Kevin Denis

Marion Le Bris

Loïc Le Guennec

Jean-Philippe Barnier

Camille Faure

Anne Gouge

Haniaa Bouzinba-Ségard

Anne Jamet

Daniel Euphrasie

Beatrice Durel

Loic Le Guennec

Nicolas Barois

Philippe Pelissier

Philippe C Morand

Mathieu Coureuil

Frank Lafont

Olivier Join-Lambert

Xavier Nassif

Sandrine Bourdoulous
email: sandrine.bourdoulous@inserm.fr

Targeting Type IV pili as an antivirulence strategy against invasive meningococcal disease

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INTRODUCTION

Type IV pili, produced by numerous pathogenic bacteria, are filamentous bacterial appendages present in both Gram-negative and Gram-positive bacteria [START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF] . These attributes mediate an extraordinary array of functions, including motility, DNA uptake, bacterial aggregation or adherence to abiotic surface or host cells, and are essential in bacterial pathogenesis [START_REF] Berry | Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives[END_REF] . They are made of the assembly of pilin subunits into a long helical structure, translocated through a pore in the outer membrane. Most type IV pili are highly dynamic, as they can rapidly retract and elongate, a process enabling twitching motility [START_REF] Craig | Type IV pili: paradoxes in form and function[END_REF][START_REF] Kolappan | Structure of the Neisseria meningitidis Type IV pilus[END_REF] . Their dynamic extension/retraction cycle centers on two cytoplasmic ATPases with antagonistic functions, highly conserved among the very widespread bacteria with retractable pili, implicating a fundamentally conserved mechanism of type IV pilus biology [START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF] . Due to their prominent display on the surfaces of many bacterial pathogens and their vital role in virulence, type IV pilus structures are particularly attractive targets for the design of therapies.

In pathogenic Neisseria meningitidis (meningococcus), type IV pili are key virulence factors.

They are made of the assembly of pilus subunits (PilE) and of other less abundant "minor" pilins that play an important role in fine-tuning type IV pilus function [START_REF] Helaine | PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells[END_REF][START_REF] Mikaty | Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress[END_REF][START_REF] Brown | Systematic functional analysis reveals that a set of seven genes is involved in fine-tuning of the multiple functions mediated by type IV pili in Neisseria meningitidis[END_REF] . Meningococci, which normally reside asymptomatically on the human nasopharyngeal mucosa, are pathogenic when they gain access to the bloodstream as they can provoke two devastating diseases, purpura fulminans and meningitis, rapidly causing death or permanent disability, despite prompt antibiotics treatments [START_REF] Stephens | Epidemic meningitis, meningococcaemia, and Neisseria meningitidis[END_REF] . Circulating meningococci adhere to the endothelium lining microvessels through a direct binding of type IV pili with endothelial receptor complexes [START_REF] Coureuil | Meningococcus Hijacks a beta2-adrenoceptor/beta-Arrestin pathway to cross brain microvasculature endothelium[END_REF][START_REF] Bernard | Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization[END_REF][START_REF] Maissa | Strength of Neisseria meningitidis binding to endothelial cells requires highlyordered CD147/beta2-adrenoceptor clusters assembled by alpha-actinin-4[END_REF] . Bacteria then rapidly proliferate, forming aggregated bacterial colonies at the endothelial cell surface, and promote signaling events that contribute to vascular alterations [START_REF] Coureuil | Meningococcus Hijacks a beta2-adrenoceptor/beta-Arrestin pathway to cross brain microvasculature endothelium[END_REF][START_REF] Coureuil | Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium[END_REF] . Colonization of brain microvessels is a prerequisite to cross the blood-brain barrier, allowing bacteria to reach the meninges where they multiply, causing meningitis, whereas, skin lesions occur secondary to meningococcal colonization of dermal vessels, which can eventually lead to purpura fulminans [START_REF] Coureuil | A journey into the brain: insight into how bacterial pathogens cross blood-brain barriers[END_REF] . Skin lesion extension correlates with irreversible endothelial ischaemic injury with extravasation of blood cells into the dermis and necrosis. Circulating bacteria elicit systemic inflammation characterized by massive activation of both macrophages in the reticuloendothelial system and circulating leukocytes. Through the synthesis and secretion of proinflammatory cytokines, endothelial cells also contribute to both local and systemic inflammation, resulting in septic shock and multiple organ failure [START_REF] Sokolova | Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP-and tyrosine kinases in invasion and inflammatory cytokine release[END_REF][START_REF] Melican | Adhesion of Neisseria meningitidis to dermal vessels leads to local vascular damage and purpura in a humanized mouse model[END_REF] . High levels of inflammatory cytokines including Interleukin-6 (IL-6), IL-8 and TNFα in patient serum closely correlate with the clinical severity of the disease [START_REF] Girardin | Tumor necrosis factor and interleukin-1 in the serum of children with severe infectious purpura[END_REF][START_REF] Waage | The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome[END_REF] . Importantly, type IV pilus-dependent adhesion of meningococci to human microvasculature is determinant to the triggering of both vascular purpuric lesions and NMICROBIOL-17122366 Revised version p. 4 inflammation [START_REF] Melican | Adhesion of Neisseria meningitidis to dermal vessels leads to local vascular damage and purpura in a humanized mouse model[END_REF][START_REF] Join-Lambert | Meningococcal interaction to microvasculature triggers the tissular lesions of purpura fulminans[END_REF] , and a prerequisite to sustained bacteremia responsible for sepsis and subsequent lethality [START_REF] Capel | Peripheral blood vessels are a niche for blood-borne meningococci[END_REF] .

Here, we demonstrate that trifluoperazine and related phenothiazines, a family of compounds previously used in human medicine to treat psychotic disorders, block the functions carried by type IV pili in different bacterial pathogens, by inducing pilus retraction within minutes.

Using N. meningitidis as a model organism, we show that these compounds regulate piliation most likely by acting on the Na + -NQR complex, which drives the selective translocation of Na + across the inner membrane, thereby building a sodium motive force. We further showed that establishment of a sodium gradient is required for piliation. In vivo, these compounds suppress the consequences of type IV pilus-dependent pathogenesis, thus opening a new era for the treatment of infections due to type IV pilus-expressing bacteria.

NMICROBIOL-17122366 Revised version p. 5

RESULTS

Trifluoperazine blocks the functions carried by bacterial type IV pili

When grown in broth for two hours, meningococci form bacterial aggregates due to interbacterial interactions, a phenotype carried by type IV pili in capsulated bacteria [START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF][START_REF] Helaine | PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells[END_REF] . We fortuitously observed that trifluoperazine, initially used in vitro as a calmodulin antagonist, rapidly induced the dispersion of bacterial aggregates (Fig. 1a). This effect was dose-dependent (Fig. 1a and1b), and it occurred within minutes (Fig. 1c and Video 1). Trifluoperazine first induced a reduction in bacterial twitching motility, shortly followed by the dispersal of the bacterial aggregates (Fig. 1d and Video 2). In contrast to trifluoperazine treatment, addition of bactericidal antibiotics used in the treatment of meningococcaemia, such as third generation cephalosporin cefotaxime or aminoglycoside gentamicin, poorly affected aggregation (Fig. 1a). Although trifluoperazine showed some antimicrobial activity against N. meningitidis, this was observed at later time point (24h after treatment) and was not as efficient as antibiotics (Supplementary Fig. 1a). Furthermore, treatment with trifluoperazine poorly affected bacterial growth during the first hour of treatment (Supplementary Fig. 1b) while it rapidly and reversibly dispersed bacterial aggregates (Supplementary Fig. 2).

The dispersal effect of trifluoperazine was observed with meningococcal strains belonging to different sequence types and to different capsular serogroups (Supplementary Fig. 3a).

Trifluoperazine was also able to reduce twitching motility and aggregates of strains of the related species, Neisseria gonorrhoeae, which causes gonorrhea (Supplementary Fig. 3B and Video 3). This effect was not limited to Neisseria species, as treatment with trifluoperazine also reduced by 40 % the pilus-dependent twitching motility of the opportunistic pathogen Pseudomonas aeruginosa PAO1, whereas it did not affect its swarming behaviour, which requires functional flagella (Supplementary Fig. 4a). Furthermore, trifluoperazine also induced bacterial dispersion from PAO1 biofilms, as shown by the loss of the 3D community (Supplementary Fig. 4b).

Trifluoperazine belongs to a large family of phenothiazine derivatives belonging to three groups: the aliphatic compounds, the piperidines and the piperazines (Supplementary Table 1). All piperazine and piperidine compounds tested induced the dispersal of liquid-grown meningococcal aggregates, in a dose-dependent manner, but with varying efficiency (Supplementary Fig. 5). By contrast, no effect was observed with the aliphatic compounds in this range of concentrations excepted for promazine. Thioridazine was the most effective compound in vitro, promoting the dispersal of bacterial aggregation at concentrations 10-fold lower than trifluoperazine, without bactericidal effect (Supplementary Fig. 6).

NMICROBIOL-17122366 Revised version

p. 6 These data demonstrate that trifluoperazine and related phenothiazines induced the dispersal of meningococcal aggregates by a mechanism independent from bactericidal activity.

Phenothiazines regulate the piliation status of meningococci

We then examined the effect of trifluoperazine and thioridazine on the piliation status of meningococci. Wild type (WT) meningococci treated with cefotaxime or gentamicin for 30 minutes expressed type IV pili on their surface similarly to non-treated bacteria (Supplementary Fig. 7a).

Conversely, few type IV pili were observed on bacteria treated for 30 min with trifluoperazine or thioridazine (Fig. 2a), suggesting that these compounds induced fiber dissociation or retraction.

Interestingly, trifluoperazine and thioridazine did not affect aggregation on a retraction-defective PilT-deleted mutant (ΔPilT) (Fig. 1c, Video 4 and Supplementary Fig. 7b), and it did not affect type IV pilus surface expression in the ΔPilT mutant (Fig. 2a and Supplementary Fig. 7c). Consistently, trifluoperazine did not significantly affect expression levels of the major pilin subunit PilE, nor of the ATPase PilT (Supplementary Fig. 7d). However, in contrast to non-treated bacteria that form large bundles of type IV pili at the bacterial surface, trifluoperazine treatment induced the enrichment of PilE within the bacterial body, most likely in the inner membrane that is the reservoir for pilin subunits [START_REF] Pelicic | Type IV pili: e pluribus unum?[END_REF] (Fig. 2b).

The above results strongly suggested that trifluoperazine favoured pilus retraction over pilus biogenesis. To confirm this hypothesis, we monitored bacterial liquid growth in the presence of trifluoperazine. This treatment inhibited the formation of aggregates by wild type bacteria, whereas it poorly affected aggregation of the PilT mutant strain (Supplementary Fig. 8), thus ruling out a possible effect on pilus biogenesis. Besides PilT, the outer membrane proteins PilC also play a major role in controlling pilus retraction by antagonising pilus retraction [START_REF] Morand | Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins[END_REF] . To address whether antagonizing pilus retraction would provide resistance to the effect of trifluoperazine, we used a strain overexpressing PilC1 (PilC1ind). Similarly to the ΔPilT mutant, trifluoperazine did not affect the inducible aggregation of a PilC1ind strain (Supplementary Fig 7e ), nor did it regulate the expression level of PilC1 (Supplementary Fig. 7d). These results demonstrated that trifluoperazine interferes with pilus dynamics.

To characterize the mechanisms by which these compounds affected meningococcal piliation, we generated strains resistant to the bactericidal activity of trifluoperazine. Four clones were forming bacterial aggregates in suspension and were resistant to the dispersal effect of both trifluoperazine and thioridazine (Supplementary Fig. 9). All clones had genetic alterations in one of 6 genes encoding for the sodium pumping NADH:ubiquinone oxidoreductase complex (Na + -NqrA-F) (Supplementary Table 2), which is crucial for establishing an electrochemical Na + gradient across the NMICROBIOL-17122366 Revised version p. 7 inner membrane [START_REF] Juarez | Insights into the mechanism of electron transfer and sodium translocation of the Na(+)-pumping NADH:quinone oxidoreductase[END_REF] . In addition, all clones had alterations in genes encoding either the glycosyltransferase lgtE or the UDP-glucose 4-epimerase GalE, both essential for incorporation of galactose into meningococcal lipopolysaccharide (LOS). Changes in LOS structure is a previously reported adaptive mechanism to osmotic pressure [START_REF] Jeworrek | Effects of specific versus nonspecific ionic interactions on the structure and lateral organization of lipopolysaccharides[END_REF][START_REF] Lloret | Ionic Stress and Osmotic Pressure Induce Different Alterations in the Lipopolysaccharide of a Rhizobium meliloti Strain[END_REF] , suggesting that mutations affecting LOS structure might compensate for alterations in Na + transport due to mutations in Nqr subunits.

Interestingly, meningococcal isogenic ΔNqrA-F mutants, devoid of individual nqr genes and with wild type GalE, were poorly piliated and aggregative (Fig. 2c), thus linking piliation and Nqr-mediated Na + pumping activity. We therefore hypothesized that trifluoperazine and thioridazine might affect sodium gradient through the inner membrane. Enrichment of broth with NaCl from 6,4 g/L (close to blood sodium level) to 21,4 g/L did not affect the aggregation for the WT meningococci (Fig. 2D).

However, NaCl inhibited the dispersal effect of both trifluoperazine and thioridazine, in a dosedependent manner. This effect was specific of the Na + ions and independent from an osmotic stress, as addition of KCl up to 22,4 g/L had no inhibitory effect (Fig. 2D). Altogether, these results strongly suggested that trifluoperazine and thioridazine rapidly affect piliation by altering the Na + pumping activity of Na + -NQR.

Phenothiazines reduce bacteria-induced endothelial cell injury in vitro

Next, we analysed the effect of these phenothiazines on initial adhesion to human endothelial cells and microcolony formation, as both rely on expression of type IV pili. When pre-treated with trifluoperazine, bacteria poorly adhered and/or formed bacterial colony at the surface of primary human endothelial cells isolated from dermal microvessels HDMECs (Supplementary Fig. 10). More interestingly, when applied to meningococcal microcolonies already established at endothelial cell surface, trifluoperazine or thioridiazine treatment for 30 min disrupted the preformed microcolonies, thus allowing the release of adherent bacteria. This effect was dose-dependent and was observed on human endothelial cells isolated from microvessels, either form dermis (HDMEC) (Fig. 3a and3b) or from brain (hCMEC/D3) (Supplementary Fig. 11). The effect of both phenothiazines was further confirmed by scanning electron microscopy analysis. Whereas bacteria formed large 3D colonies in control conditions, only few and dispersed bacteria remained at the endothelial cell surface after treatment (Fig. 3d). As expected, the two phenothiazines did not induce the dispersal of microcolonies formed on endothelial cells by the ΔPilT mutant (Fig. 3a and3c). In contrast to phenothiazines, addition of gentamicin or cefotaxime poorly induced the dispersal of microcolonies formed by wild type meningococci (Fig. 3a and3b).

We further analyzed the effect of trifluoperazine on the colonization of human brain vessels, using an ex vivo meningococcal infection model of fresh human frontal brain tissues, as described NMICROBIOL-17122366 Revised version p. 8 previously [START_REF] Bernard | Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization[END_REF] . Upon infection, meningococci replicated as microcolonies adjacent to VE-cadherinpositive endothelial cells (Fig. 3e). Consistent with in vitro cellular models, treatment of infected brain sections with trifluoperazine for 30 min induced the dispersal of meningococcal microcolonies, reducing by 80% the vascular colonization of the human brain vessels (Fig. 3e and3f).

Blood flow playing a pivotal role in stabilizing barrier function [START_REF] Seebach | Regulation of endothelial barrier function during flow-induced conversion to an arterial phenotype[END_REF] , we then addressed the impact of these phenothiazines on endothelial cell dysfunctions promoted by adherent bacteria, using HDMECs grown under laminar shear stress. As previously observed, control conditions showed meningococci-induced dismantlement of the continuous VE-cadherin staining at the endothelial cell junctions, as well asformation of gaps between cells (Fig. 4a). Cefotaxime, although reducing bacterial colonization, had no protective effect on vascular cell junction integrity. In contrast, trifluoperazine, alone or in combination with cefotaxime, induced the dispersion of most bacterial colonies and restored VE-cadherin continuity at intercellular junctions (Fig. 4a). Hence, in contrast to antibiotic treatment, trifluoperazine rapidly prevents the deleterious signalling events leading to altered cell junction integrity.

To assess a potential protective effect of phenothiazines on basement membrane integrity, HDMECs were grown on a fluorescent gelatin matrix (Gelatin-FITC) (Fig. 4b). In the absence of infection, <5% of the basement membrane was degraded. Because it takes 16 to 24h to observe matrix degradation, the time for proteinases expression, bactericidal treatment was applied after 5 hours of infection to avoid bacterial overload and subsequent artefactual cell death. In these conditions, degradation of the basement membrane induced by meningococcal infection, observed 24h post-infection, was reduced in a dose-dependent manner (40 to 50 %) upon treatment with trifluoperazine or thioridazine (Fig. 4b-d). Altogether, these results demonstrated that trifluoperazine and thioridazine induce a cytoprotective effect on endothelial cells, by preserving the integrity of their endothelial cell junctions and basement membrane.

Phenothiazines reduce bacterial colonization of human blood vessels in vivo

We investigated the potential effect of phenothiazines on endothelial colonization by meningococci in vivo, using a validated humanized mouse model of meningococcal infection using severe combined immunodeficiency (SCID) mice grafted with human skin [START_REF] Bernard | Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization[END_REF][START_REF] Melican | Adhesion of Neisseria meningitidis to dermal vessels leads to local vascular damage and purpura in a humanized mouse model[END_REF][START_REF] Join-Lambert | Meningococcal interaction to microvasculature triggers the tissular lesions of purpura fulminans[END_REF] . To mimic the conditions in patients with meningococcemia, grafted mice were infected with 5x10 6 bacteria intravenously, leading to an average bacteremia of 5x10 5 to 10 6 bacteria/ml 1h post-infection (Supplementary Fig. 12). Two hrs post-infection, bacteria had already adhered to human skin graft blood vessels; occasionally, bacterial aggregates filled the entire vessel and platelet aggregation was observed in close proximity of bacteria. Four hrs post-infection, bacteria massively colonized the human dermal vasculature with massive platelet aggregation and the integrity of the basement membrane was altered, as assessed NMICROBIOL-17122366 Revised version p. 9 by staining of collagen IV, a major basement membrane constituent (Supplementary Fig. 12). To address the effect of the two phenothiazines in vivo, the compounds were administrated intraperitoneally 2 hours after infection and mice were sacrificed 4h post-infection (Fig. 5a). Whereas bacteremia was similar in control and treated mice (Fig. 5b), meningococcal vascular colonization was reduced by 50 to 55 % in the presence of trifluoperazine (40 mg/kg) or thioridazine (4 mg/kg), whith both number of colonized vessels and extent of bacterial colonization being decreased (Fig. 5cd). The effect of trifluoperazine was stronger when administrated at 40 mg/kg than 24 mg/kg, or at 30 min post-infection compared to 2h, indicating that early treatment improved efficacy (Supplementary Fig. 13a-f). These results demonstrated the potent action of these two phenothiazines on vascular colonization by meningococci in vivo.

Phenothiazines reduce the formation of vascular lesions

We therefore examined whether reduced meningococcal colonization of the human vessels promoted by phenothiazines could prevent vascular dysfunction. First, we observed decreased platelet and red blood cell aggregation in skin grafts of phenothiazine-treated mice (Fig. 5c and5e).

This effect was dose-dependent (Supplementary Fig. 13c and13f). In contrast to infected control grafts, in which massively colonized human dermal vessels presented signs of compromised endothelial integrity, phenothiazine treatment preserved the integrity of the endothelial cell junctions, as assessed by the continuous staining of two adherens junction markers, PECAM-1/CD31 and VE-cadherin (Fig. 5c and supplementary Fig. 13c). Second, trifluoperazine reduced by 50 to 80% the transcription of several human inflammatory cytokines induced by infection, such as IL-6, CXCL8/IL-8, CXCL1/GROα or CCL2/MCP-1 or of prostaglandin G/H synthase (PTGS2/Cox2) responsible for production of inflammatory prostaglandins (Supplementary Fig. 13g). Finally, whereas infection induced the release of TNFα, IL-6 and IL-8 in mice sera, either trifluoperazine or thioridazine treatment reduced by 45 to 60% the serum level of these pro-inflammatory factors (Fig. 5f). These data demonstrated that, by decreasing vascular colonization, phenothiazines reduce vascular alteration, thrombosis and inflammation during meningococcemia.

Thioridazine improves the outcome of meningococcal infection

We therefore addressed whether vessel clearance by thioridazine improved the outcome of infected mice (Fig. 6a). In control mice, infection with 10 6 bacteria intravenously led to an average bacteremia of 5x10 6 bacteria/ml at 4h post-infection. Bacteremia was sustained at an average of 10 6 to 5x10 [START_REF] Mikaty | Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress[END_REF] bacteria/ml at 18h and 48h post-infection and 85 % of the infected control mice did not survive by day 2 (Fig. 6b and6c). Upon treatment with thioridazine 2h after infection, while the bacteremia was NMICROBIOL-17122366 Revised version p. 10 similar to that of control mice at 4h post-infection, it was reduced down to 5x10 3 to 5x10 [START_REF] Kolappan | Structure of the Neisseria meningitidis Type IV pilus[END_REF] bacteria/ml at 18h and eventually dropped to <10 2 bacteria/ml at 48h post-infection (Fig. 6b). 0nly 40% of the thioridazine-treated mice died 48h post-infection (Fig. 6c). As observed at 4h postinfection, in contrast to infected control grafts in which human dermal vessels were massively colonized, thrombosed and presented signs of compromised endothelial integrity at time of death (between 18h and 48h post-infection), thioridazine had largely reduced colonization and subsequent signs of thrombosis and preserved vascular integrity at 72h post-infection (Fig. 6d). These data demonstrated the improvement of survival outcome with the use of thioridazine in an in vivo model of meningococcal infection.

Thioridazine has adjunctive effects over antibiotic treatment

Since antibiotics are a key element for medical treatment of meningococcemia, we analysed whether thioridazine could provide a beneficial effect when administrated with antibiotics. When applied 2h post-infection at doses recommended for patients with suspected acute bacterial meningitis (200 mg/kg/day), cefotaxime exerted a strong bactericidal effect as bacteremia dropped to less than 10 2 bacteria/ml at 4h post-infection (Fig. 6a and6b) and as it totally prevented mice lethality (Fig. 6c). It reduced by 90 % vascular colonization by meningococci (Fig. 6e and6f). However, thrombosis (Fig. 6E and 6F) was increased in cefotaxime-treated mice and serum concentration of human inflammatory cytokines TNFα, IL-6 and IL-8 were not significantly reduce 4h post-infection (Fig. 6g). When administrated with cefotaxime, thioridazine reduced by 70% the signs of thrombosis (Fig. 6e and6f) and reduced vascular inflammation (Fig. 6g). 72h post-infection, whereas cefotaxime-treated grafts still presented signs of vascular alterations and thrombosis, associated with massive infiltration of polymorphonuclear neutrophils, these effects were prevented by the addition of thioridazine (Fig. 6d), therefore demonstrating that thioridazine had adjunctive effects over antibiotic treatment by reducing alteration of blood vessels and the overwhelming inflammatory response.

Collectively, these results demonstrate that targeting type IV pili, a major meningococcal virulence factor, with some phenothiazines exert a protective effect on the pathophysiological events of meningococcemia. Their use in combination with antibiotics may reduce incidence of vascular alteration, intravascular coagulation and inflammatory disorders in meningococcal diseases.

NMICROBIOL-17122366 Revised version p. 11

DISCUSSION

Much investigation has been conducted in recent years to improve treatments of infectious diseases, especially in the light of incomplete action, adverse effects, and increasing resistance to antibiotics. This implies the development of pharmaceuticals acting on previously unutilized conserved targets [START_REF] Levy | Antibacterial resistance worldwide: causes, challenges and responses[END_REF] .

Recently, bacterial virulence properties have been revisited as attractive targets for the development of new therapeutic agents [START_REF] Marra | Targeting virulence for antibacterial chemotherapy: identifying and characterising virulence factors for lead discovery[END_REF][START_REF] Felise | An inhibitor of gram-negative bacterial virulence protein secretion[END_REF] . In such a perspective, type IV pili represent particularly appealing virulence factor targets. They are associated with a remarkable array of properties ranging from motility to electric conductance, and are arguably one of the most widespread virulence factor since distinctive proteins dedicated to their biogenesis are found in most known species of prokaryotes 2 .

This study demonstrates that trifluoperazine and thioridazine rapidly impair functions carried by type IV pilus structures, including twitching motility, aggregation, or adherence to inert or cell surfaces in model bacterial pathogens: Neisseriae species and Pseudomonas aeruginosa. In an in vivo model of meningococcal infection, when administered after infection at doses compatible with use in humans, thioridazine reduces bacterial colonisation of blood vessels, as well as vascular alterations, thrombosis and inflammation, the hallmarks of invasive meningococcal infections. Furthermore, in agreement with our recent observation that colonization of blood vessels by piliated meningococci is required for sustained bacteremia responsible for sepsis and subsequent lethality [START_REF] Capel | Peripheral blood vessels are a niche for blood-borne meningococci[END_REF] , thioridazine reduces bacteremia and increases mice survival. In association with antibiotics, it reduces vascular inflammatory and thrombotic responses that were shown to be highly detrimental and correlated with the severity of the disease in patients [START_REF] Girardin | Tumor necrosis factor and interleukin-1 in the serum of children with severe infectious purpura[END_REF][START_REF] Waage | The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome[END_REF][START_REF] Van Deuren | Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections[END_REF] . Altogether, our results demonstrate in vivo the efficacy of these compounds in reducing the pathogenic consequences of a bacterial infection relying on type IV pilus expression.

Before extensive use as neuroleptics, phenothiazines were first reported as antimicrobial agents against Gram-positive and Gram-negative bacteria [START_REF] Amaral | Phenothiazines, bacterial efflux pumps and targeting the macrophage for enhanced killing of intracellular XDRTB[END_REF] . However, phenothiazines, and more particularly thioridazine, have only modest antimicrobial activity, at concentrations and time scale beyond those impacting type IV pilus functionality [START_REF] Mazumder | Trifluoperazine: a broad spectrum bactericide especially active on staphylococci and vibrios[END_REF] . In consistence with previous observation that phenothiazines potently inhibit efflux pumps in both human and microorganisms [START_REF] Amaral | Comparison of multidrug resistant efflux pumps of cancer and bacterial cells with respect to the same inhibitory agents[END_REF] , our data strongly suggest that phenothiazines target Na + -NQR. This protein complex, highly conserved among numerous non-pathogenic and pathogenic bacteria, catalyzes the oxidation of NADH and the reduction of quinone. The released energy drives a sodium motive force through the translocation of Na+ across the inner cell membrane. In many pathogenic bacteria, Na+ gradient is an entry site for electrons into the respiratory chain towards ATP synthesis, or to sustain ionic homeostasis, nutrient transports, flagellum rotation and other essential processes [START_REF] Juarez | Insights into the mechanism of electron transfer and sodium translocation of the Na(+)-pumping NADH:quinone oxidoreductase[END_REF][START_REF] Reyes-Prieto | Origin and evolution of the sodium -pumping NADH: ubiquinone oxidoreductase[END_REF] . In Vibrio cholerae, Na + -NQR was NMICROBIOL-17122366 Revised version p. 12 shown as physiologically ubiquitous, including motility and formation of bacterial monolayers, an initial step in the build-up of biofilms [START_REF] Van Dellen | Genetic analysis of Vibrio cholerae monolayer formation reveals a key role for DeltaPsi in the transition to permanent attachment[END_REF][START_REF] Minato | Roles of the sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) on vibrio cholerae metabolism, motility and osmotic stress resistance[END_REF] . The recent approach used to identify genes essential for meningococcal pathogenesis unraveled a role of the nqr complex for colonization of the human blood vessels [START_REF] Capel | Peripheral blood vessels are a niche for blood-borne meningococci[END_REF] . However, no link with type IV pili functionality had been reported so far. How this sodium gradient affects type IV pilus dynamics remains to be explored. The mechanisms promoting the ATPase-driven elongation/retraction cycles remains elusive. Model system suggests that pilus retraction proceeds as pilin subunit monomers are disassembled from the base of an extended type IV pilus polymer and dispersed into the membrane for later reuse in pilus elongation [START_REF] Craig | Type IV pili: paradoxes in form and function[END_REF][START_REF] Morand | Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins[END_REF] . Retraction can occur in two distinct speed modes, an unusual motor property conserved between different bacterial species [START_REF] Maier | A force-dependent switch reverses type IV pilus retraction[END_REF][START_REF] Clausen | High-force generation is a conserved property of type IV pilus systems[END_REF] , and switching between retraction and elongation occurs at different time scales [START_REF] Maier | A force-dependent switch reverses type IV pilus retraction[END_REF] . In N. gonorrhoeae, oxygen depletion triggers speed switching from the high to the low speed mode, resulting in the disassembly of gonococcal microcolonies within minutes [START_REF] Kurre | Oxygen depletion triggers switching between discrete speed modes of gonococcal type IV pili[END_REF] ; such effect is most likely occurring by controlling proton motive force and, in the end, changes in the pilus-pilus interaction force [START_REF] Dewenter | Oxygen governs gonococcal microcolony stability by enhancing the interaction force between type IV pili[END_REF] . As the sodium gradient generated by the Na + -NQR can provide an entry site for electron into the respiratory chain for ATP synthesis [START_REF] Juarez | Insights into the mechanism of electron transfer and sodium translocation of the Na(+)-pumping NADH:quinone oxidoreductase[END_REF][START_REF] Reyes-Prieto | Origin and evolution of the sodium -pumping NADH: ubiquinone oxidoreductase[END_REF] , phenothiazines might affect proton motive force resulting in the switch of pilus retraction mode. Furthermore, one might not exclude the possibility that periplasmic Na + fine-tunes the activity of proteins promoting pilus stability [START_REF] Brown | Systematic functional analysis reveals that a set of seven genes is involved in fine-tuning of the multiple functions mediated by type IV pili in Neisseria meningitidis[END_REF] . Further studies will be required to fully characterize the mode of action of sodium gradient on pilus dynamics.

In our mice model, trifluoperazine was effective in vivo on bacterial clearance and vascular lesions at doses 10 fold higher than recommended for chronic administration in humans, and we observed some toxicity in septic animals even in the presence of cefotaxime. However, thioridazine was very well tolerated and effective at dose regimen compatible with human use. It is therapeutically safe with few side effects when used with moderation, as proven by the 60 plus years it has been in use [START_REF] Amaral | Thioridazine: A Non-Antibiotic Drug Highly Effective, in Combination with First Line Anti-Tuberculosis Drugs, against Any Form of Antibiotic Resistance of Mycobacterium tuberculosis Due to Its Multi-Mechanisms of Action[END_REF] . We showed that it reduced vascular lesions and improved mice survival in the absence of antibiotic treatment when administrated early during the infection. These tests should also be extended to other related phenothiazines, such as mezoridazine or promazine, which were also very effective in vitro. Although it is difficult to predict from mice study how effective thioridazine and other related compounds might be on the outcome of an infection in human, we believe that it could prevent some of the deleterious consequences of the infections by bacterial pathogens relying on type IV pili expression. The role of Tfp in adhesion to host cells, aggregation, biofilm formation is crucial for pathogenesis of numerous human pathogens, such as enteropathogenic Escherichia coli EPEC [START_REF] Bieber | Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli[END_REF] , P. aeruginosa resistance [START_REF] Persat | Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa[END_REF] or nontypeable Haemophilus influenza [START_REF] Novotny | Antibodies against the majority subunit of type IV Pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS-dependent manner and confer therapeutic resolution of experimental otitis media[END_REF] . Interfering with type IV pilus-mediated host cell interaction therefore represents an NMICROBIOL-17122366 Revised version p. 13 attractive strategy to modify the course of diseases induced by these pathogens. Due to the wellconserved set of proteins involved in type IV pilus biosynthesis in Gram-negative bacteria, it is most likely that phenothiazines may also act on numerous other type-IV piliated pathogenic bacteria.

Taken together, through an unexpected regulation of bacterial type IV pili expression by phenothiazines, this work reveals a new approach to reduce meningococcal vascular colonization and subsequent vascular lesions and lethality, as well as the benefit of such molecules as adjuvant therapy for the treatment of invasive meningococcal disease. This provides proof of concept that compounds with an activity against bacterial type IV pili could be used in addition to antibiotics to prevent and treat bacterial diseases.

NMICROBIOL-17122366 Revised version p. 14

METHODS

Antibodies and Reagents

The list of the antibodies used in this work is provided in supplementary Table 3. mAb raised against PilE (20D9) and polyclonal antiserum raised against meningococcal 2C4.3 strain were described

Bacterial strains

Nm 2C4.3 (formerly clone 12) is a piliated capsulated Opa -Opc -variant of the serogroup C meningococcal clinical isolate 8013 [START_REF] Nassif | Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells[END_REF] . The isogenic derivative mutants ΔPilT, where the pilT gene was interrupted by an erythromycin-resistance cassette and the isogenic strain harbouring an IPTGinducible pilC1 gene (pilC1 ind ) were previously described [START_REF] Morand | Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins[END_REF][START_REF] Pujol | The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion[END_REF] . Transposon insertions in individual nqr genes were recovered from the genome-wide collection of N. meningitidis transposition mutants [START_REF] Geoffroy | Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis[END_REF] and isogenic 2C4.3 mutants were generated by transformation. Z5463 (formerly C396) is a capsulated, piliated OpaA + Opc + meningococcal clinical isolate of serogroup A [START_REF] Achtman | Purification and characterization of eight class 5 outer membrane protein variants from a clone of Neisseria meningitidis serogroup A[END_REF] . FAM20 is a capsulated, piliated, Opa -, Opc -strains of serogroup C 47 . Meningococci were cultured as described previously [START_REF] Hoffmann | Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis[END_REF] . Briefly, before experiments, bacteria were grown overnight on GCB solid medium (4% GC medium base; Difco), 1% agar, 0.4% glucose, 0.2 mg/ml thiamine, 0.0005% Fe(NO3)39H2O, 0.01%

L-glutamine at 37°C under 5% CO2. Several colonies were then selected and grown in DME supplemented with 0.1% BSA for 2h and were finally diluted to 10 7 bacteria/ml suspension for cell infection.

Three different strains of Neisseria gonorrhoeae were used: the reference strain MS11 and two other strains FAT1.1 and SB2ST11. Bacteria were grown for approximately 36h on chocolate agar plates.

Several colonies were then selected and grown for 2h in DMEM supplemented with 0.1% BSA and adjusted to an optical density (OD) of 0.1 before experiment. The strain Pseudomonas aeruginosa PAO1 and its derivative expressing the fluorescent protein mCherry were kindly provided by Dr Mylène Robert-Genthon (CEA Grenoble, France). PAO1 strains were grown in LB medium (supplemented with Tetracycline to maintain m-Cherry expression) overnight at 37°C on a shaker before experiments.

Bacterial growth assays

NMICROBIOL-17122366 Revised version p. 15

Growth of Nm 2C.43 in the presence of trifluoperazine or thioridazine was measured at 600 nm at intervals of 1h up to 6h with a spectrophotometer. To measure bactericidal activity, a bacterial suspension of Nm 2C4.3 at optical density (OD) 0.1 was distributed in 24-well plate (1ml/well). After 1 hour of incubation, trifluoperazine, thioridazine, gentamicin, cefotaxime or PBS alone as control were added. After 15 min treatment, serial dilutions were performed in physiological water and 100 µl of each dilution were plated on GCB solid media (BD, Difco GC media) containing supplements, incubated overnight and the colonies were counted.

Bacterial aggregation assays

Bacteria were grown in liquid culture for 2h to form bacterial aggregates before the indicated treatment, i.e. increasing concentrations of trifluoperazine (10 to 40 µM), thioridazine (1 to 10 µM), gentamicin (150 µg/ml) or cefotaxime (20 µg/ml) for 30 minutes. When indicated, bacteria were grown in liquid culture for 2h to form bacterial aggregates, then NaCl was added to reach concentrations varying from 6,4 g/L to 21,4 g/L, before treatment with trifluoperazine or thioridazine. Bacterial aggregates were visualized using a phase-contrast microscope. Quantification analysis of the size of bacterial aggregates was performed using ImageJ software.

To assess P. aeruginosa twitching and swarming motility, P. aeruginosa PAO1 strain was grown for 24h in low-agar (0.5%) medium enabling bacterial swarming (dependent on flagellum) and in highagar (1%) medium, enabling type IV pilus dependent twitching motility, as previously described [START_REF] Burrows | Pseudomonas aeruginosa twitching motility: type IV pili in action[END_REF] . P. aeruginosa PAO1 strain expressing mCherry was grown for 3 days on a solid surface under flow (2 dynes/cm 2 ) to promote biofilm maturation and growth of the three-dimensional community and trifluoperazine (100 µM) was applied for 2 h under flow. Bacteria were fixed and the fluorescent biofilm was analysed by confocal microscopy at 20x and 100x. Surface of the fluorescent biofilm (in pixel ²) was measured using ImageJ software.

Generation and whole genome sequencing analysis of bacterial resistant strains

To generate strains resistant to the action of phenothiazines, one clone of N. meningitidis 2C.43 was selected by performing 3 passages on GCB solid medium, bacteria were grown in the presence of 40 µM trifluoperazine over 3 passages, then 10 8 were plated GCB solid medium containing 50 µM trifluoperazine. Out of the 13 recovered resistant clones, 4 clones were still forming aggregates in suspension, and these aggregates were now resistant to the dispersal effect of both trifluoperazine (40 µM) and thioridazine (4 µM). Genomic DNA was extracted using DNeasy Blood and Tissue Kit.

Genomic libraries were prepared using a Nextera XT kit, multiplexed and sequenced on an Illumina MiniSeq instrument (2x150 paired-end sequencing). Single nucleotide polymorphisms (SNPs) and small indels were assessed using Snippy v3.

Cell culture and infection

Primary Human Dermal Microvascular Endothelial Cells (HDMEC) isolated from the dermis of juvenile foreskin and adult skin (different locations) and their specific endothelial cell growth medium were purchased from PromoCell. hCMEC/D3, engineered in the team of Pierre-Olivier Couraud at the Institut Cochin, is a fully differentiated human brain endothelial cell line derived from brain capillaries, which recapitulates the major phenotypic features of the blood-brain barrier [START_REF] Weksler | The hCMEC/D3 cell line as a model of the human blood brain barrier[END_REF] .

HCMEC/D3 cells were grown in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% Fetal Bovine Serum "Gold", 10 mM HEPES (PAA Laboratories GmbH, Pasching, Austria), 1%

Penicillin-Streptomycin, 1% chemically defined lipid concentrate (Invitrogen Ltd, Paisley, UK), 1.4μM hydrocortisone, 5 μg.ml -1 ascorbic acid and 1 ng.ml -1 bFGF (Sigma-Aldrich, St. Louis, MO). They were tested for mycoplasma contamination. When indicated, HDMECs were split on channel slides (µ-slide I Luer from ibidi), grown for 2 days to reach confluency and then subjected to a laminar flow shear stress (10 dynes/cm 2 ) for 4 days (ibidi Pump System, ibidi). To infect cells, bacteria were precultured in prewarmed cell culture medium for 1h 30 min at 37°C under 5% CO 2 atmosphere. The OD 600 was adjusted to 0.1 and cells were then overlaid with bacteria for 30 min (MOI of 100). Unbound bacteria were removed by three washes in cell culture media and infection was allowed to proceed for 1h at 37°C 5% CO 2 before treatment with trifluoperazine or thioridazine for 20 min. Cells were then washed and fixed in 4% paraformaldehyde for immunofluorescence analysis.

Immunofluorescence microscopy and live imaging

HDMEC or hCMEC/D3 cells were grown to confluence on Thermanox coverslips (Thermo Fischer Scientific) or ibidi device. After the indicated treatments and/or infection, cells were washed, fixed in 4% paraformaldehyde and immunostaining was performed with the indicated primary and secondary antibodies. Image acquisitions were performed on a Leica DMI6000. Quantification were done using ImageJ software (NIH). For time lapse video microscopy, bacteria grown for 2h in liquid culture medium or on cells, and image acquisitions before and after addition of trifluoperazine were performed overtime, at one frame per second for 20 minutes, on a Leica DMI6000 with Yokogawa CSU-X1M1 system and CoolSnap HQ2 (Photometrics). Image sequences were processed with ImageJ software to create the videos.

NMICROBIOL-17122366 Revised version p. 17

Matrix degradation assay

Thermanox coverslips (Thermo Fischer Scientific) were washed in ethanol 70%, dried then coated with poly-L-lysine 1 mg/ml for 20 min at room temperature. After one wash in sterile PBS, 0.5% glutaraldehyde was added for 15 min at room temperature. After three washes in PBS, gelatin-FITC (G13187 from Invitrogen) was added at 0.2 mg/ml for 10 minutes at room temperature in the dark.

Coverslip were then washed with sterile PBS and treated with 5 mg/ml sodium borohydride for 3min.

After three washes in sterile PBS, 10 5 HDMECs were seeded per well in their PromoCell culture medium and incubated overnight at 37°C 5% CO 2 . The day after, cells were infected with Neisseria meningitidis 2C4.3, as described above. Infection was allowed to proceed for 6h before gentamicin treatment at 150 mg/ml for 1h. After three washes in cell culture medium, trifluoperazine or thioridazine was added for 20 min, washed with the culture medium and were incubated overnight in cell culture media containing 15 mg/ml Gentamicin. Cells were then fixed in 4% Paraformaldehyde for 10 min, stained with phalloidin and DAPI for 1 hour and mounted in glycergel (DAKO). Image acquisitions were performed on a Leica DMI6000 with Yokogawa CSU-X1M1 system and CoolSnap HQ2 (Photometrics). Quantifications were performed using ImageJ software (NIH).

Scanning Electron Microscopy analysis (SEM)

HDMECs grown on glass coverslips were infected with meningococci for 1 h and treated with trifluoperazine or thioridazine, as described above, fixed with 4% paraformaldehyde for 10 min, treated with 1% osmium tetroxide in water, in the dark for 1 h and dehydrated with increasing ethanol concentration baths before drying with a critical point drier (Quorum Technologies K850, Elexience, France). Dry coverslips were mounted on stubs, coated with 5 nm platinum (Quorum Technologies Q150T, Elexience, France) and observed with a Zeiss SEM Merlin Compact VP (Zeiss, France).

Transmission electron microscopy

Bacteria were grown in liquid culture medium for 2h, Trifluoperazine (or control vehicle) was added at 40 µM for 20 min, bacterial suspensions were then fixed in 4% paraformaldehyde for 10 min, centrifuged at 1000 rpm for 5 minutes and the bacterial pellets washed in PBS. After negative staining with 1% phosphotungstic acid, bacteria were analysed by transmission electron microscopy, using a JEOL 1011 microscope.

NMICROBIOL-17122366 Revised version p. 18

Stochastic Optical Reconstruction Microscopy (STORM)

Bacteria were grown in liquid culture medium for 2h, Trifluoperazine (or control vehicle) was added at 40 µM for 30 min, then bacterial suspensions were fixed in 4% paraformaldehyde/0.2% glutaraldehyde for 10 min, centrifuged at 850 g for 5 min and the bacterial pellets washed in PBS, permeabilized with PBS/0.05% saponin, washed, blocked with PBS/BSA 2% for 10 min, labeled with the anti-PilE antibody in PBS/BSA 2%/saponin 0.05%-for 1h, washed, then incubated with goat antimouse Alexa Fluor 647 F(ab')2 secondary antibody fragments (Life Technologies, A-21237) (1:500) for 1h. Labeled preparations were centrifuged on microscope slides using a cytospin, post-fixed in PBS/formaldehyde 3,6% for 15 min, washed in PBS then mounted on slide with cavities (Marienfield)

in Abbelight dSTORM super resolution buffer and sealed with a twinsil 22 silicon-glue (Rotec) as previously described [START_REF] Maissa | Strength of Neisseria meningitidis binding to endothelial cells requires highlyordered CD147/beta2-adrenoceptor clusters assembled by alpha-actinin-4[END_REF] . Imaging was performed on a Leica SR GSD 3D system, with a 160x oil (NA 1.43) objective. Fluorescence was collected onto an EMCCD camera iXon Andor technology, providing an effective pixel size of 100 nm and processed with the LAS X software (Leica).

Approximately 50,000 frames were recorded for each acquisition with 20 ms exposure time, EM gain of 250 and the number of photons per pixels set to 50. 2D reconstructions were obtained from stacks (16 planes) with 50 nm axial step size, using LAS X software (Leica).

Infection of human brain tissues

The effect of trifluoperazine on the colonization of human brain vessels, using an ex vivo meningococcal infection model of fresh human frontal brain tissues was performed as described previously [START_REF] Bernard | Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization[END_REF] . In this setting, histological and anatomical characteristics of the brain vessels were conserved. Meningococci incubated with tissue sections established specific tight association with brain vessels, reminiscent of neuropathological findings in patients with meningococcal meningitis and adhesion relying on the expression of type IV pili [START_REF] Bernard | Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization[END_REF] . Fresh human brain sections, provided by control and were then gently washed horizontally 5 times and fixed in PAF 4% for 10 min at RT.

Adherent meningococci were detected by immunofluorescence analysis. Brain sections were incubated with the anti-human CD31/PECAM-1 mouse monoclonal antibody and a rabbit polyclonal serum anti-Nm 2C4.3 strain for 2h in PBS/BSA 0.1%. Alexa-conjugated Phalloidin and DAPI were added to Alexa-conjugated secondary antibodies for 1h. After additional washing, coverslips were mounted in glycergel (Dako). Entire samples were scanned using a Lamina multilabel slide scanner (PerkinElmer) and were further analysed using confocal microscopy (spinning disk Leica, 63x).

Quantification analysis of the images (n=50 per section) was performed using ImageJ software (NIH).

Results are presented as a vascular colonisation index corresponding to the area occupied by the fluorescently labelled bacteria in relation to the human vessel area delineated by the anti-PECAM-1/CD31 staining, from 2 independent sections per condition.

Infection of SCID mice grafted with human skin

Six week old CB17/Icr-Prkdc scid (Severe Combined Immunodeficiency: SCID) female mice were obtained from Janvier Labs (Saint-Berthevin, France). Human skin tissues were obtained from adult patients undergoing plastic surgery in the Service de Chirurgie Plastique et Reconstructive of the Groupe Hospitalier Saint-Joseph (Paris, France). In accordance with the French legislation, patients were informed and did not refuse to participate in the study. Experimental procedures were performed as previously described [START_REF] Join-Lambert | Meningococcal interaction to microvasculature triggers the tissular lesions of purpura fulminans[END_REF] , in accordance with the guidelines of the Institut National de la bacterial challenge, trifluoperazine (40 mg/kg), thioridazine (4 mg/kg) or vehicle as a control were administrated intraperitoneally and mice were sacrificed at 4h after infection. When used alone or in combination with cefotaxime, thioridazine was administrated subcutaneously and cefotaxime (200 mg/kg) was administrated intraperitoneally to avoid precipitation. Cefotaxime was re-administrated subcutaneously at 18h and 42h after bacterial challenge. To assess bacteremia in infected animals, 10 μl of blood was sampled using a heparinized hematocrit glass tube 1h after injection by puncture in the lateral tail vein, or 4h after injection by intracardiac puncture on animal sacrificed by intraperitoneal injection of a lethal dose of ketamine and xylazine. 10 µl of blood was used for bacterial counts performed by plating serial dilutions of blood on GCB agar plates. Results were expressed in colony-forming units (CFU) per ml of blood. The remaining blood was centrifuged (3000 rpm for 20 min at 4°C) and sera were collected and frozen at -80 °C. Human skin grafts were carefully collected in RNA later (Sigma-Aldrich), half of the graft was cut into small pieces using sterile cutter, before lysis in TRI-Reagent for RNA preparation. The rest of the tissue was washed in PBS and fixed overnight at 4 °C in 4% paraformaldehyde in phosphate buffer. After washing in phosphate buffer, the specimens were embedded in OCT and then frozen at -80°C. 7 µm thick sections of the dermis were immobilised on superfrost TM plus microscope slides and proceeded for hematoxylin and eosin staining or immunofluorescence analysis. Sections were incubated with the primary antibodies for 1h in PBS/BSA 3% then DAPI was added to Alexa-conjugated secondary antibodies for 1h. After additional washing, coverslips were mounted in glycergel (Dako), entire samples were scanned using a Lamina multilabel slide scanner (PerkinElmer) and were further analysed using confocal microscopy (spinning disk Leica, 63x). Quantification analysis of the images was performed using ImageJ software (NIH). Results are presented as a vascular colonisation or thrombosis index corresponding to the area occupied by the fluorescently labelled bacteria or platelets in relation to the human vessel area delineated by the anti-collagen IV (n= 30 to 50 vessels per graft from 3 to 7 independent grafts per condition).

Quantitative Real-Time PCR analysis

Tissues or cells were kept at -80°C with TRI-Reagent (Sigma Aldrich). RNA were extracted with the TRI-Reagent phenol/chloroform method; cDNAs were synthesized using Superscript II reversetranscriptase kit (Life Technologies). RT-qPCR were performed using SyberGreen system (Roche Diagnostics) and the endothelial cell marker gene PECAM-1/CD31 was used to normalize the expression of the other genes. Relative changes in mRNA expression were calculated using the comparative cycle method (2 -ΔΔCt ). The list of primers used in this work is provided in supplementary

Multiplex electrochemiluminescent immunoassays

The serum concentrations of human inflammatory cytokines were quantified by electrochemiluminescence using multiplex assay kits from Meso Scale Discovery. 25 µl of serum were added to the 96-well MULTI-SPOT plates and the assays were processed following the manufacturer's instructions. Plates were read on the multiplexing imager Sector S600 (Meso Scale Discovery).

The serum concentrations of cytokines produced might vary between experiments, depending on the skin grafts (different donors, the number of connected vessels contained within the graft) and/or the levels of bacteremia. However, the inhibitory effects of phenothiazines was quite similar among experiments, with a reduced inflammation varying from 50 to 70%.

Statistical analysis

Statistical significance was assessed with the Student's t-test and one way-ANOVA by Prism software.

previously 9 .

 9 DAPI, Trifluoperazine (T8516), Fluphenazine (F4765), Prochlorperazine (P9178), Perphenazine (P6402), Thioridazine (T9025), Mesoridazine (M4068), Chlorpromazine (C8138), Promazine (P6656), Triflupromazine (1686003) and Levomepromazine (L0500000) were purchased from Sigma Aldrich. Phalloidin coupled to Rhodamin or Alexa Fluor 633 were purchased from Interchim and Invitrogen, respectively. Gentamicin was purchased form Gibco and Cefotaxime from Mylan.

  Prof. Fabrice Chretien (Poincaré Hospital and Institut Pasteur, Paris), were obtained from frontal lobe specimens of macroscopically and histologically normal brain (confirmed by a neuropathologist) of individuals referred to the Department of Forensic Medicine for unexplained out-of-hospital sudden death (consent forms ML1094, PFS 10-008, ClinicalTrials.gov NCT00320099 from The Institutional Review Boards of the Poincaré Hospital, Versailles-Saint Quentin University and the French "Agence de la Biomédecine"). After freezing of the brain tissue with isopentane cooled in liquid nitrogen, the sections (7 µm thick) containing leptomeninges, cortical ribbon and the underlying white matter were immobilised on superfrost TM plus microscope slides and stored at -80°C. Defrosted sections were rehydrated in PBS for 5 min and incubated for 1h with medium containing 0.1% BSA prior to infection with suspensions of bacteria (2x10 7 bacteria in 150µl of medium containing 0.1% BSA) for 1h at 37°C. Sections were then treated for 30 min with Trifluoperazine 30 µM or PBS alone as a NMICROBIOL-17122366 Revised version p.19

  Santé et de la Recherche Médicale and were conformed to the European ethical legislation (Directive 2010/63/EU). The experimental protocol was approved by the Animal Experimentation Ethics Committee of the Université Paris Descartes (consent forms CEEA34.O.J.L.039.12 and 2018012515596498). Briefly, mice were prepared for transplantation by shaving the hair of the back and abdominal areas after an intraperitoneal injection of ketamine 100 mg.kg -1 and xylazine 10 mg.kg -1 . A skin flap was created and a full thickness human skin graft was placed onto the wound bed. The transplants were held in place with 6-0 nonabsorbable monofilament suture materials, and the flap was then sutured above the transplant. Four to six weeks after human skin transplantation, grafted mice were randomized into control and treated groups for meningococcal infection experiments. N. meningitidis strains were grown overnight at 37 °C on GCB agar plates prepared without iron and supplemented with 15 μM deferoxamine (Desferal, Novartis) with appropriate antibiotic when required. Bacterial colonies were harvested and cultured in RPMI with 1% bovine serum albumin medium and 0.06 μM deferoxamine with gentle agitation to reach the exponential phase of growth. Bacteria were then resuspended in physiological saline solution. Mice were infected intravenously with 100 µl (5x10 6 bacteria) of this bacterial suspension. Ten mg of human holotransferrin (R&D Systems) was administered intraperitoneally just before infection. 2h after NMICROBIOL-17122366 Revised version p.20

Figure 1 .

 1 Figure 1. Trifluoperazine inhibits bacterial aggregation (a,b) N. meningitidis 2C4.3 was grown in liquid culture for 2h to form bacterial aggregates and was then treated with increasing concentrations of trifluoperazine (10 to 40 µM), gentamicin (150 µg/ml) or cefotaxime (20 µg/ml) for 30 minutes. Bacterial aggregates were visualized using a phase-contrast microscope. Images are representatives of 4 independent experiments performed in triplicate (scale bars = 200 µm) (b) Quantification analysis of the size of bacterial aggregates was performed using ImageJ software. (Mean ± SEM, n=3 experiments, *P <0.05, **P <0.01, ns non-significant ns1 P=0.24, ns2 P=0.30 ; ns3 P=0.14, One-way ANOVA Dunnett multiple comparison test. (c) Nm 2C4.3 wild type strain and its isogenic derivative mutant ΔPilT grown in liquid culture for 2h to form bacterial aggregates were treated with 50 µM trifluoperazine. Time lapse phase-contrast video microscopy was performed to visualize the effect of trifluoperazine on bacterial aggregates over time. Images were taken at the indicated time points of the video (see supplementary video 1) (scale bars = 200 µm). This experiment was repeated a minimum of 10 times with similar results. (d) The effect of trifluoperazine treatment (50 µM) on twitching motility and bacterial aggregation was analysed overtime on Nm 2C4.3 bacterial aggregates using time lapse phase-contrast video microscopy and movement tracking analysis (Imaris software). ~3 min after addition of the drug, twitching was progressively lost, followed by the dispersion of the bacterial aggregates (~6 min after addition of the drug). This experiment was performed twice with similar results.

Figure 2 .

 2 Figure 2. Trifluoperazine and Thioridazine regulate the piliation status of meningococci (a) Nm 2C4.3 wild type and the isogenic ΔPilT mutant grown in liquid culture for 2h to form bacterial aggregates were treated with trifluoperazine (40 µM), thioridazine (4 µM) or PBS (Control) for 20 minutes, then fixed and stained with an anti-PilE antibody to visualize type IV pili and DAPI to visualize bacteria. Images are representatives of 4 independent experiments (scale bars = 20 µm). (b) Following treatment with trifluoperazine 40 µM for 20 minutes, bacteria were fixed and stained for pilin PilE and analysed by an optical super-resolution (~20 nm) microscopy technique based on singlemolecule localization (dSTORM) on a Leica SR GSD. 50,000 frames were recorded for each acquisition, reconstructed using the Leica Application Suite (LAS X) core module and merged with the bright-field images of the same fields. Images are representatives of 3 independent experiments (Scale bars = 2 µm). (c) Nm 2C4.3 wild type and the isogenic ΔNqrA-F mutants were grown in liquid culture for 2h to form bacterial aggregates, then fixed and stained with an anti-PilE antibody to visualize type IV pili and DAPI to visualize bacteria. Images are representatives of 2 independent experiments (scale bars = 20 µm). (d) N. meningitidis 2C4.3 was grown in liquid culture for 2h to form

Figure 3 :

 3 Figure 3: Trifluoperazine and Thioridazine release bacteria from compact microcolonies formed on human endothelial cells (a-c) HDMECs were infected with Nm 2C4.3 wild type and the isogenic ΔPilT mutant for 1h30, treated with trifluoperazine (10-40 µM), thioridazine (1-8 µM), gentamicin (150 µg/ml) or cefotaxime (20 µg/ml) for 30 minutes. (a) Cells were fixed and stained for cellular actin (phalloidin, red) and bacteria (anti-2C4.3, green) and analysed by fluorescent microscopy. Images are representatives of 4 independent experiments performed in triplicate (scale bars = 200 µm) (b-c) Quantification of the bacterial colonization by the wild type (b) and the isogenic ΔPilT mutant (c) was performed on 30 fields using ImageJ software. (b) Mean ± SEM, n=3 to 4 independent experiments, *P <0.05; **P <0.01; ***P<0.001; ns non-significant ns1 P=0.36, ns2 P=0.99 ; ns3 P=0.49, One-way ANOVA Dunnett multiple comparison test. (d) HDMEC were infected with Nm 2C4.3 for 1h30, treated with trifluoperazine (40 µM), thioridazine (4 µM) or PBS (Control) for 30 minutes, fixed, and analysed once by scanning electron microscopy (scale bars = 20 µm). (e) Serial brain sections from the same donorwere infected with Nm 2C4.3 for 1h30 to promote in situ adhesion to human brain vessels. Brain section were then treated with trifluoperazine (30 µM) for 30 min, washed, fixed and stained for the endothelial cell junction marker VE-cadherin (green), bacteria (red) and nuclei (blue), and the whole section was scanned using a Lamina scanner (Perkin Helmer). (f) Bacterial colonisation within the brain sections assessed by immunofluorescence analysis was quantified using ImageJ software as the ratio between bacterial surface colonisation in relation to the lumen surface of the vessels. 50 vessels per brain section were analysed. Mean of n=2 brain sections from 2 different donors.

Figure 4 :

 4 Figure 4: Trifluoperazine and Thioridazine reduces bacteria-induced endothelial cell injury and matrix degradation (a) HDMECs, grown under laminar shear stress (5 dynes/cm 2 ) for 5 days, were infected with Nm 2C4.3 wild type strain for 2h, treated with trifluoperazine (40 µM, 30 min), cefotaxime (20 µg/ml, 2h) or a combination of both. Cells were then fixed and stained for bacteria (red) and VE-cadherin (green), and analysed by confocal microscopy. Images are representatives of 3 independent experiments (scale bars = 50 µm). (b-d) HDMECs grown on fluorescent matrix (gelatin-FITC), were infected for 5h treated with increasing concentration of trifluoperazine (0-50 µM), thioridazine (0-4 µM) and 150 µg/ml gentamycin to prevent bacterial overload and subsequent cell death, due to

Figure 5 :

 5 Figure 5: Trifluoperazine and Thioridazine induces bacterial clearance from human blood vessels in vivo and reduces signs of thrombosis, vascular injury and inflammation. (a-f) Mice grafted with human skins were infected intravenously with Nm 2C4.3 wild type strain (5x10 6 bacteria). 2h after bacterial challenge, mice received 40 mg/kg trifluoperazine, 4 mg/kg thioridazine intraperitoneally or vehicle as a control. Mice were sacrificed at 4h post-challenge. This experiment was performed twice with n=3 or 4 mice per group, using skin from two different donors (n= indicate the number of mice per group). (a) Schematic representation of the different protocols used in vivo. (b) Control bacteremia 2h post-infection. Mean ± SEM. (c) Shown are representative images of the histological and immunofluorescence analysis of the skin grafts isolated from mice either non-infected or infected and treated with 40 mg/kg trifluoperazine, 4 mg/kg thioridazine at 2h post-challenge, or vehicle as a control. Top panel: Hematoxylin and eosin (HE) staining of the skin grafts, presenting signs of platelet and red blood cell aggregation in infected vessels. Middle panel: immunofluorescence analysis of the skin grafts showing the human vessels (Collagen IV, red) bacteria (green), platelet aggregation (pink) and nuclei (DAPI, blue). Lower panels: vascular integrity was assessed by immunofluorescence using markers of the endothelial cell junctions (PECAM-1 or VE-Cadherin, green) and bacteria (red). (d) Bacterial colonisation within the skin grafts assessed by immunofluorescence analysis was quantified using ImageJ software as the ratio between bacterial surface colonisation in relation to the lumen surface of the vessels. 30 to 50 vessels per mouse were analysed. Mean ± SEM, ***P<0.001; Two-tailed Student's t-test. (e) Thrombus formation within the skin grafts was assessed by immunofluorescence analysis and quantified using ImageJ as the ratio between thrombus surfaces in relation to the lumen surface of the vessels. 30 to 50 vessels per mouse were analysed. Mean ± SEM, *P<0.05, **P<0.01; Two-tailed Student's t-test. (f) Production of human cytokines (TNFα, IL-6, IL-8) in the mice sera was quantified with multiplex electrochemiluminescent immunoassays. 1 point correspond to one mice. Mean ± SEM from 2 independent experiments, *P <0.05, **P <0.01, ***P <0.001, ns non-significant, ns non-significant ns1 P=0.36, ns2 P=0.38 ; ns3 P=0.11, One-way ANOVA Tukey's multiple comparison test.

Figure 6 :

 6 Figure 6: Thioridazine improves the outcome of meningococcal infection alone or in combination with antibiotics.

(

  Figure 1

Table 4 .

 4

NMICROBIOL-17122366 Revised version p.

21

Acknowledgments

We thank Mylène Robert-Genthon and Fabrice Chretien for providing P. aeruginosa PAO1 strain and brain tissues; Maryline Favier of the histology facility, Alain Schmitt and Thomas Guilbert of the imaging facility, Karine Bailly and Muriel Andrieu of the cytometry facility of the Institut Cochin for their expert technical help. K.D. and L.LG. were supported by a doctoral fellowship from la Region Ile de France and the Fondation pour la Recherche Médicale, respectively. This work was supported by a collaborative research grants from the Agence Nationale de la Recherche of France (ANR-14-IFEC14-0006) to S.B. and X.N. in the framework of the Infect-ERA joint transnational call (European funding for infectious diseases research), by the Société d'Accélération du Transfert de Technologie (ANR-10-SATT-05-01) to S.B. and by grant ANR 10-EQPX-04-01 to F.L.

Data availability

Data supporting the findings of this study are available within the paper and its supplementary information files. The sequences of the SNP variants resistant to the action of phenothiazines are available at NCBI's BioProject database under accession number PRJNA481885 (http://www.ncbi.nlm.nih.gov/bioproject/481885).

NMICROBIOL-17122366 Revised version p. [START_REF] Jeworrek | Effects of specific versus nonspecific ionic interactions on the structure and lateral organization of lipopolysaccharides[END_REF]

Additional information

The authors declare no competing financial interests. Log CFU/mL


Lambert Stéphane

Agathe Furet

Julien Lorentz

Jean Philippe Jarrin

Pascal Villard

ROCKFALL PROTECTION WALLS MADE OF INTERCONNECTED CONCRETE BLOCKS: SMALL-SCALE EXPERIMENTS AND NUMERICAL MODELING

Keywords: protection structure, experiments, small-scale, dissipation

This article investigates the impact response of a recently developed type of rockfall protection structure, with reduced footprint and consisting of interconnected concrete blocks. This investigation is based on small-scale experiments and numerical simulations. The experiments concerned structures with different designs and exposed to impact under various conditions. Mechanisms associated with the structure response are addressed in terms of displacement and energy dissipation. General trends are derived in order to propose an optimized design.

INTRODUCTION

Passive protection structures forming a nearly vertical obstacle to the propagation of rockfall, such as embankments or flexible barriers, generally confront designers with footprint constraints associated with the space required for the structure itself augmented by the space required for its normal operation. This footprint constraint is particularly strong when dealing with transportation corridors at the toe of steep slopes or in narrow valleys [START_REF] Lambert | An experimental investigation of the response of slender protective structures to rockfall impacts[END_REF]. This study deals with a specific type of rockfall protection structure with reduced footprint and consisting in interconnected concrete blocks forming a wall, typically 1-m in thickness (see Fig. 1, left). In order to develop this technological solution, improve this structure type and propose design recommendations, a study involving small-scale experiments, real-scale experiments and numerical modelling has been initiated. This article focuses on the small-scale experiments conducted in the lab with the aim of investigating the influence of the design configurations, on the main mechanisms governing the structure response.

EXPERIMENTS

The experiments were conducted in the lab on a structure at a 1/4 scale. The structure design is slightly simplified compared to the real-scale one but considers its main features. The wall consisted of 4 horizontal rows of 9 or 10 concrete blocks, each being 33kg in mass, 0.38m in length and 0.20m in width and height (Fig. 2). Iron rods and plates interconnected contiguous blocks in the vertical and horizontal directions respectively, in a similar manner as in the realscale structure. As shown in Fig. 2 (left) the mechanical assembly accounts for a certain slack, resulting from the space between adjoining concrete blocks, as well as the space around the rods and the space between the plates and the rods.

The experiments consisted in impacts in the middle of the wall and at the various heights from the soil, by a 35-kg in mass projectile with a spherical head and travelling horizontally at a velocity up to 7.7 m/s at impact (max. kinetic energy of 1030 J). High-speed cameras allowed tracking the displacement with time of different points of the structure. The final geometry of the structure was determined thanks to photogrammetry.

The first configuration tested concerned a simple wall. The three following configurations also included two partition walls, placed perpendicular to the wall on the side opposite to the impact. These configurations differed one from each other by the distance between the partition walls (1 and 1.6 m) and the size of the partition wall (either made of 1.5 and 3 concrete blocks).

NUMERICAL MODEL

A 3d numerical model of the small-scale structure has been developed with FLAC3D [START_REF]FLAC3D -Fast Lagrangian Analysis of Continua in 3-Dimensions, Ver. 6.0 User's Manual. Itasca[END_REF] in the aim of providing a better understanding of the physical phenomena involved, in particular by addressing the energy dissipation issue [START_REF] Furet | Simulation of impacts on a rockfall protection wall made of interconnected concrete blocks[END_REF] (see Fig. 1, right). The objective with this model is to reproduce the global structure behavior in terms of displacement. Consequently, the constitutive model for the concrete blocks was kept simple. For instance, the model accounted for energy dissipation by plastic strain but not for blocks breakage. No additional numerical damping was considered in the simulations.

Data from the literature were complemented with results from specific static tests to calibrate the various parameters related to the concrete blocks, steel rods and plates, as well as those related to the soil/block and block/block interfaces. Specific experiments also included impact tests on single blocks. The model was then validated against the results from the impact experiments, to compute the contribution of the different energy dissipative mechanisms occurring during impact.

RESULTS

Impacts on the walls led to a localized deformation of the wall, exhibiting a post-impact V shape. As illustrated in Fig. 4 (a), this deformation was associated with a significant increase in spacing between blocks on the structure back face as well as with the rupture of concrete blocks in the impact direction, in a very few cases. In these later cases, the impact resulted in the ejection of some small pieces of concrete. This phenomenon can be avoided by adding fibers in the concrete, as done for the real-scale structures. It is worth highlighting that none of the experimental conditions led to the structure fall over. The length of the wall displaced during the impact (length of the V shape) appeared variable depending on the impact conditions and structure configuration.

The films revealed that during the impact, the wall deformation in the impact axis could be described according to three main mechanisms: (i) wall sliding at its base, (ii) translation along the horizontal axis and in the impact direction of the block with respect to the underlying ones, and (iii) tilting of the wall. This latter mechanism is reversible, contrary to the other ones. It is associated to a temporary lift up of the structure on the impact side. The amplitude of each of these mechanisms was found to be strongly variable depending on the impact conditions (kinetic energy and impact height) as well as on the structure configuration. As expected, both the sliding and the deformation along the vertical axis increase with the impact energy (Tab. 1). Also, the deformation along the vertical axis significantly increases when the impact height increases. As for the wall configuration, the experiments revealed that partition walls tended to limit the basal sliding but to favor tilting.

The numerical model revealed efficient in mimicking the structure deformation. Combined with experimental data, it allowed computing the energy dissipated by friction at the base and plastic strain induced in the impacted block. These two mechanisms were found to be responsible for the dissipation of at least 40% of the impact energy, depending on the impact conditions and structure configuration. Nevertheless, due to experimental bias and modeling simplification it was not possible to compute precisely all the dissipative mechanisms.

CONCLUSION

In order to investigate the impact response of walls consisting of interconnected concrete blocks, small-scale experiments were conducted varying the wall designs and impact conditions. In parallel, a numerical model of the wall was developed. The experiments allowed drawing some conclusions regarding the prevailing deformation mechanisms. The beneficial effect of partition walls on the structure response has been evidenced. In parallel, the proposed modeling approach revealed appropriate for modeling the impact response of these articulated and complex structures. In the end, the experiments suggests some general directions for improving the design of these rockfall protection walls. Nevertheless, extrapolation of these results to real-scale structures is not possible due to similitude issues. For this reason, reals-scale experiments and numerical simulations will be conducted in view of deriving design guidelines.

Fig. 1

 1 Fig. 1 The Bloc Armé® structure consists of a wall made of concrete blocks, interconnected via vertical bars and horizontal plates: design drawing (left) and numerical model (right).

Fig. 2

 2 Fig. 2 Building of the wall, showing the blocks, rods and plates (left). Simple wall about to be impacted (right).

Fig. 3

 3 Fig. 3 Example of a wall equipped with large partition walls spaced 1.6 m: views from the side (left) and from the top (right). Dimensions in cm.

Fig. 4

 4 Fig. 4 Wall after impact: experiments (a) and modelling (b). Simple wall case.

Univ. Grenoble Alpes, INRAE,

rue de la papeterie,

Saint Marin d'Hères, France, stephane.lambert@inrae.fr 2 Géolithe, 181 Rue des Bécasses, 38920 Crolles, France, agathe.furet@geolithe.com[START_REF] Furet | Simulation of impacts on a rockfall protection wall made of interconnected concrete blocks[END_REF] Géolithe Innov, 181 Rue des Bécasses, 38920 Crolles, France, julien.lorentz@geolithe.com 4 Géolithe, 181 Rue des Bécasses, 38920 Crolles, France, jean-philippe.jarrin@geolithe.com 5 Univ. Grenoble Alpes, CNRS, Grenoble INP, 3SR, 38000 Grenoble, France, pascal.villard@univ-grenoblealpes.fr


Daniel Jacob

François Ehrenmann

Philippe Chaumeil

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The Maggot tool allows users to describe the Metadata of their datasets produced within a collective (research unit, platform, multi-partner project, etc.), thus making it possible • to answer certain questions of the Data Management Plan (DMP) concerning the organization, documentation, storage and sharing of data in the data storage space, • to meet certain data and metadata requirements, referred to by the Open Research Europe (ORE) in accordance with the FAIR principles.

Designed to foster good data management, with « data sharing » in mind.

Daniel Jacob (1) , François Ehrenmann (2) , Philippe Chaumeil (2) (1

) INRAE UMR Biologie du Fruit & Pathologie (2) INRAE UMR BioGECO
The use of dictionaries has no other purpose to facilitate the entry of metadata, entry which can be long and repetitive in generalist data warehouses (such as repository based on Dataverse).

Dictionaries allow you to record multiple information necessary to define an entity, such as the names of people or even the funders. its information, once entered and saved in a file called a dictionary, can be subsequently associated with the corresponding entity. Metadata thus produced can be associated with corresponding data directly in the storage space which is then scanned and then referenced in a database. We can thus carry out a search on the metadata in order to find one or more sets of data. Only metadata is accessible by default.

Use of dictionaries to target the CV by mixing thesaurus and ontologies

SKOSMOS Thesaurus

©

  Copyright 2022-2023 Contact : daniel.jacob@inrae.fr Source : github.com/inrae/pgd-mmdt/ Documentation : https://inrae.github.io/pgd-mmdt/ List of well-chosen and limited CVs according to a reference (e.g. Data Document Initiative) List of ontologies to choose according to your domain Dictionary of people Built the web interface according to the specifications

  https://pmb-bordeaux.fr/maggot/