samples were analyzed using and inverted Leica DRMIBe microscope and a 20 X phase contrast objective. Image acquisitions were performed every 3 min for 200 hrs. The mean velocity of migration was measured for all tracks followed for at least 5 hours. The root square of MdSD over time was plotted over time and fitted by linear regression. The slopes of the linear fit were compared using an ANCOVA test (linear model). The median cell surface was quantified as the mean of the surface for three time points (25%, 50% and 75%) of the whole cell track and dispersion measured by the Median absolute dispersion (MAD).

Invasion assays

Tissue culture Transwell inserts (8 μm pore size; Falcon, Franklin Lakes, NJ) were coated for 3 hours with 10 μg of Matrigel following the manufacturer's instructions (Biocoat, BD Biosciences, San Jose, CA). Inserts were placed into 24-well dishes containing 500 μl of RPMI medium supplemented with 1% fetal calf serum. 5 × 10 4 melanoma cells were added to the upper chamber in 250 μls of serum-free RPMI medium. After 24 hours, transmigrated cells were scored by bright field microscopy. Experiments were performed at least three times, each with duplicate samples.

Image processing and statistical analysis

For the quantification of the number of adhesion structures in C2.7 cells, a semi-automated protocol was developed using Icy software 34 . Spinning-disk fluorescent microscopy planes were used to detect vinculin structures using HK means thresholding and overlaid binary masks obtained from the threshold projections of F-actin labeled images (Max-entropy method). FAs were detected as spots positive for both vinculin mCherry and actin structures using Wavelet Spot Detector. The number of adhesions was analyzed using Dunn's multiple comparisons test. The statistical analysis of cell motility was performed in the R software. Medians were compared using a Wilcoxon rank sum test and dispersion by Median absolute dispersion (MAD) parameter.

Microfluidics cell adhesion assay

Analysis of cell detachment under shear stress was based on previous works 35 Acquisition was performed using a 20 X objective using phase contrast and fluorescence illumination (excitation 480 ± 20 nm, emission 527 ± 30 nm). Fluorescent images were acquired before and after flushing to differentiate between target and control cells. Phase contrast images were acquired every 200 ms. Fold enrichment was defined as the ratio between of attached labeled and unlabeled cells.

REFERENCES TO METHODS

Introduction

Shigella is a highly specialized bacteria that causes bacillary dysentery. Humans are its only known natural host and a very low bacterial load is sufficient to trigger disease (DuPont et al. 1989). All Shigella species harbor a large virulence plasmid of ~200 kb that encodes key virulence factors (Sansonetti et al. 1983), including a Type III Secretion System (T3SS) and a set of effector proteins that are translocated into the host cytosol. Unlike other pathogenic bacteria, Shigella does not express any known constitutive adhesin. However, adhesion is induced through the activity of the T3SS that activates binding of the bacterial surface protein IcsA to host cell receptors (Brotcke Zumsteg et al. 2014;Skoudy et al. 2000). Prior to contact with the cell body, Shigella is first captured by filopodia, a process mediated by the T3SS tip complex components (Romero et al. 2012). Upon contact with the host membrane, bacterial effector proteins are injected into the host cytoplasm (van der Goot et al. 2004). This initial contact depends on the IpaB-CD44 interaction (Skoudy et al. 2000) and on lipid rafts (Lafont et al. 2002). Once the translocon is formed, injection of first wave of effector proteins that remodel the cytoskeleton takes place within minutes, leading to the formation of actinrich foci (Enninga et al. 2005). IpaA, one of these Shigella type III effectors, is involved in bacterial invasion through its interaction with the focal adhesion proteins vinculin (Tran Van Nhieu, Ben-Ze'ev, and Sansonetti 1997) and talin (Valencia-Gallardo et al. 2019). This interaction is mediated by the presence of three Vinculin Binding Sites (VBS) at the IpaA C-terminal end (Park et al. 2011;Izard, Tran Van Nhieu, and Bois 2006). Recent evidence indicates that IpaA VBS3 also interacts with a partially unfolded conformer of talin (Valencia-Gallardo et al. 2019). Since talin is known to mediate integrin inside-out activation [START_REF] Changede | Nascent Integrin Adhesions Form on All Matrix Rigidities after Integrin Activation[END_REF], IpaA may trigger integrin activation through its interaction with talin.

It has been reported that injection of Shigella type III effector proteins in intestinal cells leads to downregulation of antimicrobial peptides in the absence of bacterial invasion [START_REF] Sperandio | Virulent Shigella Flexneri Subverts the Host Innate Immune Response through Manipulation of Antimicrobial Peptide Gene Expression[END_REF]. Also, T3S apparatus contact and / or injection of effector proteins in lymphocytes dampens the immune response (Pinaud et al. 2017a;Konradt et al. 2011a). Here, we describe how injection of IpaA by bacteria that transiently interact with host cells enhances Shigella capture associated with inside-out integrin activation. Priming of cells by IpaA injection in a "kiss-and-run" mechanism provides new insights into the Shigella invasion process.

Results

Construction of functional 4-Cys-tagged IpaA

To track the injection of IpaA into host cells, we generated a construct corresponding to full length IpaA fused to a teracystein motif (IpaA-4C; Figure 1A; Experimental procedures). This construction allows IpaA-4C detection based on fluorescein-based biarsenical dye (FlAsH) labeling, as previously reported for other Shigella and Salmonella T3SS effectors (Enninga et al. 2005;[START_REF] Van Engelenburg | Quantification of Real-Time Salmonella Effector Type III Secretion Kinetics Reveals Differential Secretion Rates for SopE2 and SptP[END_REF].

We first confirmed expression of IpaA-4C in an ipaA mutant strain by fluorescence microscopy analysis. As observed for the translocon components IpaB and IpaC prior to secretion, IpaA-4C showed a predominant localization at one bacterial pole (Figure 1B; Enninga et al., 2005: Collet et al., 2018). To determine if IpaA-4C was functional, we performed cell invasion assays. Shigella ipaA mutant expressing IpaA-4C showed the same invasion levels of a WT strain, indicating full complementation (Figure 1C).

IpaA injection in host cells in the absence of bacterial invasion

To track IpaA during bacterial early capture and invasion steps, Shigella expressing IpaA-4C was incubated with HeLa cells. Because of the low levels of Shigella invasivity in vitro, invasion assays were performed at high multiplicity of infection (MOI ~1000, Exp. procedures). As expected from previous immunofluorescence localization studies, IpaA-4C was observed to localize in membrane ruffles at bacterial entry foci (Figure 2A (Tran [START_REF] Van Nhieu | Modulation of Bacterial Entry into Epithelial Cells by Association between Vinculin and the Shigella IpaA Invasin[END_REF]. Surprisingly, all cells, including cells devoid of bacteria, showed increased levels of IpaA-4C signal, as compared to cells incubated with a mxiD mutant strain (Figure 2B), indicating that IpaA translocation occurred in the absence of bacterial invasion by "kissing-and-running" bacteria. IpaA-4C showed a diffuse labeling in the cell cytosol and also formed clusters at the cell apical side (Figure 2C). These IpaA clusters were consistently observed in all cells challenged with ipaA / IpaA-4C but not in cells incubated with the mxiD / IpaA-4C strain (Figure 2C). The median size of the clusters observed was of 0.34 μm 2 (Figure 2D).These observations indicate that IpaA persists in host cells following injection and suggest its association with a cell component, triggering its clustering at the cell apical membrane.

Injected IpaA promotes inside-out integrin activation

Talin interaction with integrin leads to integrin inside-out activation associated with the formation of integrin nano-clusters [START_REF] Changede | Nascent Integrin Adhesions Form on All Matrix Rigidities after Integrin Activation[END_REF]. Since IpaA was shown to bind to talin, presumably stabilizing a partially-unfolded talin conformer (Valencia-Gallardo et al. 2019), we reasoned that the IpaA clusters observed at the apical membrane could be linked to integrin clusters following IpaA-and talin-mediated insideout activation. To test this, we used an antibody that binds to activated integrin following cell challenge with Shigella strains expressing various fragments of IpaA.

As shown in Figure 3, in non-infected cells and cells incubated with the T3S-defficient mxiD mutant strain, active integrins showed a diffuse distribution, with clusters usually detected at one cell edge but not overall the cell body (Figure 3; NT; mxiD). In cells challenged with WT Shigella, however, clusters were consistently observed throughout the apical cell surface, resembling those present in cells with manganese-induced integrin activation (Figure 3; WT, Mn 2+ ). This change in active integrin distribution was dependent on IpaA, since no such integrin redistribution could be detected when cells were challenged with an ipaA mutant strain (Figure 3). We next evaluated the contributions of IpaA VBSs in integrin activation. Neither bacteria lacking all three VBSs nor VBS3 showed increased clustering at the cell body, (Figure 3; dVBS1-3, dVBS3).

VBS3 alone was shown to be sufficient to restore active integrin redistribution (Figure 3; dVBS1-2).

Although a qualitative change in active integrin distribution was observed, the overall levels of active integrin were not significantly different between cells challenged with an ipaA mutant strain and a wild-type strain, when analyzed by immunofluorescence microscopy (Figure 4A). The lack of significant differences might be due to the detection limits of the procedure used to detect active integrins with respect to the low amounts of IpaA injected by bacteria. To overcome this limitation and further confirm that intracellular IpaA triggered integrin inside-out activation, cells were transfected with constructs containing various IpaA subdomains and analyzed integrin activation.

Consistent with previous observations, cells expressing all three VBSs had an increased level of active integrins, reaching overall levels similar to manganeseactivated cells (Figure 4B; VBS1-3, +Mn 2+ ). In contrast, cells lacking VBS3 had levels of active integrin similar to non-transected cells (Figure 4B; VBS1-2; NT). These results indicate that VBS3 mediates integrin activation and clustering.

Priming of epithelial cells by injected IpaA-4C facilitates de novo

Shigella invasion

Previous reports have shown the important role of IpaA in bacterial invasion and the contribution of IpaA VBSs. Given that Shigella injection of IpaA into host cells leads to the general activation of integrins on the cell surface, including cells devoid of bacteria, we tested whether "kiss-and-run" injection of IpaA primes cells for de novo invasion by extracellular bacteria. A microfluidics setup was implemented to assess de novo bacterial capture by primed cells. HeLa cells plated inside the microfluidic chamber were incubated with bacteria for 30 mins to allow for T3 effector injection (priming).

Then, samples were washed with buffer to remove bacteria that were not associated with cells and a bacterial suspension was perfused under a constant flow into the chamber. Using this procedure, few rare events of bacterial capture were observed in primed HeLa cells (Figure 5A). In contrast, such capture events were not observed in not-primed cells. This suggests that bacterial capture was promoted by prior bacterial priming.

To better quantify de novo bacterial capture, we performed a high-thoroughput twowave invasion assay. In the first, priming wave, cells were incubated in 6-well plates with Shigella ipaA mutant expressing different IpaA constructs. After washing, cells were challenged with a second wave of wilt-type Shigella. Samples were fixed and invasion levels were quantified by immunostaining. We used GFP-expressing Shigella in the second wave challenge to differentiate bacteria from the two infection waves.

As shown in Fig. 5B, priming with Shigella WT resulted in a 3-fold increase in de novo bacterial capture over cells primed with the T3 secretion-deficient mxiD strain. However, when cells were primed with an ipaA mutant strain, no increase in bacterial capture was observed indicating that IpaA was required for priming. Bacteria expressing IpaA constructs lacking all three VBSs or VBS3 alone failed to restore de novo capture.

Remarkably, priming of bacteria expressing IpaA VBS3 alone was sufficient to restore the capture levels (Figure 5B). These results indicate that IpaA VBS3 mediates host cell priming to increase de novo bacterial capture and invasion.

Discussion

Here, we report that Shigella is able to prime epithelial cells for invasion through a kissand-run mechanism. While the effects of injected T3 effectors on immune function of lymphocytes and the downregulation of antimicrobial peptides production were previously observed in the absence of bacterial association (Konradt et al. 2011b;Pinaud et al. 2017b;[START_REF] Sperandio | Virulent Shigella Flexneri Subverts the Host Innate Immune Response through Manipulation of Antimicrobial Peptide Gene Expression[END_REF], the impact on bacterial invasion of epithelial cells has not been reported previously.

We show here that IpaA VBS3 is crucial for an efficient integrin clustering and priming of epithelial cells. Since no such effects were observed with IpaA VBS1-2, the IpaA VBS3dependent priming is likely be due to its unique ability to bind to talin (Valencia-Gallardo et al. 2019). Our results suggest that VBS3 mediates integrin inside-out activation, which might in turn affect integrin clustering and concomitant membrane lipid composition. Indeed, integrins and focal adhesion proteins are associated with lipid rafts in adhesion structures (Head, Patel, and Insel 2014). These lipid rafts coalesce after integrins engage with fibronectin, permitting the rapid recruitment of talin at FAs [START_REF] Fuentes | Coordinated Mechanosensitivity of Membrane Rafts and Focal Adhesions[END_REF]. This process is mediated by integrin-dependent Src-RhoA signaling, leading to activation of formins and consequent actin nucleation [START_REF] Kalappurakkal | Integrin Mechano-Chemical Signaling Generates Plasma Membrane Nanodomains That Promote Cell Spreading[END_REF], implicating signaling pathways also involved in Shigella invasion (Dumenil et al., 1999;[START_REF] Alto | Identification of a Bacterial Type III Effector Family with G Protein Mimicry Functions[END_REF][START_REF] Mounier | The IpaC Carboxyterminal Effector Domain Mediates Src-Dependent Actin Polymerization during Shigella Invasion of Epithelial Cells[END_REF]. Inside-out integrin activation could represent a strategy exploited by Shigella to promote capture of additional bacteria, since the association of the T3SS and translocation of protein effectors depends on the presence of lipid rafts enriched in cholesterol and sphingolipids (van der Goot et al. 2004). Furthermore, these membrane domains are also enriched in the hyaluronan receptor CD44, which associates with the T3SS tip complex protein IpaB [START_REF] Oliferenko | Analysis of CD44-Containing Lipid Rafts: Recruitment of Annexin II and Stabilization by the Actin Cytoskeleton[END_REF]). The clustering of integrins might also directly play a role, since IpaB, together with IpaC and ipaD, have also been reported to associate with integrins [START_REF] Watarai | Interaction of Ipa Proteins of Shigella Flexneri with alpha5beta1 Integrin Promotes Entry of the Bacteria into Mammalian Cells[END_REF].

Talin and vinculin recruitment at entry sites mediated by IpaA VBSs, together with vinculin activation, could promote the coalescence of lipid rafts at bacterial injection sites. Such process could explain the formation of IpaA clusters at the host cell surface (Park et al. 2011;Izard, Tran Van Nhieu, and Bois 2006;Valencia-Gallardo et al. 2019).

Additionally, IpaA activity as RhoA activator could also play a role in promoting integrin clustering (Demali, Jue, and Burridge 2006).

A similar mechanism of cooperative invasion has been reported in Salmonella and was shown to be dependent on the presence of Salmonella-induced membrane ruffles, (Lorkowski et al. 2014;[START_REF] Misselwitz | Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion[END_REF]. Lorkowski et al. showed that additional bacteria invaded preferentially at sites where an initial invasion event took place. In the present study, although bacteria was observed to bind to Shigella-induced ruffles, binding of additional bacteria elsewhere in the cell body was also observed, indicating that membrane ruffling is not necessary to induce de novo bacterial capture by kissand-run bacteria.

Shigella priming of host cells might also have a favorable impact on other pathogens, such as Yersinia spp [START_REF] Martens | Interactions of Commensal and Pathogenic Microorganisms with the Intestinal Mucosal Barrier[END_REF]. The intestinal milieu is densely populated by bacteria, and bacterial capture is unlikely to be restricted to Shigella. Thus, the promotion of generalized bacterial capture might contribute to coinfections involving Shigella and other enteric pathogens, as is evidenced by the high rate of co-infections in dysentery-endemic areas (Andersson et al. 2018).

Experimental procedures

Generation of IpaA-tetracysteine Shigella strain

IpaA tetracystein construction (IpaA-4C) was generated by polymerase chain reaction using the 5'-TGCCCGGGCTGCTGCATGGAACCGTAAAAGGGCGAATTCTGCAGA-3',

Discussion

Shigella is a highly efficient pathogen, despite being non-motile and lacking "conventional" adhesion mechanisms, ingestion of a very low dose is sufficient to trigger dysentery symptoms. The fact that the acquisition of the pINV plasmid allows E. coli strains to become invasive and that a convergent loss of motility is observed in different strains, suggest that advantages conferred by motility and constitutive adhesion are fully compensated by proteins carried in the pINV plasmid.

IpaA is a highly conserved protein and strains lacking this protein, and specifically its VBSs, are highly deficient for invasion. Identification of the mechanisms through which this protein exerts its function can lead to a better understanding of invasion strategies not only in Shigella, but also in other pathogens, since pathogens often target similar processes.

Because of their biochemical similarity to talin VBSs (i.e. being amphipathic helices with some functionally homologous residues at their contact sites and binding to vinculin in their VD1 domain in a similar fashion), previous studies of isolated IpaA VBS1 and VBS3 have attributed their functional activity to disruption of vinculin headtail interactions, leading to its activation. VBS2 role was thought to act in conjunction with VBS1 to lock IpaA to vinculin. This would suggest a functional redundancy for IpaA VBS1-2 and VBS3, which seems to be unlikely. It is therefore logical to speculate that IpaA VBSs convey additional functionalities.

In search for novel functions for VBS3, the host laboratory performed a screening for unknown binding partners through a double-hybrid assay, yielding talin as a potential candidate.

Previous studies have shown that IpaA VBS2 binds to the second bundle of VD1, suggesting a binding model where IpaA VBS1 binds to the first bundle of VD1 and IpaA VBS2 to the second bundle, stabilizing the interaction. However, under this scheme a binding site for VBS3 would have to be somewhere else in the vinculin molecule. Solid-phase assays also suggested the unveiling of additional binding sites outside the VD1 vinculin domain when it is incubated with IpaA VBS 1-3, a finding that has not been previously reported for any vinculin-interacting molecule.

IpaA VBS3 binding to talin

First described as a Vincnulin Binding Site, IpaA VBS3 binding to talin is unexpected, as no amphpatic -helices have been reported to be hybrid talin/vinculin binders.

Comparison with other VBSs and residue substitution analysis indicate that hydrophobic interactions are relevant for targeting hydrophobic grooves of both vinculin and talin. However, the residues K498 and R489 account for talin-specific interactions, as suggested by VBSs alignment and affinity analysis. This analysis also suggests that talin and vinculin hybrid binding properties could be a more general property of VBSs, as the same surface is involved in binding to both targets. Furthermore, Talin VBSs 46, 9, 6 and 50; as well as IpaA VBS1 were observed to bind to talin H1-H4 with varying affinity degrees. It is unlikely that this functional similarity is the result of domain duplication, since their aminoacid sequences are remarkably different and their relative positions inside their respective helical bundles is different in some cases. Talin H5, the helix that is replaced by IpaA VBS3, is the fifth helix of a 5-helix bundle and so it is H46. However, talin VBSs 9 and 6 are part of a 4-helix bundle, whilst talin H50 is the fourth helix of a 5-helix bundle.

The fact that hybrid talin-binding VBSs target talin H1-H4 fragment but are unable to bind to the 5-helix bundle indicates hybrid VBSs are unable to displace H5 helix. This would make a partially-unfolded talin conformer the target of these hybrid VBSs. As it is described in the introduction, talin rod unfolds upon force application and there is evidence that talin bundles that contain H1-H4 (Bundle R1) and H46 (Bundle R10) unfold under a similar force range (Yao et al. 2016). In addition, there is modeling and experimental evidence suggesting that some talin bundles have stable intermediates before becoming completely unfolded (Mykuliak et al. 2018), which supports the existence of an R1 bundle with a displaced H5 helix. However, most of the experimental studies performed so far have explored only complete unfolding of helical bundles and lack the sensitivity to detect single-helix displacements. If a talin conformer with its fifth helix is displaced exists, it would most likely be located in cell regions where force exertion is low. In agreement with this hypothesis, we detected the presence of GFP-labeled VBS3 along filopodia. Along filopodia, retraction forces are driven by actin fibers' retrograde flow and are driven by cortical actin dynamics [START_REF] Bornschlögl | Filopodial Retraction Force Is Generated by Cortical Actin Dynamics and Controlled by Reversible Tethering at the Tip[END_REF], thus generating low-range forces on protein linkers such as talin. It is important to note that this labeling was located in talin-enriched regions devoid of vinculin, whereas vinculin signal was detected towards the cell interior, a region where force exertion would be higher.

Based on the high structural similarity between IpaA VBS3 and talin H46 and on the high affinity at which both of them bind to talin H1-H4, we propose talin H46 and perhaps other talin VBSs mediate talin-talin interactions that might result in talin oligomers. The role of these hypothetical talin oligomers requires further studies. It has been reported that the amount of force that can be transmitted by filopodia is limited by the strength of the link between actin fibers and the membrane at the filopodia tip. Talin is recruited at filopodial tips by myosin X and connects actin fibers to integrin [START_REF] Lagarrigue | A RIAM/lamellipodin-talin-integrin Complex Forms the Tip of Sticky Fingers That Guide Cell Migration[END_REF]. It is therefore possible that under a specific force range at which partially-unfolded talin conformers exist, endogenous hybrid VBSs stabilize talin-talin interactions to increase the linkage strength between actin fibers and integrins at the filopodial tips, thus modulating the force exerted by filopodia.

Application of increasing forces along the filopodia might disrupt the partiallyunfolded conformation and lead to a full exposure of VBSs and to vinculin recruitment towards the cell body (Figure 18A).

GFP tagged IpaA VBS3 localizes to filopodial tip and shaft adhesions and its enriched in nascent adhesions. During Shigella invasion, it is likely that its role involves the stabilization of early and low force adhesions. In particular, targeting of nascent and filopodial adhesion structures by Shigella is relevant for bacterial invasion for a number of reasons. Being Shigella's first contact with host cells through filopodia, it would be relevant for it to promote its adhesion once the initial contact is established through the T3S apparatus. Indeed, GFP-tagged IpaA VBS3 was detected at bacterial contact sites with filopodial tips. As mentioned before, the limiting factor during in filopodial retraction force lies in the linkage between actin fibers and the membrane. VBS3 stabilization of low-force adhesions at initial contact sites might strengthen this linkage.

Vinculin supra-activation: a novel interaction mode

All the previously reported VBSs interact with the first domain of vinculin, as has been described for talin, α-actinin (Philippe R. J. Bois et al. 2005[START_REF] Bois | The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin[END_REF], α-catenin [START_REF] Peng | α-Catenin Uses a Novel Mechanism to Activate Vinculin[END_REF]) and isolated IpaA VBSs ( [START_REF] Van Nhieu | Vinculin Binding in Its Closed Conformation by a Helix Addition Mechanism[END_REF]Park, Valencia-Gallardo, et al. 2011;Izard, Tran Van Nhieu, and Bois 2006). Moreover, the classical mode of activation by talin requires mechanical stretching of talin to reveal its cryptic VBSs and it does not involve vinculin oligomerization. This is why the generation of vinculin trimers upon incubation with an IpaA subdomain containing VBSs 1-3 is unexpected. The only mechanism described so far involving vinculin multimerization is by vinculin binding to phosphatidylinositol 4,5 -bisphosphate, which induces homotypic and heterotypic interactions through the vinculin tail domain. This interaction is proposed to contribute to vinculin activation and turnover at focal adhesions [START_REF] Chinthalapudi | Lipid-Directed Vinculin Dimerization[END_REF][START_REF] Chinthalapudi | Differential Lipid Binding of Vinculin Isoforms Promotes Quasi-Equivalent Dimerization[END_REF].

Structural modeling of vinculin/IpaA VBS1-3 equimolar complex, based on this crosslink data, suggest the existence of two conformers. In one model IpaA VBS3 binds to the interface between vinculin subdomains VD1 and VD2. This finding alone is outstanding, since no binding sites have been reported to target this region. The second conformer, in addition to contain a VBS3 binding site at the interdomain interface, importantly involves the rearrangement of a conformational change in the orientation between both domains. This finding is also highly relevant, since no rearrangements induced by VBSs in vinculin head domain have ever been reported. This suggests a novel mechanism of action by IpaA VBSs when forming part of the same polypeptide, as opposed to the apparent functional redundancy previously reported when studied separately.

On the other hand, modelling of vinculin interacting with a construct devoid of IpaA VBS3, IpaA VBS1-2, yielded no significant difference in the conformation of vinculin domains VD1 and VD2. IpaA VBS1-2 was also unable to generate high-order vinculin trimers. Thus, vinculin trimer formation and conformational changes require the presence of VBS3 as part of the subdomain.

It is likely that the vinculin/IpaA VBS1-3 modeled structures represent intermediate states between inactive vinculin and the formation of vinculin trimers devoid of IpaA VBS1-3. Under this hypothesis, binding of IpaA would not only disrupt head-tail interactions, but would also trigger conformational changes in vinculin head, unveiling novel binding sites that mediate vinculin-vinculin interactions, perhaps by disrupting interactions between VD1 and VD2 domains. It is reasonable to assume that vinculin multimerization domains are located in the first two vinculin head domains, since timer formation of full-length vinculin is recapitulated when using vinculin fragments containing only VD1 and VD2. Although no structural modeling of vinculin trimers were generated, it is likely that IpaA VBSs binding pockets become either unavailable or are allosterically modified by vinculin head rearrangement, since no IpaA VBS1-3 was detected in complex with vinculin trimers. If this is the case, it is also likely that affinities for VBSs (e.g. talin, α-actinin, vinexin) from other proteins change when trimers are formed, as they also bind to VD1. This would lead to a force-dependent differential scaffolding of adhesion proteins. This mechanism of regulation is completely different from the "classical" activation described so far, which is why we propose the new term "supra-activation" when referring to this process (Figure 19B).

IpaA-induced conformational changes in vinculin could be proxies of an endogenous process. It is important to remember that vinculin, like talin, lies at the core of adhesion structures, a mechanosensitive structure [START_REF] Case | Integration of Actin Dynamics and Cell Adhesion by a Three-Dimensional, Mechanosensitive Molecular Clutch[END_REF]. Both talin and vinculin share also an α-helical bundle structure and both are tethered at their extremes: talin to integrin and F-actin, and vinculin to VBSs and F-actin. Being its tail domain tethered to the fibers, the physical stress could be transmitted to the head domain and lead to conformational changes in the rest of the molecule, in an analogous manner to talin unfolding. This could result in the unveiling of cryptic binding sites, perhaps not only to other vinculin molecules, but also to other proteins.

The fact that the trimer is devoid of VBSs might also mirror a physiological process. Supra-activation might induce a change in binding affinities for binding partners under the canonical active state. This might contribute to regulate the scaffolding of adhesions proteins at different levels of tension. Indeed, there is evidence that other proteins involved in adhesions can experience conformational changes (X. [START_REF] Hu | Molecular Stretching Modulates Mechanosensing Pathways[END_REF]). However, direct evidence for trimer formation in vivo and under physiological conditions is still required.

Modelling of vinculin interacting with a construct devoid of IpaA VBS3, IpaA VBS1-2, yielded no significant difference in the conformation of vinculin domains VD1 and VD2. IpaA VBS1-2 was also unable to generate high-order vinculin trimers. Thus, vinculin trimer formation and conformational changes require the presence of VBS3 as part of the subdomain. It is likely that the interaction of VBS3 with the VD1-VD2 interface is crucial to trigger vinculin trimer formation. [START_REF] Carisey | Vinculin Regulates the Recruitment and Release of Core Focal Adhesion Proteins in a Force-Dependent Manner[END_REF]). Although it is known that constitutively active vinculin and VD1 domains retard talin refolding due to stabilization of unfolded talin VBSs, they also eventually disappear. It is remarkable that adhesion structures in cells transfected with IpaA VBS1-3 resist the effect of actomyosin relaxation by Rho kinase inhibitor, and in some cases adhesions even increase in size, a phenomenon that surpasses the effect of constitutively-active vinculin. This observation further supports a novel force-independent vinculin activation mechanism by IpaA VBSs.

IpaA VBSs effects on focal adhesion increase of stability also impact cell adhesion strength. Microfluidic-adhesion assay also indicates that cell adhesion is enhanced in cells transfected with IpaA VBS1-3 and this effect is dependent on its effects through vinculin. Notably, the difference in resistance to cell detachment between control cells and IpaA VBS1-3 transfected cells is more marked at early time points after initial contact with the substrate. Both cell populations have similar detachment levels at later time points. This would suggest that IpaA VBS1-3 does not have an effect on the adhesion strength that the cell can exert, but it rather triggers force-independent maturation of adhesion structures. These observations suggest IpaA VBS1-3 decreases the amount of force required to trigger adhesion formation by bypassing steps that normally require actomyosin contraction.

It is important to note that neither conformational changes nor the striking cell adhesion effects take place when IpaA VBS 1-2 constructs were analyzed. The results observed for this construct were more in accordance with the canonical activation of vinculin. This stresses the role of IpaA VBS3 in supra-activation and further supports in vivo effects of conformational changes induced by IpaA VBS1-3 on vinculin head observed in vitro.

Although there is no direct evidence of trimer formation in vivo, the hypothesis that IpaA-mediated supra-activation of vinculin bypasses the requirements of force application is strongly supported by the experimental results.

IpaA VBSs role during Shigella invasion

IpaA VBSs mutants have a low invasion ability, thus highlighting the importance of recruiting vinculin and talin to bacterial entry sites. Shigella is first captured by filopodia (Romero et al. 2011), mediated by the interaction between T3 apparatus and filopodial tips. After contact, injection of IpaA might contribute to the stabilization of semi-stretched talin through IpaA VBS3 for an efficient filopodial capture to take place. After filopodial retraction and engagement of increasing forces, talin might completely stretch, leading to IpaA VBS3 release and allowing IpaA interaction with vinculin (Figure 20).

Bacterial anchoring to cytoskeleton is highly important, since in paralell to IpaA secretion T3SS effector proteins IpgB1 and IpgB2 promote the formation of membrane ruffling and bacteria could ultimately be pushed outwards. However, Shigella requires to do this in the absence of a counterforce. IpaA induction of conformational changes on vinculin (supra-activation) that would otherwise require force is thus an elegant solution to this problem.

On the other hand, the observation of injection-only events and the presence of IpaA clusters at the cell surface devoid of bacteria suggest a cooperative invasion process, similar to the one reported for Salmonella, where additional bacteria are captured by ruffles induced by previous bacteria (Lorkowski et al. 2014). However, it is likely that bacteria undergoing this kiss-and run mechanism result in a priming of the cell surface, rendering competent the host surface for bacterial invasion or stimulating the formation of filopodia to increase filopodial capture, as Shigella IpaA VBSs do.

Bacterial adhesins and flagella are often the target of immune response. In the intestinal lumen the constant shedding of antibodies that target bacterial surface proteins can prevent the adhesion of both commensal and pathogenic bacteria [START_REF] Kline | Bacterial Adhesins in Host-Microbe Interactions[END_REF]. Although Shigella infections do induce serum and mucosal antibody responses, protection against reinfections is not always conferred [START_REF] Mani | Status of Vaccine Research and Development for Shigella[END_REF]. In addition to bacterial effector proteins that dampen the immune response, the lack of bacterial targets such as adhesins and flagella might be part of Shigella pathogenic strategy. In order to compensate this, targeting the natural host adhesion machinery to promote bacterial uptake is an elegant strategy.

As evidenced in previous studies, Shigella depends on a cholesterol-and sphingolipid-enriched membrane composition to trigger T3S interaction and translocation (van der Goot et al. 2004). This composition is also present in lipid rafts enriched at cellular adhesions (Head, Patel, and Insel 2014). It is thus plausible that IpaA-mediated induction of adhesion-like structures at invasion sites is a strategy to also locally modify membrane composition to promote bacterial contact through T3S apparatus. It is known that talin interaction with integrins can lead to integrin insideout activation, and activated integrins are associated with lipid rafts, and so are several of the molecules involved in adhesion formation [START_REF] Changede | Integrin and Cadherin Clusters: A Robust Way to Organize Adhesions for Cell Mechanics[END_REF]. This strategy would result in the double function of promoting bacterial adhesion to cell surface and anchoring to host cytoskeleton.

The observation of increase in integrin activation levels in cells transfected with IpaA VBS1-3 supports the hypothesis that IpaA interaction with vinculin and talin promotes inside-out activation. Also, the observation of integrin clustering on the surface of cells injected with kiss-and-run bacteria suggests that Shigella injection-only strategy might have an impact in the sensitivity of cells to bacterial capture. Of note, similar strategy of cooperative invasion has been reported in Salmonella (Lorkowski et al. 2014), Furthermore, Shigella sensitization of host cells might also have a favorable impact in other pathogens. The intestinal milieu is densely populated by bacteria, and bacterial capture is unlikely to be restricted to Shigella. The promotion of unrelated bacterial capture by Shigella might explain the high rate of coinfections of enteric pathogens observed in areas of endemic disease, where coinfection reaches 65% of the cases (Andersson et al. 2018).

Finally, other pathogens might trigger similar vinculin and talin targeting. Rickettsia protein Cell Antigen 4, has been described to harbor two VBSs [START_REF] Park | The Rickettsia Surface Cell Antigen 4 Applies Mimicry to Bind to and Activate Vinculin[END_REF], and Chalmydia protein TarP contains a segment with three VBSs in a similar spatial arrangement to that of IpaA [START_REF] Thwaites | Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane[END_REF]. However, their mechanism of action remains to be further explored. On the biochemical side, we describe a new role of IpaA VBS3 in targeting a semistretched talin conformer. We also show a novel mechanism of vinculin activation and induction of conformational changes in vinculin head, pointing towards a potentially unreported physiological mode of action for vinculin on adhesion structures, to which Shigella has evolved to take advantage of. This "supraactivation" mechanism, mediated by IpaA VBS1-3, would have the particularity of not requiring the exertion of force to form adhesion-like structures around bacterial foci, This aspect is key for a free-floating bacteria to anchor to its host cytoskeleton, while at the same time inducing membrane ruffling.

On the last part of this work, we show that kiss-and-run injection of effector proteins, and particularly of IpaA, promotes a cooperative invasion. Although IcsA has been reported to act as an inducible adhesin upon bile salt contact, no constitutive receptor has been identified. On the contrary, interaction between the tip complex proteins and lipid rafts components (cholesterol and sphingolipids) and enriched proteins (CD44 and integrins), has been shown to be essential for invasion. Both the recruitment of focal adhesion proteins to bacterial entry sites and the stimulation of RhoA activity induced by IpaA might contribute to the priming of the host surface, enriching it with lipid rafts, and promoting the adhesion and invasion of additional bacteria.

Altogether, IpaA highjacking molecular mechanisms reveal a highly adapted strategy for invasion. First, priming of cells to promote the capture of additional bacteria by talin recreuitment to the host membrane and triggering inside-out activation and modifying membrane lipid composition. And second, its ability to take advantage of focal adhesion proteins' mechanosensing properties to promote bacterial anchoring in the absence of counterforces. Funcionally, IpaA would first bind to partiallyunfolded talin in order to stabilize filopodial adhesions upon bacterial contact. As filopodia retract and the amount of force increases, IpaA would be released from talin due to further talin unfolding and would be free to supra-activate vinculin, leading to the formation of force-independent adhesion-like structures.

It is likely that other pathogens take advantage of similar mechanisms, being cytoskeleton and membrane components common virulence targets. Of note, Salmonella can also promote a cooperative invasion dependent on the formation of membrane ruffles (Lorkowski et al. 2014). However, the mechanism behind this phenomenon is different, since the homolog of IpaA in Salmonella, SipA, has a low degree of homology with IpaA at its C-terminus and does not possess VBSs [START_REF] Kaniga | Identification of Two Targets of the Type III Protein Secretion System Encoded by the Inv and Spa Loci of Salmonella Typhimurium That Have Homology to the Shigella IpaD and IpaA Proteins[END_REF].

However, other pathogens also harbor VBSs in their effector proteins. Such is the case of Rickettsia protein Cell Antigen 4, where two VBSs have been reported [START_REF] Park | The Rickettsia Surface Cell Antigen 4 Applies Mimicry to Bind to and Activate Vinculin[END_REF]) and of Chalmydia protein TarP, which also harbors three VBSs in a highly similar arrangement to Shigella VBSs [START_REF] Thwaites | Vinculin Interacts with the Chlamydia Effector TarP Via a Tripartite Vinculin Binding Domain to Mediate Actin Recruitment and Assembly at the Plasma Membrane[END_REF]. Although reported to bind to and activate vinculin, no studies have been done to analyze noncanonical vinculin activation. However, filopodial capture and formation of adhesionlike structures have been observed at entry sites (Ford et al. 2018), which suggest a similar mechanism might take place.

  mueven las tenazas del papá del camarón el pescado que Dios comió la serpiente que devoró un conejo el conejo que siempre se burló del coyote el coyote que tragó un panal de avispas la miel que brota de mis senos

  FIGURE 1. SHIGELLA DIARRHEA MORTALITY RATE ................................................................................................ FIGURE 2. SHIGELLA WHOLE-GENOME PHYLOGENETIC TREE .............................................................................. FIGURE 3. COMPARATIVE MAP OF THE SHIGELLA VIRULENCE PLASMID ............................................................. FIGURE 4. STRUCTURE OF THE INTESTINAL MUCOSA........................................................................................... FIGURE 5. STRUCTURE OF THE SHIGELLA T3SS ..................................................................................................... FIGURE 6. REGULATION OF THE SECOND WAVE OF EFFECTORS .......................................................................... FIGURE 7. DYNAMICS OF ACTIN FILAMENTS......................................................................................................... FIGURE 8. MYOSIN II AND Α-ACTININ IN ACTIN FILAMENT CONTRACTION .......................................................... FIGURE 9. INTEGRINS LIGANDS AND INTRACELLULAR DYNAMICS ....................................................................... FIGURE 10. MATURATION OF ADHESION STRUCTURES ........................................................................................ FIGURE 11. MODEL OF THE MOLECULAR CLUTCH HYPOTHESIS ........................................................................... FIGURE 12. STRUCTURAL MODEL OF TALIN .......................................................................................................... FIGURE 13. VINCULIN STRUCTURE AND ACTIVATION ........................................................................................... FIGURE 14. FOCAL ADHESION ORGANIZATION ..................................................................................................... FIGURE 15. CATCH-BOND MECHANISM SCHEME ................................................................................................. FIGURE 16. PEDESTALS FORMED BY EPEC AND EHEC ........................................................................................... FIGURE 17. FILOPODIAL CAPTURE ........................................................................................................................ FIGURE 18. IPAA VINCULIN BINDING SITES ........................................................................................................... FIGURE 19. MODEL OF IPAA VBSS INTERACTION WITH FOCAL ADHESION PROTEINS ....................................... FIGURE 20. MODEL FOR IPAA ACTION ON TALIN AND VINCULIN .......................................................................

  SHIGELLA EFFECTOR PROTEINS .............................................................................................................. TABLE 2. PILI/FIMBRIAE OF PATHOGENIC GRAM-NEGATIVE BACTERIA ............................................................... TABLE 3. NON-EXHAUSTIVE LIST OF ADHESION FACTORS AND HOST RECEPTORS OF PATHOGENIC BACTERIA .. disease characterized by fever, abdominal pain, cramps, and diarrhea with bloody and mucoid stools. This disease is commonly caused by Entamoeba histolytica or by Shigella species, which are respectively termed amoebic dysentery and bacillary dysentery ("WHO | Dysentery" 2010). Although both diseases cause a similar set of symptoms, the underlying mechanisms are different.

Figure 2 .

 2 Figure 2. Shigella whole-genome phylogenetic tree. Strains from S. flexneri (SF), S. sonnei (SS), S. boydii (SB) and S. dysenteriae (SD) are clustered in 5 different monophyletic lineages (S1-S5). Reproduced from (Sahl et al. 2015).

Figure 2 .

 2 Figure 2. Shigella whole-genome phylogenetic tree

Figure 3 .

 3 Figure 3. A comparative gene map of the Shigella virulence plasmid. Black ring shows the GC content of the reference pSs_046 sequence. The following purple, pale green, teal, khaki, and blue rings show comparisons between pSs_046 and the virulence plasmids of S. boydii str. BS512, S. boydii str. Sb227, S. dysenteriae str. Sd197, S. flexneri F2a str. 301 (pCP301) and S. flexneri F5a (pWR501), respectively. Outer ring represents annotations of genes or genetic clusters based on function: known virulence factor genes (red); plasmid replication, transfer and maintenance genes (black); transposon, phage-borne and insertion sequence elements (orange); genes encoding hypothetical proteins (teal); S. sonnei-specific

Figure 3 .

 3 Figure 3. Comparative map of the Shigella virulence plasmid

Figure 4

 4 Figure 4 depicts the spatial segregation of epithelial cell types. Stem cells are located at the depth of the crypts and as daughter cells are produced and differentiate, they migrate upwards. Four distinct cell types are produced from this stem cell population: absorptive, goblet, enteroendocrine and Paneth cells. Adsorptive cells, or enterocytes, are the most abundant population of cells and have microvilli on their apical side; Goblet cells secrete mucus; Enteroendocrine cells secrete peptides and catecholamines, whilst Paneth cells secrete antibacterial proteins. As the intestinal epithelium is renewed, the old cells are extruded into the intestinal lumen at the villi apical extremities or the crypts' mouth. (For a review on the mechanisms of cell renewal see[START_REF] Crosnier | Organizing Cell Renewal in the Intestine: Stem Cells, Signals and Combinatorial Control[END_REF]).

Figure 4 .

 4 Figure 4. Structure of the intestinal mucosa in the jejunum (left) and in the colon (right). IEL: Intraepithelial lymphocytes; IEC: Intraepithelial cells; AMP: Antimicrobial peptides. Modified from (Mowat and Agace 2014)

Figure 4 .

 4 Figure 4. Structure of the intestinal mucosa

Figure 5 .

 5 Figure 5. Structure of the Shigella T3SS. Tip components shown in yellow and green. Needle components in blue-greens, transmembrane base dark purple, inner membrane export apparatus light purple, C-ring and ATPase complex in light blues. OM, outer membrane. IM, inner membrane

Figure 6 .Figure 6 .

 66 Figure 6. Regulation of the second wave of effectors. A. Inactive T3SA. B. Active T3SA. Prior to activation of T3 secretion, the IpgC chaperone associates with IpaB and IpaC. After activation, IpaB and IpaC are secreted, releasing IpgC, which may then interact with the MxiE transcriptional activator. This allows the transcription of genes harboring a MxiE box. Modified from (Campbell-Valois and Pontier 2016)

Figure 7 .

 7 Figure 7. Dynamics of actin filaments. A. Actin polymerization. B. Actin regulation. (1) Arp2/3 recruitment (2) Actin branching (3) Formin elongation of actin fibers (4) Ena/VASP elongation of actin fibers (5) Filament capping (6) Depolimerization of actin filaments. C. Formin and Ena/VASP nucleation and elongation activity.

Figure 7 .

 7 Figure 7. Dynamics of actin filaments

Figure 8 .

 8 Figure 8. Myosin II and α-actinin in actin filament contraction. Myosin II polymerizes into bipolar filaments, with the motor domains walking in opposing actin filaments cross-linked by α-actinin. Modified from (Svitkina 2018).

Figure 8 .

 8 Figure 8. Myosin II and α-actinin in actin filament contraction

Figure 9 .

 9 Figure 9. Integrins ligands and intracellular dynamics. Different αβ pairs and their ligands (Modified from Hynes 2002).

Figure 9 .

 9 Figure 9. Integrins ligands and intracellular dynamics

Figure 10 .Figure 10 .

 1010 Figure 10. Maturation of adhesion structures. Modified from (www.mechanobio.info)

Figure 11 .

 11 Figure 11. Model of the molecular clutch hypothesis. Disengaged molecular clutch results in actin treadmilling behaviour towards the interior of the cell, generating rearward movement. Engagement, forces required to counteract membrane resistance are transmitted to the matrix and protrusions at the cell edge are generated.

Figure 11 .

 11 Figure 11. Model of the molecular clutch hypothesis

  helix bundles until they become exposed, which happens only upon force-dependent stretching of talin and bundle unfolding (Figure12B)[START_REF] Hytönen | How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism[END_REF]; (del[START_REF] Rio | Stretching Single Talin Rod Molecules Activates Vinculin Binding[END_REF]).

Figure 12 .Figure 12 .

 1212 Figure 12. A. Structural model of Talin. Depicted in blue are the Vinculin Binding sites. B. Unfolding of helix bundle. Talin bundles unfolding leads to unveiling VBSs.Modified from[START_REF] Yao | Mechanical Activation of Vinculin Binding to Talin Locks Talin in an Unfolded Conformation[END_REF]

Figure 13

 13 Figure 13. A. Vinculin structure and activation. Vinculin subdomains are indicated in colors (D1, blue; D2, cyan; D3, green; D4, yellow; Tail domain, red). Binding sites for Talin, α-catenin, PIP2, F-actin, Arp2/3, vinexin and Ponsin are indicated. B. Vinculin activation by VBSs. Vinculin head domain is colored in cyan, VD1 is indicated in red, Tail domain in green. VBS (light cyan)

Figure 13 .

 13 Figure 13. Vinculin structure and activation

Figure 14 .Figure 14

 1414 Figure 14. Focal adhesion organization. Internal organization changes with respect to traction forces and protein composition follow a layered organization. Modified from (Case and Waterman 2015)

Figure 15 .

 15 Figure 15. Catch-bond mechanism scheme. On low force, FimH binds transiently to its receptor. Upon flow application, lectin and pilin domains are separated and the high-affinity state for the lectin domain is stabilized after release of the allostericinhibitory pilin domain.

Figure 15 .

 15 Figure 15. Catch-bond mechanism scheme

Figure 16 .

 16 Figure 16. Pedestals formed by EPEC and EHEC. Scanning electron micrographs (left) and transmission electron microscopy (center and right) from EPEC and EHEC apposed to pedestals in intestinal cells.Modifed from[START_REF] Campellone | Repetitive N-WASP-Binding Elements of the Enterohemorrhagic Escherichia Coli Effector EspF(U) Synergistically Activate Actin Assembly[END_REF]

Figure 17 .

 17 Figure 17. A. Filopodial capture. B. Effector proteins during invasion (C. M.Valencia-Gallardo, Carayol, andVan Nhieu 2015)

Figure 18 .Figure 18 .

 1818 Figure 18. IpaA Vinculin Binding Sites. A. Scheme of the location of VBSs along IpaA and their aminoacid sequence. B. Alignment of the three IpaA VBSs. C.Helical wheel view of the three VBSs. Hydrophobic residues (yellow), charged residues (red and blue), polar residues (green) Modified from(Park, Valencia- Gallardo, et al. 2011)

Figure 1 .

 1 Figure 1. Talin Is Required for Shigella Invasion and Is Recruited in an IpaA-Dependent Manner (A) Representative micrographs of control cells and cells treated with anti-talin siRNA prior to bacterial challenge at 37 C for 15 min. Green, actin; red, bacterial lipopolysaccharide (LPS). The arrows point to actin ''cups'' at bacterial cell contact (CTRL) or at the periphery of the foci (siRNA talin). Scale bar, 2 mm. (B) The average intensity ± SEM of actin foci was determined at the indicated time points following bacterial challenge for control cells (empty squares) or anti-talin siRNA-treated cells (black squares). N = 3, >30 foci. (C) Cells were treated with anti-talin siRNA prior to bacterial challenge at 37 C for 15 min. The percentage of internalized bacteria ±SEM after 30 min of infection with wild-type (WT) Shigella was determined (STAR Methods). For each sample, n > 35 foci in at least three independent experiments. ****p < 0.001, Wilcoxon test. (D) Representative micrographs of cells were transfected with talin-GFP and challenged with Shigella strains for 30 min at 37 C. Red, bacterial; blue, actin; green, talin-GFP. Cells challenged with the ipaA mutant strain complemented with full-length IpaA (Bricogne et al., 2011), vector alone (2.1), IpaA DVBS3, and IpaA DVBS1-2. The arrows point to talin coat structures surrounding invading bacteria. Scale bar, 5 mm. (E) Average percentage of actin foci forming talin-coat structures induced by the indicated bacterial strain ± SD (STAR Methods). For each sample, n > 35 foci in at least three independent experiments. Chi-square test (c 2 ) with post hoc comparison (false discovery rate [FDR] correction for p value).

µ 12 .Figure 2 .

 122 Figure 2. IpaA VBS3 Interacts with Talin (A) Analysis of IpaA VBSs and talin H1-H4 interaction using 6%-18% gradient native PAGE. The talin H1-H4:IpaA VBS molar ratio is indicated, with 1 corresponding to a final concentration of 25 mM. Arrowheads point to talin H1-H4 and talin H1-H4:IpaA VBS3. (B) Isothermal titration calorimetry (ITC) analysis of the interaction between talin H1-H4 and the indicated IpaA VBSs. The estimated K D values are indicated. IpaA VBS2 showed no binding to talin. (C) Native PAGE analysis. The talin H1-H4:IpaA derivative molar ratios are indicated, with 1 corresponding to a final concentration of 25 mM.(D) SEC-MALS analysis. Indicated proteins were incubated for 60 min in column buffer prior to SEC analysis using a Superdex 200 10/300 GL increase column. Traces, normalized absorbance at 280 nm of the indicated proteins or complex species in the corresponding color. Dotted lines, molecular mass of the indicated proteins or complexes determined by MALS in the corresponding colors.

Figure 3 .

 3 Figure 3. IpaA VBS3 Forms a Compact Fold with Talin H1-H4(A) X-ray structure of the talin H1-H4:IpaA VBS3 complex. Blue, talin polypeptide chain; white, interacting residues; green, bound IpaA VBS3, with interacting residues in green. VBS3 residues are annotated according to full-length IpaA.(legend continued on next page)

Figure 4 .

 4 Figure 4. Talin Binding by IpaA VBS3 Promotes Focal Adhesion Formation Cells were trypsinized and replated onto fibronectin-coated glass coverslip for 15 min prior to fixation and processing for fluorescence staining. Samples were analyzed by spinning disk microscopy. Cells were transfected with GFP: GFP-IpaA VBS3 (VBS3), A495K, or K498E variant and talin-mCherry (talin) or mCherryvinculin (vinculin). (A) Representative fluorescence micrographs of confocal basal planes. Scale bar, 5 mm. (B) Wavelet spot detection (green) of adhesion structures in replated cells (STAR Methods). (C) Distribution of cells as a function of the number of detected VBS3-labeled adhesions. Median ± MAD: VBS3, 23 ± 10.5 (18 cells, n = 3); A495K, 2.5 ± 2.5 (14 cells, N = 3); K498E, 1 ± 1 (17 cells, N = 3); GFP, 0 ± 0 (9 cells, N = 3). ***p < 0.005 and ****p < 0.001 (Dunn's test). (D) Size distribution of focal adhesions (FAs) for talin-mCherry transfected cells. Median ± MAD: VBS3, 1.79 ± 0.76 (774 FAs, N = 3); A495K, 1.53 ± 0.51 (568 FAs, N = 3); K498E, 1.53 ± 0.51 (383 FAs, N = 3); GFP, 1.29 ± 0.39 (878 FAs, N = 3). ***p < 0.005 and ****p < 0.001 (Dunn's test). (E) Size distribution of FAs for mCherry-vinculin-transfected cells. Median ± MAD: VBS3, 2.04 ± 0.76 (629 FAs, N = 3); A495K, 1.79 ± 0.76 (595 FAs, N = 3); K498E, 1.53 ± 0.51 (480 FAs, N = 3); GFP, 1.23 ± 0.32 (482 FAs, N = 3). ****p < 0.001 (Dunn's test).

Figure 5 .

 5 Figure 5. IpaA VBS3 Targets Talin-Containing Filopodial Adhesions Cells transfected with vinculin-mCherry and talin-mCherry, GFP-IpaA VBS3, A495K, or K498E variants. (A) Representative confocal micrographs. Scale bar, 5 mm. (B and D) Distribution of the distance relative to the cell body (d) of filopodial clusters labeled with the indicated marker in cells co-transfected with GFPtalin and vinculin-mCherry (B) and VBS3 or vD1 and vinculin-mCherry (D). Median ± MAD: talin, 2.48 ± 1.81 (858 clusters, N = 3); vinculin, 1.71 ± 1.31 (252 clusters, N = 3); VBS3, 3.08 ± 2.22 (1,008 clusters, N = 3); vD1, 2.53 ± 1.79 (537 clusters, N = 3). **p < 0.01 and ****p < 0.001 (Mann-Whitney test). (C) Distribution of the distance relative to the cell body (d c ) of filopodial clusters co-labeled with the indicated markers in co-transfected cells. Median ± MAD: VBS3 + talin, 2.86 ± 1.49 (285 clusters, N = 3); VBS3 + vinculin, 1.43 ± 0.88 (128 clusters, N = 3); talin + vinculin, 2.32 ± 1.36 (209 clusters, N = 3). *p < 0.05, ***p < 0.005, and ****p < 0.001 (Dunn's test). (E) Percentage of filopodia showing VBS3 clusters. VBS3 (563 filopodia, N = 3), A495K (375 filopodia, N = 3), K498E (332 filopodia, N = 3), GFP (495 filopodia, N = 3). ****p < 0.001 (Pearson's c 2 test). (F) Dot plot of filopodial lengths. Bar, median ± MAD. VBS3, 34.5 ± 3.6 mm (656 filopodia, N = 4); talin, 24.1 ± 4.4 mm (435 filopodia, N = 4); vD1, 21.0 ± 3.6 mm (336 filopodia, N = 3); A495K, 2.6 ± 0.9 mm (345 filopodia, N = 3); K498E, 2.9 ± 1 mm (434 filopodia, N = 3); GFP + talin, 3.04 ± 0.01 mm (589 filopodia, N = 3); GFP + vinculin, 2.96 ± 0.01 mm (516 filopodia, N = 3); **p < 0.01, ***p < 0.005, and ****p < 0.001 (Dunn's test).

Figure 6 .

 6 Figure 6. IpaA VBS3 Stabilizes Shigella-Induced Cell Adhesions (A) Representative micrographs of bacterial capture by filopodia. Cells co-transfected with IpaA-VBS3 and talin-mCherry and challenged prior to bacterial challenge. Green, IpaA VBS3; red, talin; blue, bacteria; cyan, F-actin. Scale bar, 5 mm. Talin and GFP-IpaA VBS3 are detected at the filopodial tip where bacterial capture occurs, as well as in bacterial coats in invasion foci induced by WT Shigella but not the ipaA mutant. (B) Cells were challenged with ipaA mutant complemented with full-length IpaA (1,739 cells, N = 5); DVBS3: IpaADVBS3 (1,258 cells, N = 2); DVBS1-2: IpaA DVBS1-2 (1,728 cells, N = 5); A495K: IpaADVBS1-2 A495K (1,316 cells, N = 4); K498E: IpaADVBS1-2 K498E (1,291 cells, N = 4); DVBSs: IpaA DVBS1-3 (909 cells, N = 2). The number of bacteria associated with filopodia per cell was scored. ****p < 0.001 (Mann-Whitney test). (C) HeLa cells were challenged with bacteria for 30 min at 37 C, fixed, and processed for immunofluorescence staining. Representative micrographs. Red, bacteria; green, talin. Scale bar, 5 mm. (D and E) FAs were scored using automatic detection (STAR Methods). The median adhesion size (D) and median number per cell (E) are indicated for each sample. (D) Median ± MAD: IpaA, 0.4949 ± 0.3535 (81 cells, N = 3); DVBS1-2, 0.4242 ± 0.2828 (60 cells, N = 3); A495K, 0.3535 ± 0.3535 (70 cells, N = 3); K498E, 0.4242 ± 0.2828 (75 cells, N = 3); 2.1, 0.3535 ± 0.2121 (111 cells, N = 3); not infected, 1.7742 ± 0.5914 (80 cells, N = 3). (E) Median ± MAD: IpaA, 17 ± 11 (81 cells, N = 3); DVBS1-2, 29 ± 19 (60 cells, N = 3); A495K, 5 ± 5 (70 cells, N = 3); K498E, 23 ± 16 (75 cells, N = 3); 2.1, 5 ± 4 (111 cells, N = 3); not infected, 29 ± 8 (80 cells, N = 3). (D) *p < 0.05 and ****p < 0.001 (Kolmogorov-Smirnov test of probability distribution). (E) **p < 0.01 and ****p < 0.001 (Kruskal-Wallis test). (F) The median size of FAs is plotted for each cell as a function of the adhesions' number per cell.

  . IpaA VBS3 (N-TRETIFEASKKVTNSLSNLISLIGT-C, 488-512), and VBS3 variant peptides K9498A (N-TRETIFEAS KAVTNSLSNLISLIGT-C), K498E (N-TRETIFEASKEVTNSLSNLISLIGT-C), R489A K498A (N-TAETIFEASKAVTNSLSNLISLIGT-C) and A495K (N-TRETIFEKSKKVTNSLSNLISLIGT-C) were synthetized by Genscript USA Inc. Talin peptides VBS46 (N-YTKKELIE CARRVSEKVSHVLAALQ-C, 1945-1970), Talin VBS6 (N-FQDVLMQLANAVASAAAALVLKAKS-C, 664-689), Talin VBS9 (N-RGVGAAATAVTQALNELLQHVKAH-C, 765-789), Talin (N-NLKSQLAAAARAVTDSINQLITMCT-C, 1330-1357) and Talin VBS50

  IpaA injection during invasion leads to the recruitment of Vinculin to bacterial entry sites, a process mediated by the presence of Vinculin Binding Sites at the C-terminal end of IpaA. SEC-MALS and native gels were used to characterize the interaction between IpaA VBS1-3 and vinculin. Analysis of cross-linked complexes by Massspectrometry allowed the obtention of structural models for IpaA VBSs/Vinculin interactions. We also characterized the functional effects of IpaA VBSs constructs in cell adhesions combining microscopy analysis, microfluidics and migration assays to evaluate the effects of VBSs in cell adhesion and affinity. My contribution to this article was the conception and realization of experiments, generation of vinculin constructs, microscopy analysis of adhesion structures, and implementation of the microfluidics-based adhesion assay. its mode of vinculin activation likely reflects a key step during maturation of cell adhesions driven by acto-myosin contractility.

Figure 1 -

 1 FIGURES AND LEGENDS

Figure 2 -

 2 Figure 2 -Characterization of IpaA contact sites on vinculin.

Figure 3 -

 3 Figure 3 -A483 stabilizes vinculin in cell adhesions in the absence of mechanotransduction

  96-well Maxisorp (Nunc) ELISA plates were coated with 30 nM of full-length vinculin, vinculin constructs or IpaA proteins at the indicated concentrations in binding buffer(25 mM Tris PH 7.4, 100 mM NaCl and 1 mM -mercaptoethanol). Samples were blocked with PBS-BSA 2%, washed and incubated with IpaA or vinculin proteins in binding buffer containing 0.2% BSA at room temperature for one hour. After incubation, the plates were washed and incubated with an anti-IpaA (dilution 1/2000 e ) polyclonal primary antibody 3 or anti-vinculin (dilution 1/2000 e ) Vin11Vin.5 monoclonal antibody (Sigma-Aldrich) in binding buffer containing 0.2% BSA for one hour at room temperature. Plates were washed and incubated with an HRP-coupled secondary anti-rabbit or anti-mouse IgG antibody (1/32000 e ) (Jackson ImmunoResearch) for one hour. The reaction was revealed by adding 100 l of tetramethylbenzidine (Sigma-Aldrich) for 15 min, stopped by adding 50 l of 0.66N H2SO4 and the absorbance was read at 450 nm (Dynatech MR400).BN-PAGE (Blue Native -Polyacrylamide Gel Electrophoresis) protein native gel analysis and complex cross-linking 25 M of vinculin constructs were incubated with different molar ratios of IpaA proteins in a 1X

  staining C2.7 mice myoblasts cells were seeded at 2.5x10 4 cells in 25 mm-diameter coverslips. Cells were transfected with 3 g of pGFP-A524 or pGFP-A483 plasmids with 6 l JetPEI transfection reagent (Polyplus) for 16 hours following the manufacturer's recommendations. Cells were fixed in PBS containing 3.7% paraformaldehyde for 20 min at 21°C and permeabilized with 0.1% Triton X-100 for 4 min at 21°C. Cells were processed for immunofluorescence staining using the Vin11.5 anti-vinculin monoclonal antibody (ref. V4505, Sigma-Aldrich) and anti-mouse IgG antibody coupled to Alexa 546 (Jackson Research) and Phalloidin-Alexa 633 (Invitrogen), as described previously 3 . Samples were analyzed using an Eclipse Ti inverted microscope (Nikon) equipped with a 60 x objective, a CSU-X1 spinning disk confocal head (Yokogawa), and a Coolsnap HQ2 camera (Roper Scientific Instruments), controlled by the Metamorph 7.7 software.

  . 1205Lu melanocytes were transfected with the indicated constructs, then labeled with 2  calcein-AM (Life Technologies) in serum-free DMEM for 20 minutes. Cells were detached by incubation with 2  Cytochalasin D (Sigma-Aldrich) for 40 minutes to disassemble FAs, followed by incubation in PBS containing 10 mM EDTA for 20 minutes. Cells were washed in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM HEPES at pH 7.3) by centrifugation and resuspendedin the same buffer at a density of 1.5 x 10 6 cells/ml. Calcein-labeled transfected cells and control unlabeled cells were mixed at a 1:1 ratio and perfused onto a 25 mm-diameter glass coverslips (Marienfeld) previously coated with 20 g/ml fibronectin and blocked with PBS containing 2% BSA (Sigma-Aldrich) in a microfluidic chamber on a microscope stage at 37°C. We used a commercial microfluidic setup (Flow chamber system 1C, Provitro) and a Miniplus3 peristaltic pump (Gilson) to adjust the flow rate in the chamber. Microscopy analysis was performed using a LEICA DMRIBe inverted microscope equipped with a Cascade 512B camera and LED source lights (Roper Instruments), driven by the Metamorph 7.7 software (Universal imaging). Cells were allowed to settle for the indicated time prior to application of a 4 ml/min, flow corresponding to a wall shear stress of 22.2 dyn/cm 2 (2.22 Pa).

  5'-AGCCCGGGCACGAGTTCAGGAAATCCTTATTGATATTCTTTA-3' primers and cloned the plasmid pCR2.1 IpaA (Valencia-Gallardo et al. 2019), digested with SmaI. All the VBS mutants S. flexneri strains were previously reported (Valencia-Gallardo et al. 2019).

Figure 1 .

 1 Figure 1. Construction of IpaA-4C. A. Scheme of IpaA-4C construct. VBS are

Figure 2 .

 2 Figure 2. IpaA-4C is injected by kiss-and-run bacteria. A. 2D projections (Top) and 3D

Figure 3 .

 3 Figure 3. . Shigella IpaA VBS3 mediates integrin clustering. HeLa cells were

Figure 4 .

 4 Figure 4. IpaA VBSs lead to integrin activation. A. HeLa cells incubated with the

Figure 4 .

 4 Figure 4. Shigella cell priming increases bacterial capture. A. Timelapse of

Figure 19 .

 19 Figure 19. Model of IpaA VBSs interaction with focal adhesion proteins. A. Hybrid vinculin/talin VBS in talin mediates talin oligomers in semi-stretched talin molecules. B. IpaA VBSs lead to supra-activation and vinculin trimerization in the absence of force

Figure 19 .

 19 Figure 19. Model of IpaA VBSs interaction with focal adhesion proteins

Figure 20 .

 20 Figure 20. Model for IpaA action on talin and vinculin.1, IpaA stabilization of semistretched talin by IpaA stabilizes initial contact. 2, Activation and supra-activation of vinculin leads to vinculin trimer formation and promotes bacterial anchoring.

Figure 20 .

 20 Figure 20. Model for IpaA action on talin and vinculin

  Altoghether, IpaA VBS interaction with talin and a novel mechanism of vinculin activation open possibilities for unexpected regulatory processes in cell adhesion. It also points to novel ways in which other pathogens can take advantage of cell adhesion mechanisms.

Qui zuuyu' naa gate'

		No me verás morir
	Qui zuuyu' naa gate'	No me verás morir
	qui zanda gusiaandu' naa
	Naa nga jñou'
	bixhozelu'
	diidxa' yooxho' bixhozegolalu'
	guira' ni ma bisiaa ca dxi ca lii
	nisa ruuna ti guesa ma stale dxi bibani
	ti na' yaga ni jmá nabana'
	biniti lade bandaga
	Qui zuuyu' naa gate'
	ti naa nga
	ti dxumi su
	ra caniibi ru' na'
	bixhoze bendabua'
	benda ni gudó diuxi
	beenda ni bichá ruaa ti lexu
	lexu ni gudxite gueu'
	gueu' ni gubi lidxi bizu
	dxiña bizu ni rindani lu xidxe'
	xquipilu' nga naa
	ne qui zuuyu' gate'
	Neca zacuxhou' ma guirá tu zé
	qui zuuyu naa gate'
	ziuu ti xuba'
	ga'chi' lade gui'xhi' nuu lu neza
	ndaani' guidxi di' zabigueta'
	ne laa gusindani guendanabani
	ne laa gaca gueta xquendanu
	ne laa gusibani stiidxanu
	ne qui zuuyu' naa gate'
	ti zácanu nadipa'
	ti zabaninu xadxí
	ti riuunda stinu qui zati
	ti zacanu laanu ne lii
	ne ca xiiñi' xiiñinu
	ne xu guidxilayú
	ni gunibidxacha nisado'
	ne zacanu stale ladxidó'
	naaze dxiichi' xquenda binnizá
	ne qui zuuyu' gate'
	qui zuuyu' naa gate'
	qui zuuyudio'
	naa gate'

Table 1 .

 1 Shigella effector proteins

Table 1 . Shigella effector proteins. Modified from

 1

	(Killackey, Sorbara, and

Table 2 .

 2 Pili/fimbriae of pathogenic Gram-negative bacteria

Table 2 . Pili/fimbriae of pathogenic Gram-negative bacteria (modified from Bhunia 2018) Type of pili Present in

 2

	Type I	E. coli, H. influenzae, Y. pestis
	Type IV	Pseudomonas spp., Vibrio spp., Enteropathogenic E. coli, N.
		meningitides
	P pili	E. coli, H. influenzae, Y. pestis
	Bundle-forming	Enteropathogenic E. coli
	pili
	Curli	S. enterica, E. coli

Table 3 .

 3 Non-exhaustive list of adhesion factors and host receptors of pathogenic bacteria

Table 3 . Non-exhaustive list of adhesion factors and host receptors of pathogenic bacteria (

 3 modified from Bhunia 2018;[START_REF] Solanki | Host-Bacteria Interaction and Adhesin Study for Development of Therapeutics[END_REF]

	Pathogen	Adhesion factors Host receptor
	L. monocytogenes	Internalin A	E-cadherin
		Internalin B	c-Met, gC1q-R/p32
		Virulence invasion	Gp90
		protein
		Listeria adhesion	Hsp60
		protein
	Campylobacter spp.	CadF	Fibronectin
	Arcobacter	Hemagglutinin	Glycan receptor
	Enteropathogenic,	Intimin	Translocated intimin receptor
	enterohemorragic E.coli
	Y. enterocolitica	YadA	Collagen/fibronectin/laminin/β1-
			integrin
	S. aureus	Fibronectin-binding	Fibronectin
		protein
	V. cholerae	Toxin-coregulated	Glycoprotein
		pili
	N. meningitides	OpcA	Vitronectins, proteoglycans
	E. coli	OmpA	Ecgp glycoprotein
	B. pertussis	Pertactin	Not identified

  Equipe Communication Intercellulaire et Infections Microbiennes, Centre de Recherche Interdisciplinaire en Biologie (CIRB), Colle `ge de France, 75005 Paris, France 2 INSERM U1050, 75005 Paris, France 3 Centre National de la Recherche Scientifique UMR7241, 75005 Paris, France 4 MEMOLIFE Laboratory of Excellence and Paris Science Lettres, 75005 Paris, France 5 Laboratoire de Chimie des Processus Biologiques, Colle `ge de France, 75005 Paris, France 6 Centre National de la Recherche Scientifique UMR8229, 75005 Paris, France 7 Macromolecular X-ray Crystallography Core, Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA 8 Cell Adhesion Laboratory, Department of Integrative Structural & Computational Biology, The Scripps Research Institute, Jupiter, FL 33458, USA

1 9

Lead Contact *Correspondence: guy.tran-van-nhieu@college-de-france.fr https://doi.org

/10.1016/j.celrep.2018.12.091

Table 1 .

 1 Non-linear Fit Values for ITC Measurements

	Cell	Titrant	K D (Talin H1-H4)	N	DH (kcal/mol)	ÀTDS (kcal/mol)	DG (kcal/mol)	K D (vD1)
	Talin H1-H4	IpaA VBS1	15.2 ± 1.1 mM	1.22	À4.10	À2.47	À6.57	110 pM
		IpaA VBS2	NA	NA	NA	NA	NA	6.6 nM
		IpaA VBS3	174.5 ± 18.9 nM	0.89	À6.65	À2.81	À9.47	54 pM
		IpaA VBS3 K498A	163.1 ± 1.1 nM	1.18	À6.28	À2.97	À9.26	n.d.
		IpaA VBS3 K498E	300.3 ± 2.8 nM	0.73	À12.1	3.25	À8.90	n.d.
		IpaA VBS3 R489A K498A	328.9 ± 1.9 nM	0.69	À8.68	À0.16	À8.84	n.d.
		IpaA VBS3 A495K	2.93 ± 0.2 mM	1.05	À3.73	À3.81	À7.54	n.d.
		talin VBS46	60 ± 0.8 nM	0.92	À16.97	7.20	À9.77	3 nM
		talin VBS6	21 ± 3.4 mM	1.26	À14.34	7.97	À6.37	n.d.
		talin VBS9	1 ± 0.1 mM	1.29	À14.65	6.47	À8.18	n.d.
		talin VBS50	45 ± 7.5 mM	1.02	À13.23	7.31	À5.92	99 nM
		talin VBS33	no binding	NA	NA	NA	NA	n.d.
		talin VBS36	no binding	NA	NA	NA	NA	n.d.
	A483 (site 1)	talin H1-H4	115.7 ± 32.15 nM	1	À6.38	À3.07	À9.45	n.d.
	A483 (site 2)		4.6 ± 0.6 mM	1	À6.76	À5.01	À7.27	n.d.
	A524 (sites 1 + 2)		2.0 ± 0.5 mM	1.52	À3.97	À3.78	À7.76	n.d.

TABLE d

 d

	STAR+METHODS
	KEY RESOURCES TABLE Continued
	REAGENT or RESOURCE	SOURCE		IDENTIFIER
	Experimental Models: Cell Lines
	HeLa cells mouse monoclonal anti-talin clone 8d4 antibody Reverse: CTCCCGGGTCTGGTACCAGGG anti-Shigella serotype V LPS rabbit antibody CTGTCGACTGATGCCAGTGTTTCATACG REAGENT or RESOURCE Mouse Embryo Fibroblast cells Oligonucleotides Antibodies Primers targeting Human vinculin Forward:	ATCC Sigma-Aldrich Tran Van Nhieu et al., 1997 CONTACT FOR REAGENT AND RESOURCE SHARING CCL-2 d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell lines Cat# T3287-2ML; RRID:AB_535866 d METHOD DETAILS N/A B Bacterial strains SOURCE ATCC N/A IDENTIFIER This Paper N/A
	DyLight 405 AffiniPure Goat Anti-Rabbit IgG AGTCTTTC	Jackson Immunoresearch	Cat# 111-475-045
	Alexa Fluorâ 488 AffiniPure Donkey Primers targeting IpaA 483: Forward:	Jackson Immunoresearch This paper	Cat# 715-545-150 N/A
	Anti-Mouse IgG GGCGAATTCCCGGAGACACATATT
	Alexa Fluor 647 Phalloidin TAACACG Reverse: GCCGTCGACTTAA TCCTTATTGATATTCT Bacterial and Virus Strains Shigella flexneri M90T ATCATGGCCAGCAAAGG Reverse: GCGC Primers targeting IpaA 483: Forward: GCGAT	Invitrogen Park et al., 2011 This paper		Cat# A22287 N/A N/A
	Shigella flexneri AfaE GGCCGCTTAATCCTTATTGATATTC	Park et al., 2011		N/A
	Shigella flexneri IpaA -Primers targeting IpaAVTBS: Forward:	Park et al., 2011 This paper		N/A N/A
	Shigella flexneri IpaA -:2.1 ACCCGGGGATTAAGCGGCC Reverse:	This paper		N/A
	Shigella flexneri IpaA -: IpaA ACCCGGGATCCTGATTTAGTTCC	This paper		N/A
	Shigella flexneri IpaA -:DVTBS Shigella flexneri IpaA -:DVBS 1-2 CTTTTTTTGATTTTTCAAATATCGTTTCCCGTG Shigella flexneri IpaA -DVBSs AAGTAACAAACTC Reverse: GAGTTTGTTA CACGGGAAACGATATTTGAAAAATCAAAAA Primers for VTBS A495K mutation: Forward:	This paper This paper This paper This paper		N/A N/A N/A N/A
	Chemicals, Peptides, and Recombinant Proteins Primers for VTBS K498E mutation: Forward:	This paper		N/A
	Talin H1-H4 CGATATTTGAAGCTTCAAAAGAAGTAACA	This paper	SUPPLEMENTAL INFORMATION N/A
	IpaA483 ACAAACTCCCTAA Reverse: TTAGGG	This paper		N/A
	IpaA524 AGTTTGTTGTTACTTCTTTTGAAGCTT CAAATATCG	This paper	Supplemental Information includes three tables and seven figures and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.12.091. N/A
	IpaA VTBS (N-TRETIFEASKKVTNSLSNLISLIGT-C, bicistronic costruction: Forward: CGAATTCAG 488-512) Primers for pET28a-IpaA-VBS3/VTBS-talinH1-H4	This paper This paper		N/A N/A
	VTBS variant K9498A (N-TRETIFEASKA GAGGACAGCTATGCACCGAGGACACATG	This paper		N/A
	VTNSLSNLISLIGT-C) Reverse: CGAGCTCGTTACTGACGGGGCT
	VTBS variant K498E (N-TRETIFEASKEVTN CAGCACTG	This paper		N/A
	SLSNLISLIGT-C) Stealth Select Anti-Talin1 siRNA	Invitrogen		Cat# 1299003
	VTBS variant R489A K498A (N-TAETIFEASKAVTN Stealth Select Anti-vinculin siRNA	This paper Invitrogen		N/A Cat# 1299001
	SLSNLISLIGT-C) Recombinant DNA
	VTBS variant A495K (N-TRETIFEKSKKVT NSLSNLISLIGT-C) Human Vinculin-mCherry	This paper This paper		N/A N/A
	Talin peptide VBS46 (N-YTKKELIECARRVSE Human talin-GFP	This paper Laboratory of Ken Yamada	N/A N/A
	KVSHVLAALQ-C, 1945-1970) A483-GST	This paper		N/A
	Talin VBS6 (N-FQDVLMQLANAVAS GFP-A483-633	This paper This paper		N/A N/A
	AAAALVLKAKS-C, 664-689) GFP-IpaAVTBS	This paper		N/A
	Talin VBS9 (N-RGVGAAATAVTQAL talin VBS1 (H1-H4) 482-636	This paper Izard et al., 2006		N/A N/A
	NELLQHVKAH-C, 765-789) GST-IpaA 524-633	Izard et al., 2006		N/A
	Talin (N-NLKSQLAAAARAVTDSINQLITMCT-C, IpaA-VBS1 (611-633)	This paper Izard et al., 2006		N/A N/A
	1330-1357) IpaA-VBS2 (565-586)	Izard et al., 2006		N/A
	Talin VBS50 (N-QVVLINAVKDVAKALG pET28a-IpaA-VBS3/VTBS-talinH1-H4 DLISATKAA-C, 2077-2102) Software and Algorithms Critical Commercial Assays Yeast two-hybrid assay IpaA1-565 Icy Bioimaging Analysis software Deposited Data XDS	This paper This paper Hybrigenics services de Chaumont et al., 2012 Kabsch 2010	N/A N/A N/A N/A
	Shigella IpaA-VBS3/TBS in complex with the Talin	This paper		PDB: 5NL1
	VBS1 domain 488-512 (structure)

B Antibodies B Generation of expression constructs B Yeast double hybrid analysis B Protein purification B Native-PAGE analysis B SEC-MALS B Crystallization, structure determination, and crystallographic refinement B Isothermal titration calorimetry B siRNA transfection B Cell challenge with Shigella strains B Immunofluorescence microscopy analysis B Image processing and analysis d QUANTIFICATION AND STATISTICAL ANALYSIS d DATA AND SOFTWARE AVAILABILITY (Continued on next page) Cell Reports 26, 921-932.e1-e6, January 22, 2019 e1 http://xds.mpimf-heidelberg.mpg.de/ (Continued on next page) e2 Cell Reports 26, 921-932.e1-e6, January 22, 2019

  Gutierrez, E. et al. Microfluidic devices for studies of shear-dependent platelet adhesion. Lab

	635 642	3088.67 2516.36 3341.67 3413.78 3040.71 3141.53 4655.33	2.62 Score 3.44 4.11 3.79 3.43 3.53 4.45	NFTVEKMSAEINEIIR -ELLPVLISAMK ALASIDSKLNQAK -MSAEINEIIR NYVTETNADTIDKNHAIYEK -AKEVSSALSK GSPGIPGDTYLTR -AQQVSQGLDVLTAKVENAAR VGKETVQTTEDQILKR -ELLPVLISAMK NYVTETNADTIDKNHAIYEK -AKEVSSALSK IDDTSAELLTDDISDLKNNNDITAENNNIYK -	K236 K295 E554 D484 K59 K562 D598	E200 E240 K571 K366 K200 E572 K625
	616 636 643	Chip 8, 1486-1495, doi:10.1039/b804795b (2008). Exp. MH + Primary Score Peptide Sequence 4457.1 3.28 IDDTSAELLTDDISDLKNNNDITAENNNIYK -MAKMIDER 32798 1924.16 # 32670 2.61 INNKLK -ELLPVLISAMK 33042 4613.23 3.70 IDDTSAELLTDDISDLKNNNDITAENNNIYK -33042 4613.23 3.70 IDDTSAELLTDDISDLKNNNDITAENNNIYK -NLGPGMTKMAK 33042 4613.23 4.11 IDDTSAELLTDDISDLKNNNDITAENNNIYK -NLGPGMTKMAK IDDTSAELLTDDISDLKNNNDITAENNNIYK -2965.5 2.51 IDDTSAELLTDDISDLK -AAKDVTTSLSK 4586.24 4.1 IDDTSAELLTDDISDLKNNNDITAENNNIYK -AAKDVTTSLSK 3291.74 3.81 IDDTSAELLTDDISDLK -AKEVSSALSKVLSK 3944.01 3.86 IDDTSAELLTDDISDLK -AQQVSQGLDVLTAKVENAAR 3704.92 4.32 KIDAAQNWLADPNGGPEGEEQIR -ELLPVLISAMK 3291.74 3.64 IDDTSAELLTDDISDLK -AKEVSSALSKVLSK 5745.03 4.09 TIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPV 4173.16 4.98 GQGSSPVAMQKAQQVSQGLDVLTAK -4584.24 4.52 IDDTSAELLTDDISDLKNNNDITAENNNIYK -5153.62 4.56 IDDTSAELLTDDISDLKNNNDITAENNNIYK -VGKETVQTTEDQILK AKEVSSALSKVLSK 4011 3.29 NINKD -KLEAMTNSK AAKDVTTSLSK KVTNSLSNLISLIGTK NYVTETNADTIDKNHAIYEK -2641.56 4.45 AKEVSSALSKVLSK -ELLPVLISAMK 3047.64 3.99 LKVTDANIR -GILSGTSDLLLTFDEAEVR 4911.48 3.83 4536.51 4.44 SLGEISALTSKLADLRRQGK IDDTSAELLTDDISDLKNNNDITAENNNIYK -3468.66 2.62 NYVTETNADTIDKNHAIYEK -NLGPGMTKMAK 3350.71 3.48 GSPGIPGDTYLTR -GILSGTSDLLLTFDEAEVR 4173.16 5.8 GQGSSPVAMQKAQQVSQGLDVLTAKVENAAR -4584.24 4.95 IDDTSAELLTDDISDLKNNNDITAENNNIYK -5153.62 4.46 IDDTSAELLTDDISDLKNNNDITAENNNIYK -GDSPEARALAKQVATALQNLQTK -AKEVSSALSKVLSK 3469.67 3.01 NYVTETNADTIDKNHAIYEK -NLGPGMTKMAK 3604.91 3.34 VTNSLSNLISLIGTKSGTQER -ETVQTTEDQILK KLEAMTNSK AAKDVTTSLSK KVTNSLSNLISLIGTK 2965.5 2.94 IDDTSAELLTDDISDLK -AAKDVTTSLSK 5162.6 4.51 4911.48 4.06 ALAKQVATALQNLQTK 3255.72 4.00 AQQVSQGLDVLTAKVENAAR -MSAEINEIIR IDDTSAELLTDDISDLKNNNDITAENNNIYK -3651.79 3.37 NYVTETNADTIDKNHAIYEK -NSKNQGIEEALK 3542.88 3.63 IDDTSAELLTDDISDLK -ALAKQVATALQNLQTK 4173.16 5.49 GQGSSPVAMQKAQQVSQGLDVLTAKVENAAR -4584.24 4.42 IDDTSAELLTDDISDLKNNNDITAENNNIYK -5153.62 4.36 IDDTSAELLTDDISDLKNNNDITAENNNIYK -NLGPGMTKMAK 4483.2 2.64 IDDTSAELLTDDISDLKNNNDITAENNNIYK -AKEVSSALSK 5255.49 3.79 IDDTSAELLTDDISDLKNNNDITAENNNIYK -2280.32 3.69 ELLPVLISAMK -LNQAKGWLR 4911.48 4.37 AKEVSSALSKVLSK 2571.40 3.08 LKVTDANIR -ETVQTTEDQILKR 3137.65 3.41 AAKDVTTSLSK -GILSGTSDLLLTFDEAEVR 2892.69 3.97 ALAKQVATALQNLQTK -ELLPVLISAMK AAKDVTTSLSK 3252.74 3.81 VTNSLSNLISLIGTKSGTQER -ETIFEASKK NLGPGMTKMAKMIDER 3184.71 4.81 AQQVSQGLDVLTAKVENAAR -SLGEISALTSK IDDTSAELLTDDISDLKNNNDITAENNNIYK -2621.29 3.10 GSPGIPGDTYLTR -QQELTHQEHR 2387.41 3.65 DVTTSLSKVLK -ELLPVLISAMK 2553.46 3.78 ALASIDSKLNQAK -ELLPVLISAMK 4586.24 3.35 IDDTSAELLTDDISDLKNNNDITAENNNIYK -2988.61 4.48 VTNSLSNLISLIGTKSGTQER -VTDANIR # Exp. MH + Primary Score Peptide Sequence 3051.56 3.52 NYVTETNADTIDKNHAIYEK -INNKLK 3342.67 2.74 NYVTETNADTIDKNHAIYEK -AKEVSSALSK 3141.54 2.71 NYVTETNADTIDKNHAIYEK -EVSSALSK 3117.73 5.00 AKEVSSALSKVLSK -VGKETVQTTEDQILK 4612.22 4.82 IDDTSAELLTDDISDLKNNNDITAENNNIYK -3129.61 2.19 NQGIEEALKNRNFTVEKMSAEINEIIR 4583.26 3.43 AAKDVTTSLSK 2556.44 2.35 INNKLK -VGKETVQTTEDQILKR NLGPGMTKMAK 2280.32 3.35 ELLPVLISAMK -LNQAKGWLR AAKDVTTSLSK 3093.61 3.36 IDDTSAELLTDDISDLK -EVSSALSKVLSK 4612.22 2.86 NLGPGMTKMAK 3685.90 3.56 MSAEINEIIR IDDTSAELLTDDISDLKNNNDITAENNNIYK -2518.38 2.49 SKDITK -VGKETVQTTEDQILKR IDDTSAELLTDDISDLKNNNDITAENNNIYK -3184.71 4.71 AQQVSQGLDVLTAKVENAAR -SLGEISALTSK 4584.24 3.49 IDDTSAELLTDDISDLKNNNDITAENNNIYK -3092.6 3.38 IDDTSAELLTDDISDLK -EVSSALSKVLSK IDDTSAELLTDDISDLKNNNDITAENNNIYK -GQGSSPVAMQKAQQVSQGLDVLTAK -4586.25 5.2 AAKDVTTSLSK 3014.59 3.29 DITKSTTEHR -VGKETVQTTEDQILKR 3074.69 3.12 KVTNSLSNLISLIGTK -ETVQTTEDQILK 2881.63 3.77 QVATALQNLQTKTNR -ELLPVLISAMK AAKDVTTSLSK 3092.6 3.51 IDDTSAELLTDDISDLK -EVSSALSKVLSK 3310.75 4.29 GILSGTSDLLLTFDEAEVR -AVANSRPAKAAVH IDDTSAELLTDDISDLKNNNDITAENNNIYK -2708.42 3.25 SKDITK -GILSGTSDLLLTFDEAEVR 3580.72 3.88 VLQLTSWDEDAWASKDTEAMK -MSAEINEIIR 4584.24 3.59 IDDTSAELLTDDISDLKNNNDITAENNNIYK -DVTTSLSKVLK Score 3470.68 3.86 NYVTETNADTIDKNHAIYEK -NLGPGMTKMAK 3883.92 3.71 NYVTETNADTIDKNHAIYEK -ETVQTTEDQILKR 3787.88 4.12 GSSHHHHHHSSGLVPR 4584.25 4.05 AAKDVTTSLSK # Exp. MH + Peptide Sequence GQGSSPVAMQKAQQVSQGLDVLTAK SLGEISALTSK 3349.69 4.03 NYVTETNADTIDKNHAIYEK -LKVTDANIR 4655.33 4.44 IDDTSAELLTDDISDLKNNNDITAENNNIYK -Primary 5992.94 5.1 GILSGTSDLLLTFDEAEVR -IDDTSAELLTDDISDLKNNNDITAENNNIYK -IDDTSAELLTDDISDLKNNNDITAENNNIYK -3614.9 4.15 GQGSSPVAMQKAQQVSQGLDVLTAK -3349.69 4.44 NYVTETNADTIDKNHAIYEK -LKVTDANIR DVTTSLSKVLK	A524 D604 K540 D595 D604 D594 E608 D595 D598 K387 D595 K632 D361 K600 D598 D561 K579 K542 E590 D455 E554 G517 E368 E608 D595 D558 K513 D598 D598 E590 K366 E554 D594 K366 D598 D594 D594 E590 E200 D595 K542 K625 K464 K513 D598 K366 G517 K625 K276 E590 K513 E554 E554 K562 K579 E224 E608 K540 E200 D598 E590 D604 K352 K526 K366 D595 D594 D594 K530 K498 K476 D594 E147 K526 K261 D594 E554 K562 E128 E590 A483 D561 E590 E590 K352 D558	Hv D1 K173 K170 K617 K579 K366 K571 E31 K373 D618 K498 E200 K59 K617 K579 E128 D121 K373 K617 K498 K170 K444 E200 K170 E66 K170 K617 K579 E240 K219 K464 E375 K617 K498 K464 K170 K571 K281 E128 E200 E494 E66 K579 E437 E181 E200 E200 K617 D545 K173 E200 K540 K571 K572 K236 K617 K281 K579 E66 K170 K170 E437 K617 K579 E66 E240 E66 E200 K579 E66 K617 E66 K507 E128 E240 K617 E60 G12 K617 Hv D1D2 A524 K542 K625 K362 K170 E437 K542
		33322 5310.64 33334 2347.28 5328.54 3685.87 3100.63 3725.82 4655.32 3725.82 3726.02 4584.24 5153.62 33432 2138.26 33496 2315.32 4655.33 # Exp. MH + 3723.01 3161.66 4633.41 4655.32 3161.66 # Exp. MH + 3293.83 4584.24 5153.62	4.48 2.51 4.76 4.03 3.94 3.27 4.35 3.72 3.87 4.22 3.99 2.88 3.09 4.78 4.34 3.21 Primary Primary 3.54 Score 4.35 3.61 Score 4.63 4.94 3.53	VGKETVQTTEDQILKR DVTTSLSKVLK -MSAEINEIIR IDDTSAELLTDDISDLKNNNDITAENNNIYK -IDDTSAELLTDDISDLK NINKD -TIESILEPVAQQISHLVIMHEEGEVDGK AQQVSQGLDVLTAKVENAAR -KLEAMTNSK IDDTSAELLTDDISDLKNNNDITAENNNIYK -NYVTETNADTIDKNHAIYEK -ETVQTTEDQILK AAVQAAVSNLVR VENAARKLEAMTNSK AAKDVTTSLSK KVTNSLSNLISLIGTK GQGSSPVAMQKAQQVSQGLDVLTAK -DVTTSLSKVLK NYVTETNADTIDKNHAIYEK -ETVQTTEDQILK GQGSSPVAMQKAQQVSQGLDVLTAK -IDDTSAELLTDDISDLKNNNDITAENNNIYK -IDDTSAELLTDDISDLKNNNDITAENNNIYK -VLKNINKD -ELLPVLISAMK AAKDVTTSLSK -ELLPVLISAMK IDDTSAELLTDDISDLKNNNDITAENNNIYK -DVTTSLSKVLK ELLPVLISAMK IDDTSAELLTDDISDLKNNNDITAENNNIYK -GSPGIPGDTYLTR -VGKETVQTTEDQILKR ELLPVLISAMK AAKDVTTSLSK KVTNSLSNLISLIGTK Peptide Sequence TIESILEPVAQQISHLVIMHEEGEVDGK -DVTTSLSKVLK GSPGIPGDTYLTR -VGKETVQTTEDQILKR Peptide Sequence AQQVSQGLDVLTAKVENAAR -ELLPVLISAMK IDDTSAELLTDDISDLKNNNDITAENNNIYK -IDDTSAELLTDDISDLKNNNDITAENNNIYK -	K600 K625 K600 K632 E368 K562 D594 K562 K352 K614 D604 K628 K617 D594 K352 G517 E28 D595 G517 K366 D604 E590	D67 K373 E66 E31 K625 D586 E60 E200 D618 K498 E240 E200 E200 K625 E200 E60 K381 Hv D1D2 K625 Hv D1D2 D67 E200 K617 K498
	617 623	34822 2642.55 3323.71 2918.61 1670.89 3293.83	2.68 4.54 3.18 3.51 3.3	AKEVSSALSKVLSK -ELLPVLISAMK LNQAKGWLRDPSASPGDAGEQAIR -ALASIDSK KLEAMTNSKQSIAK EVSSALSKVLSK -VGKETVQTTEDQILK -VGKETVQTTEDQILK AQQVSQGLDVLTAKVENAARK -LNQAKGWLR AAKDVTTSLSK KVTNSLSNLISLIGTK	K579 K281 K579 K59 E368	E200 D274 E66 E66 K281
	618 624	38218 3685.86 Suppl. Table 1. Cross-linked residues characterized in the D1:A524 complex (XL-amino acids 3.87 NINKD -TIESILEPVAQQISHLVIMHEEGEVDGK K632 E31 3169.64 4.03 GQGSSPVAMQKAQQVSQGLDVLTAK -VENAAR K352 E368 3294.83 3.66 AQQVSQGLDVLTAKVENAAR -ELLPVLISAMK K366 E200 1977.96 3.06 GSPGIPGDTYLTR -MIDER G517 D176 1670.89 3.38 VGKETVQTTEDQILK K59 D67 2845.52 4.73 VTNSLSNLISLIGTKSGTQER -ELQEK K513 E520 2593.44 3.85 KVTNSLSNLISLIGTK -ETIFEASK K498 E490
	619 620 621 625 626 637 627 638 622 628 639 629 630 640 641	38218 3685.86 # Exp. MH + of each protein are bolded in the sequences). 3.66 NINKD -TIESILEPVAQQISHLVIMHEEGEVDGK 3278.66 3.69 VLQLTSWDEDAWASK -KLEAMTNSKQSIAK Suppl. Table 2. Cross-linked residues characterized in the D1D2:A524 complex (XL-amino K632 E28 D275 2772.45 3.78 GSPGIPGDTYLTR -AQQVSQGLDVLTAK G517 D361 3322.71 4.03 LNQAKGWLRDPSASPGDAGEQAIR -ALASIDSK K281 D374 Primary 2845.52 4.62 VTNSLSNLISLIGTKSGTQER -ELQEK K513 E523 3909.09 4 ETIFEASKKVTNSLSNLISLIGTK -GSPGIPGDTYLTR E490 G477 K381 3695.87 3.82 LNQAKGWLRDPSASPGDAGEQAIR -MSAEINEIIR K281 E240 # Exp. MH + Primary Score Peptide Sequence A524 acids of each protein are bolded in the sequences). # Exp. MH + Peptide Sequence A483 4613.23 3.68 IDDTSAELLTDDISDLKNNNDITAENNNIYK -D594 3169.64 3.79 GQGSSPVAMQKAQQVSQGLDVLTAK -VENAAR K352 Score 4655.33 5.07 IDDTSAELLTDDISDLKNNNDITAENNNIYK -D594 K625 E368 K170 NLGPGMTKMAK 3695.87 3.81 LNQAKGWLRDPSASPGDAGEQAIR -K281 E240 3339.66 3.31 NYVTETNADTIDKNHAIYEK -AKEVSSALSK D561 K571 DVTTSLSKVLK Suppl. Table 3. Cross-linked residues characterized in the D1D2 -A483 1:1 complex (XL-amino Primary Score Peptide Sequence Hv D1 34075 2342.29 3 ELLPVLISAMK -NLGPGMTKMAK E200 K170 # Exp. MH + Primary Peptide Sequence A524 Hv D1D2 3251.77 5.03 ALAKQVATALQNLQTKTNR -RQGKGDSPEAR K464 3050.56 3.57 NYVTETNADTIDKNHAIYEK -INNKLK E554 K540 2214.27 3.22 AKEVSSALSK -ELLPVLISAMK -K571 E200 MSAEINEIIR 3341.68 3.44 NYVTETNADTIDKNHAIYEK -AKEVSSALSK D558 K571 4655.33 4.86 IDDTSAELLTDDISDLKNNNDITAENNNIYK -D595 K625 acids of each protein are bolded in the sequences) E458 3251.77 4.50 ALAKQVATALQNLQTKTNR -RQGKGDSPEAR K464 D455 3340.67 3.55 NYVTETNADTIDKNHAIYEK -AKEVSSALSK E558 K571 3824.86 2.64 GSPGIPGDTYLTR -KIDAAQNWLADPNGGPEGEEQIR G517 3665.8 3.75 KIDAAQNWLADPNGGPEGEEQIR -MSAEINEIIR K387 E240 3341.68 4.19 NYVTETNADTIDKNHAIYEK -AKEVSSALSK E554 DVTTSLSKVLK K571 D389
	631
						40 42 44 46

Cell Reports 26, 921-932, January 22, 2019

CellReports 26, 921-932, January 22, 2019 923

Cell Reports 26, 921-932, January 22, 2019

Cell Reports 26, 921-932, January 22, 2019

Cell Reports 26, 921-932, January 22, 2019

CellReports 26, 921-932.e1-e6, January 22, 2019 e5

ACKNOWLEDGMENTS

We thank Dr. Olivera Francetic for careful reading of the manuscript and Dr. David Stroebel (IBENS) for his help with ITC measurements. This work benefited from the facilities of Plateforme de Mesures d'Interaction des Macromole ´cules (PIM), I2BC, and Plateforme de Proteomique (Ecole Normale Superieure). We acknowledge SOLEIL for provision of synchrotron radiation facilities (proposal ID 20150780), and we would like to thank the Proxima 1 team for assistance in using the beamline. This work was supported by grants from INSERM, CNRS, and Colle `ge de France to the CIRB, as well as a grant from PSL Idex project ''Shigaforce.'' C.V.-G. is a recipient of a PhD fellowship from Memolife Labex and a post-doctoral fellowship from PSL Idex. N.C. is a recipient of an IRG Marie-Curie fellowship. D.-I.A.-S. is funded by CONACYT and the Labex Memolife international PhD program. ACKNOWLEDGEMENTS The authors thank Gauthier Mercante for technical help, Philippe Mailly for help with image analysis and René-Marc Mège for insightful discussions and reading of the manuscript. This work was supported by grants from Inserm, CNRS and Collège de France to the CIRB, as well as grant from the PSL Idex project "Shigaforce". DIAS and BCC are recipients of a PhD fellowship from a CONACYT scholarship. CV-G and DIAS also received support from the Memolife Labex. HK and LM were supported by Swiss National Science Foundation (grant no. SNF 200021 160188) and LM by the Knut And Alice Wallenberg Foundation (grant no. KAW 2016.0023).

DATA AND SOFTWARE AVAILABILITY

Crystallographic structure of the IpaA-VBS3 / talin H1-H4 complex was deposited on the Protein Data Bank, with PDB ID: 5NL1. e6 Cell Reports 26, 921-932.e1-e6, January 22, 2019

List of abbreviations

DECLARATION OF INTERESTS

The authors declare no competing interests.

AUTHOR CONTRIBUTIONS

CV-G and DIAS conceived and performed most of the experimental works, data analysis and wrote the manuscript. BCC and BM analyzed TIRF experiments. CB-N performed the SEC-MALS analysis. CB and NQD performed and analyzed experiments with melanocytes with the help of DJ and AM. BCC, AA and JF provided technical help for cell adhesion and microfluidics experiments. CM and JCR designed and performed the LC-MS analysis. DBL analyzed the cross-linked mass spectrometry data. HK and LM generated structural models. GTVN designed the project and wrote the manuscript. Suppl. Movie 4. 1205Lu melanoma cells 1205Lu melanoma cells were transfected with the indicated constructs. Cells were perfused in a microfluidic chamber and allowed to adhere for 20 min prior to application of shear stress reaching 22.2 dynes.cm -2 . The elapsed time is indicated in seconds.

Article 3

Overview

The aim of the Article 3 is to look into the functional role of IpaA in the context of

Priming assays

All S. flexneri strains were cultivated in TCS medium at 37°C under agitation (200 rpm).

A fresh culture was grown to mid-exponential phase (OD600=1) from an overnight preculture. Bacteria were washed three times on PBS and resuspended on EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM HEPES at pH 7.3). HeLa cells were plated on coverslips the day before at 300,000 cells per well. Before infection, HeLa cells were washed 3 times in EM buffer and incubated with bacterial suspensions for 45 min. Cells were washed thoroughly three times and the second wave of Shigella strains was incubated with the cells for 45 min. Cells were washed and fixed using 3.7% PFA and processed for immunofluorescence staining as previously described (Izard, Tran Van Nhieu, and Bois 2006) .

FlAsH Bacterial imaging

Bacteria grown to mid-exponential phase were washed three times with PBS, and incubated in TCS with the FlAsH reagent at 2 μM final concentration (Invitrogen). Prior to contact with HeLa cells, bacteria were washed three times in PBS and resuspended in EM buffer. HeLa cells were incubated with suspensions of FlAsH-labeled bacteria for 30 min followed by washing with PBS. After incubation, cells were fixed with PFA 3.7% and washed for 30 min at 37°C in PBS-BAL wash buffer (Invitrogen), permeabilized and stained with anti-LPS and Alexa647-phalloidin.

Flow cytometry

HeLa cells were plated on 6-well plates at 200,000 cell/well a day before transfection with plasmids encoding GFP-IpaA VBS1-2 (A524) or GFP-IpaA VBS1-3 (A483) using JetPRIME (Invitrogen). On the day of transfection, cells were trypsinized, washed and fixed using PFA 4%. Cells were blocked with PBS+FCS 1% and incubated with antibody anti-activated β1 integrin clone HUTS-4 conjugated with Alexa-647 (Merck-Milipore).

Cells were washed and analyzed using a FACS Aria.

Timelapse adhesion video

We used a commercial microfluidic setup (Flow chamber system 1C, Provitro) and a Miniplus3 peristaltic pump (Gilson) to adjust the flow rate in the chamber. Microscopy analysis was performed using a LEICA DMRIBe inverted microscope equipped with a Cascade 512B camera and LED source lights (Roper Instruments), driven by the Metamorph 7.7 software (Universal imaging). Bacteria were grown as described above and injected into the microfluidics setup, followed by an incubation of 15 minutes, followed by washing with EM buffer. The second wave of bacteria was injected into the chamber at a flowrate of 1ml/min and images were acquired at a 100ms interval.