Pascale Cossart

Alice Lebreton

A trip in the "New Microbiology" with the bacterial pathogen Listeria monocytogenes

Keywords: Listeria monocytogenes, non-coding RNA, cellular microbiology, endocytosis, chromatin

Listeria monocytogenes is a food-borne pathogen causing an opportunistic disease called listeriosis. This bacterium invades and replicates in most cell types, due to its multiple strategies to exploit host molecular mechanisms. Research aiming at unravelling Listeria invasion and intracellular lifestyle has led to a number of key discoveries in infection biology and cell biology. In this review, we report on our most recent advances in understanding the intimate crosstalk between the bacterium and its host, resulting from in-depth studies performed over the past five years. We specifically highlight new concepts in RNA-based regulation in bacteria and discuss important findings in cell biology, including a new role for clathrin and an atypical mitochondrial fragmentation mechanism. We also illustrate the notion that bacterial infection regulates host gene expression at the chromatin level, contributing to an emerging field called patho-epigenetics. This review corresponds to the lecture given by one of us (P.C.

Introduction

The causative agent of listeriosis, Listeria monocytogenes, has led to a number of discoveries in various biological fields. It was identified by E.G.D. Murray in 1924, as a gram-positive bacillus responsible for epidemic cases of mononucleosis in laboratory animals, and named Bacterium monocytogenes [START_REF] Murray | A disease of rabbits characterised by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n.sp.)[END_REF]. It was later on shown to cause sporadic cases of meningitis in humans [START_REF] Burn | Characteristics of a New Species of the Genus Listerella Obtained from Human Sources[END_REF], before being recognized as an opportunistic, food-borne pathogen of human, cattle and wild animals [reviewed in 3]. L. monocytogenes can live freely in the environment, but infects also a wide range of animal hosts including arthropods as well as cold and warm-blooded vertebrates. In mammalian hosts, this bacterium can cross the intestinal, foeto-placental and blood brain barriers, allowing its dissemination throughout the organism (Fig. 1A). Even though listeriosis is not a major health concern for immunocompetent individuals, it can constitute a life-threatening disease in the elderly and in immunocompromised patients; it also has serious outcomes in pregnant women, causing still-birth or frequently lethal neonatal infections [reviewed in 4].

L. monocytogenes has been broadly used by immunologists for its ability to induce a cellmediated immune response, while antibodies play no role in recovery from infection [START_REF] Lane | Requirement of thymus (T) lymphocytes for resistance to listeriosis[END_REF][START_REF] Mackaness | Cellular resistance to infection[END_REF]. It has also become a tool for anti-tumour immunotherapies, and is in pre-clinical trials as a live-attenuated vaccine for cancer treatment [START_REF] Rothman | Live-attenuated Listeriabased immunotherapy[END_REF]. Listeria atypical immunological properties derive in part from its ability to replicate intracellularly [reviewed in 8]. Indeed, this facultative intracellular bacterium can enter and multiply in the cytosol of most human cell types and spread to neighbouring cells, using an arsenal of virulence factors that target diverse cellular components and subsequently hijack various host cell functions (Fig. 1B). For the past 30 years, unravelling the intracellular lifestyle of L. monocytogenes has contributed to a significant number of advances, not only in microbiology and cellular microbiology, by identifying virulence factors and characterizing their functions, but also in cell biology, by elucidating cellular pathways that had remained elusive and giving rise to the discovery of novel molecular mechanisms [reviewed in 9].

The major bacterial effectors responsible for bacterial entry into host cells, escape from the phagocytic vacuole, intracellular motility, as well as their cellular partners, are now well characterized [reviewed in [START_REF] Cossart | Illuminating the landscape of host-pathogen interactions with the bacterium Listeria monocytogenes[END_REF][START_REF] Stavru | Cell biology and immunology of Listeria monocytogenes infections: novel insights[END_REF]. Nonetheless, the past five years of research have brought additional knowledge about the multiple strategies used by Listeria to subvert its host functions and programmes. We review here our most recent achievements in the understanding of the hostbacterial crosstalk; we are not aiming at being exhaustive, but instead wish to place significant advances into a broader perspective, including that of work by others. We report how highresolution transcriptomics of the bacterium allowed the identification of unconventional mechanisms of bacterial gene expression; we discuss how bacterial-induced phenotypes enabled us to reconsider our appreciation of a number of central cellular functions; last, we highlight progress in deciphering the host response to infection and its manipulation by Listeria, in particular at the onset of innate immune defences at the chromatin level. As ever, the use of L. monocytogenes as a model proves an outstanding tool to generate and establish novel concepts in infection biology, in microbiology and in cell biology.

Discovery of unconventional mechanisms regulating bacterial gene expression.

The bacterial transcriptional landscape examined at high resolution.

Upon infection, the onset of the host-bacterium dialogue translates into a drastic remodelling of both prokaryotic and eukaryotic gene expression programmes. Initial transcriptome studies had focused on Listeria grown in vitro [START_REF] Milohanic | Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA[END_REF][START_REF] Severino | Comparative transcriptome analysis of Listeria monocytogenes strains of the two major lineages reveals differences in virulence, cell wall, and stress response[END_REF]. Later on, high-resolution transcriptomics of bacteria grown in in vitro, ex vivo and in vivo, using tiling microarrays, have provided us with a detailed

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Trends in Cell Biology 2014 4 insight into the reshaping of the bacterial transcriptional landscape upon switch from saprophytism to virulence [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]. This study has allowed a comprehensive re-annotation of the genome of the L. monocytogenes strain EGD-e [START_REF] Glaser | Comparative genomics of Listeria species[END_REF] and the establishment of the first genome-wide operon map. Novel transcriptional units have been defined, in particular antisense RNAs (asRNA), some of which spanning several open reading frames (ORF). 29 novel non-coding RNAs (ncRNA) of less than 500 nucleotides have also been uncovered, adding up to the 21 previously known small RNAs (sRNA). Remarkably, cross-species comparison has revealed that several sRNAs are absent in the non-pathogenic species Listeria innocua and display similar expression features as protein-coding virulence genes. Moreover, deletion of some of these sRNA genes impaired virulence in a mouse listeriosis model [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF][START_REF] Mraheil | The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages[END_REF], suggesting that sRNAs can participate in the control of virulence. Last, analysis of the transcriptomes of deletion mutants of two major regulators of Listeria virulence genes, PrfA and σ B [START_REF] Kazmierczak | Contributions of Listeria monocytogenes sigmaB and PrfA to expression of virulence and stress response genes during extraand intracellular growth[END_REF], has highlighted that σ B plays a key role in switching on the expression of virulence genes in the intestinal lumen, while PrfA triggers the expression of effectors required for intracellular lifestyle when the bacteria are grown in blood [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF].

This pioneering work in Listeria transcriptomics using tiling arrays was followed by a number of others, which, by analyzing a variety of growth conditions and techniques, contributed to drawing an accurate picture of the bacterial transcriptional landscape and its fine-tuning at different stages of the infection process. For instance, the transcriptional profiling of L. monocytogenes recovered from the spleen of infected mice provided important information regarding the metabolic status of the bacterium in vivo, the regulatory networks at play in virulence, and allowed the identification of novel candidate virulence genes [START_REF] Camejo | In vivo transcriptional profiling of Listeria monocytogenes and mutagenesis identify new virulence factors involved in infection[END_REF].

An additional degree of resolution in Listeria transcriptomes was reached with the use of RNA-Seq, which allowed the mapping of transcription start sites in both L. monocytogenes and L. innocua [START_REF] Wurtzel | Comparative transcriptomics of pathogenic and non-pathogenic Listeria species[END_REF]. This analysis, performed on bacteria grown in several different growth conditions, has enabled the annotation of additional sRNAs and asRNAs, in addition to all 5'-UTRs. Public access and navigation through these results are facilitated by the Listeria browser (http://www.weizmann.ac.il/molgen/Sorek/listeria_browser/). Thanks to an increasing number of high-resolution transcriptomic studies [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF][START_REF] Mraheil | The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages[END_REF][START_REF] Wurtzel | Comparative transcriptomics of pathogenic and non-pathogenic Listeria species[END_REF][START_REF] Behrens | Ultra Deep Sequencing of Listeria monocytogenes sRNA Transcriptome Revealed New Antisense RNAs[END_REF][START_REF] Oliver | Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs[END_REF], referenced Listeria ncRNAs now include 154 trans-acting sRNAs, 46 cis-regulatory RNAs and 104 asRNAs [START_REF] Bécavin | Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Variations Underlying Differences in Pathogenicity[END_REF]. These ncRNAs constitute a pool of putative regulatory mechanisms for bacterial gene expression, most of which remain to be characterized. Detailed study of some of these transcripts has revealed an amazing versatility in their regulatory properties, which we will illustrate below.

Novel riboswitch-mediated regulations.

Among the new ncRNA elements discovered in the above-described studies, some had been previously annotated in databases as putative riboswitches. Riboswitches are cis-regulatory RNA elements, which act most often as sensors of metabolites, but can also bind a variety of specific ligands including metal ions or cyclic-di-GMP. Upon ligand binding, a conformational change in the structure of the riboswitch leads to premature transcription termination, translation arrest, or both. Interestingly, the L. monocytogenes LysRS riboswitch has been shown to act not only as a lysine-dependent regulator of the transcription of the downstream gene, but also as a transcription terminator for the upstream gene [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF].

Another novel type of riboswitch is the previously named sRNA Rli39 (Fig. 2A). Instead of being classically located in a 5'-untranslated region (UTR), this vitamin B 12 -dependent riboswitch is located at the 3'-end of a gene, and in antisense orientation to the downstream gene pocR. In Salmonella, PocR is a transcriptional regulator for genes of the propanediol utilisation pathway -a process requiring B 12 as a cofactor. PocR also positively regulates its own expression in the presence of propanediol. In-depth analysis of this locus in Listeria has revealed that the B 12 -

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dependent riboswitch controls the expression of an asRNA to pocR, aspocR [START_REF] Mellin | A riboswitch-regulated antisense RNA in Listeria monocytogenes[END_REF]. In absence of B 12 , the riboswitch adopts an anti-terminator structure, which allows transcription of aspocR and thus blocks that of pocR. Conversely, the transcription of aspocR terminates in presence of B 12 . Altogether, this elegant system allows the transcription of pocR -and thereby that of PocRregulated genes -only when both propanediol, the substrate of the pathway, and vitamin B 12, the cofactor of propanediol catabolic enzymes, are present in the growth medium.

While riboswitches usually directly regulate their targets in cis, the role of the short transcripts terminated upon ligand binding to the riboswitch had remained unexplored; their study has revealed that one such transcript acts in trans in the regulation of prfA [START_REF] Loh | A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes[END_REF]. Transcription termination by a Sadenosylmethionine (SAM) -dependent riboswitch can generate a small RNA, SreA, as a function of the concentration in methionine (Fig. 2B). Independently of SAM binding, base-pairing of SreA to the 5'-UTR of the prfA transcript blocks its translation. Conversely, PrfA controls the transcription of SreA, thus creating a negative feedback loop on prfA expression. This transregulation of prfA expression by SreA adds to the previously described cis-acting thermosensor [START_REF] Johansson | An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes[END_REF], which unmasks the ribosome binding site (RBS) at 37°C.

Based on these two studies and on bioinformatics predictions, we anticipate that many other ncRNAs may be regulated by riboswitch-dependent mechanisms; the extent of this new level of control in bacterial regulatory networks remains to be assessed.

The "excludon" concept.

As early as the release of the first high-resolution transcriptome of L. monocytogenes, a class of very long asRNAs (lasRNA) had been highlighted, which displayed a dual function as both an asRNA and an mRNA [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]. The participation of these long asRNAs (lasRNA) in a novel type of regulation has been confirmed and extended, giving rise to the concept of the excludon (Fig. 2C) [START_REF] Wurtzel | Comparative transcriptomics of pathogenic and non-pathogenic Listeria species[END_REF][START_REF] Sesto | The excludon: a new concept in bacterial antisense RNAmediated gene regulation[END_REF]. Excludon loci harbour a peculiar topology, where two transcriptional units encoding proteins with mutually exclusive functions display a divergent orientation. In this context, the very long 5'-UTR of one gene can span over the divergent transcriptional unit (sometimes, covering several ORFs of an operon) and inhibit its expression, while its coding part is potentially translated. It is highly possible that the excludon concept holds true not only in bacteria, but also in other organisms.

A PNPase-dependent CRISPR system in Listeria.

Even though RNA-mediated regulation of gene expression is an expanding field, not all small RNAs have a function as transcriptional regulators. For instance, clustered regularly interspaced short palindromic repeats (CRISPR) have been described to constitute a prokaryotic adaptive immunity against exogenous nucleic acids. An additional role of these sRNAs in the virulence of pathogenic bacteria is emerging [reviewed in [START_REF] Hatoum-Aslan | Impact of CRISPR immunity on the emergence and virulence of bacterial pathogens[END_REF][START_REF] Louwen | The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria[END_REF]. CRISPR processing and function depend on the nuclease or helicase activities of proteins encoded by CRISPR-associated (cas) genes. In addition to classical type I and II CRISPR-cas systems, all L. monocytogenes strains harbour a RliB-CRISPR, which has previously been shown to play a role in virulence [START_REF] Toledo-Arana | The Listeria transcriptional landscape from saprophytism to virulence[END_REF]. Unlike other CRISPRs, RliB adopts a non-canonical secondary structure and is devoid of cas genes [START_REF] Sesto | A PNPase dependent CRISPR System in Listeria[END_REF]; however, part of the processing of RliB-CRISPR to its mature form depends on Listeria polynucleotide phosphorylase (PNPase). RliB is functional for defence foreign DNA, in a PnpAdependent manner, provided cas genes from a canonical type I CRISPR-cas system are present elsewhere in the genome. The PnpA-dependent regulation of the activity of RliB-CRISPR points to a more versatile mode of action of CRISPR than classically illustrated.

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Trends in Cell Biology 2014

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Cell biology processes explored in light of Listeria infection.

Most initial studies of L. monocytogenes infectious process have focused on host cytoskeleton rearrangements occurring during entry, intra-and inter-cellular movements [START_REF] Pizarro-Cerda | Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells[END_REF][START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF]. During entry, which occurs by a zipper mechanism, activation of the cell surface receptors E-cadherin and Met by bacterial proteins, InlA and InlB respectively, triggers cell-signalling cascades, which lead to the recruitment of components of the host cell cytoskeleton at the bacterial entry site [reviewed in [START_REF] Cossart | Endocytosis of viruses and bacteria[END_REF][START_REF] Pizarro-Cerda | Entry of Listeria monocytogenes in mammalian epithelial cells: an updated view[END_REF] (Fig. 3A,B). This promotes engulfment of the bacterium into membrane extensions, closing of the phagocytic cup and formation of an internalisation vacuole. After escape from the vacuole, Listeria is able to navigate through the host cytosol, via the recruitment of the actin-nucleating complex Arp2/3 by the surface-exposed bacterial protein ActA [reviewed in 33]. By doing so, ActA stimulates the polymerisation of a branched actin filament network at the rear end of the bacterium. These filaments generate so-called actin comet tails, which propel the bacterium across the cytoplasm and eventually as protrusions into neighbouring cells.

New advances have been recently made in understanding how the host cytoskeleton is subverted by Listeria. As in past studies, analysis of these nano-filaments not only provides insights into the intracellular bacterial lifestyle, but also improves our understanding of the mechanistic role of several cytoskeletal components.

Clathrin function reconsidered.

It has long been thought that clathrin was only involved in the endocytic uptake of small cargo, such as nutrients and signalling molecules [START_REF] Conner | Regulated portals of entry into the cell[END_REF]. The heavy and light clathrin chains can assemble into triskelions and form a polyhedral lattice around budding membrane vesicles [START_REF] Fotin | Molecular model for a complete clathrin lattice from electron cryomicroscopy[END_REF]. This dogma was reconsidered when it appeared that clathrin was also involved in the entry of L. monocytogenes, other bacteria and large viruses using a zipper mechanism [START_REF] Ehrlich | Endocytosis by random initiation and stabilization of clathrin-coated pits[END_REF][START_REF] Veiga | Listeria hijacks the clathrindependent endocytic machinery to invade mammalian cells[END_REF][START_REF] Veiga | The role of clathrin-dependent endocytosis in bacterial internalization[END_REF]. The clathrin-mediated endocytosis machinery can therefore also participate in the internalization of large particles, where it acts as an actin network organizer [reviewed in 39]. Clathrin is involved not only in endocytic pathways, but also in pedestal formation by enteropathogenic E. coli (EPEC), a hallmark of E. coli colonisation of the intestine [START_REF] Veiga | Invasive and adherent bacterial pathogens co-Opt host clathrin for infection[END_REF].

The hierarchical recruitment of clathrin adaptor proteins and actin their was then thoroughly explored, during the entry of L. monocytogenes into host cells, or during pedestal formation [START_REF] Bonazzi | Clathrin phosphorylation is required for actin recruitment at sites of bacterial adhesion and internalization[END_REF]. In both cases, the adaptor Dab2 is required for the formation of clathrin-coated pits, and the light chain of clathrin recruits the actin-binding factor Hip1R. Bacterial adhesion induces the tyrosine phosphorylation of clathrin heavy chain, a key event in the internalisation process. In parallel, the recruitment of myosin VI by Dab2, and its progression along actin filaments, provides a pulling force for Listeria internalization (Fig. 3A).

The function of clathrin as a hub promoting actin cytoskeletal rearrangements has been further extended to other events in cell biology; indeed, the same machinery is involved in the formation of adherens junctions between epithelial cells [START_REF] Bonazzi | A common clathrin-mediated machinery co-ordinates cell-cell adhesion and bacterial internalization[END_REF]. Altogether, these findings have led to a paradigm shift: clathrin should no longer be considered as solely involved in endocytosis.

Control of Listeria entry and actin dynamics by a lipid phosphatase.

During endocytosis of bacterial pathogens, phosphoinositides play a crucial role in local signalling events at membranes by recruiting cellular effectors involved in entry and activating signalling cascades that control actin dynamics [reviewed in 43]. During InlB-dependent invasion, phosphorylation of phosphatidyl-inositol bisphosphate (PI 4,5 P 2 ) to trisphosphate (PI 3,4,5 P 3 ) by the phosphatidylinositol 3-kinase (PI3K) p85/p110 promotes Listeria internalisation, through the activation of Rac1 and WASP family members, and stimulation of actin polymerisation [START_REF] Bierne | WASP-related proteins, Abi1 and Ena/VASP are required for Listeria invasion induced by the Met receptor[END_REF][START_REF] Ireton | A role for phosphoinositide 3kinase in bacterial invasion[END_REF][START_REF] Seveau | A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway[END_REF]

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(Fig. 3A). A novel player in this pathway, the phosphatidylinositol phosphatase OCRL, has recently been shown to have an opposite effect, consistent with its biochemical activity, which reversed PI3K action. When recruited at the site of bacterial adhesion (Fig. 3B), OCRL restricts bacterial entry by dephosphorylating PI 3,4,5 P 3 , thereby inducing actin depolymerisation downstream of Met [START_REF] Kühbacher | Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection[END_REF].

Septins: New partners of actin filaments and link with autophagy.

Among other components of the cytoskeleton, septins, a family of GTP-binding proteins which can assemble into heteropolymeric filaments, had been found associated with the Listeria phagosome [START_REF] Pizarro-Cerda | Distinct protein patterns associated with Listeria monocytogenes InlA-or InlBphagosomes[END_REF]. SEPT9, SEPT2 and SEPT11 localize at the site of Listeria as well as Shigella entry [START_REF] Mostowy | Septins regulate bacterial entry into host cells[END_REF] (Fig. 3A). Surprisingly, SEPT2 is required for bacterial invasion, whereas SEPT11 restricts it, suggesting that different septins may play opposite roles in the tuning of actin-based molecular events [START_REF] Mostowy | Septins regulate bacterial entry into host cells[END_REF][START_REF] Mostowy | Septin 11 restricts InlB-mediated invasion by Listeria[END_REF]. Mechanistically, septins influence the availability of Met at the cell surface and modulate the interaction of InlB with its receptor Met, probably by anchoring Met to the actin cytoskeleton [START_REF] Mostowy | A role for septins in the interaction between the Listeria monocytogenes invasion protein InlB and the Met receptor[END_REF].

More generally, septin recruitment seems to be a general pattern wherever actin polymerisation is taking place [START_REF] Mostowy | Cytoskeleton rearrangements during Listeria infection: clathrin and septins as new players in the game[END_REF]. For instance, septin rings assemble around Listeria and Shigella actin comet tails [START_REF] Mostowy | Entrapment of intracytosolic bacteria by septin cage-like structures[END_REF]. Quite strikingly, septins can also entrap Shigella into highly stable cage-like structures. This process is dependent on N-WASP recruitment by bacterial surface protein IcsA and actin polymerisation. Encaged Shigella are not motile, and are therefore also restricted in their ability to spread from cell to cell. Septin cage formation and autophagy appear as inter-dependent processes [START_REF] Mostowy | Entrapment of intracytosolic bacteria by septin cage-like structures[END_REF][START_REF] Ogawa | Escape of intracellular Shigella from autophagy[END_REF]. Caging has not been observed for Listeria; interestingly, Listeria has been shown to escape autophagy thanks to the activity of its surface protein ActA [START_REF] Yoshikawa | Listeria monocytogenes ActAmediated escape from autophagic recognition[END_REF], which has led to the hypothesis that ActA allows the bacterium to evade both septin caging and autophagy. One should note that Listeria has also evolved a distinct, redundant mechanism to escape autophagy, via the surfaceexposed protein InlK. This member of the internalin family disguises intracytosolic bacteria from autophagic recognition by interacting with the host major vault protein (MVP) [START_REF] Dortet | Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy[END_REF].

Together with a number of studies in other systems, the above-reported studies contributed to highlight septins as scaffolds for protein recruitment in numerous biological processes, including host-microorganism interactions. Septins also constitute diffusion barriers for subcellular compartmentalization. Due to their central functions, septins are increasingly considered as the fourth component of the cytoskeleton [reviewed in 57].

Three-dimensional architecture of actin comet tails.

The two-dimensional structure of actin comet tails has been described very early on, both by optical and electron microscopy [START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF][START_REF] Mounier | Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2[END_REF], and thorough insight into comet tail composition has been provided by proteomics [START_REF] Van Troys | The actin propulsive machinery: the proteome of Listeria monocytogenes tails[END_REF]; however, the three-dimensional organization of these structures remained elusive. The use of cryo-electron tomography has recently revealed the 3D-structure of these tails at the resolution of individual filaments [START_REF] Jasnin | Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails[END_REF] (Fig. 3C). In comet tails as well as in other cellular actin-based assemblies (stress fibres and filopodia), actin is bundled into parallel, hexagonally-packed filaments interspaced by regular 12-13 nm spacing. Together with a report proposing that tangential actin filaments act as primers for explosive actin-network growth [START_REF] Achard | A "primer"-based mechanism underlies branched actin filament network formation and motility[END_REF], this work has contributed to an explanatory model for the initiation of comet-tail assembly and ensuing actin-based motility.

Mitochondrial dynamics: A novel fission mechanism.

In addition to exploring the structure and dynamics of the cell cytoskeleton, research on L.

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Trends in Cell Biology 2014 8 monocytogenes has contributed to deepening our knowledge of several other central cellular processes. For instance, it has led to the identification of an atypical mechanism of fragmentation of the mitochondrial network, which is induced upon Listeria infection [START_REF] Stavru | Listeria monocytogenes transiently alters mitochondrial dynamics during infection[END_REF] (Fig. 3D,E). This fragmentation, which is triggered upon calcium influxes due to membrane pores created by the major virulence factor of L. monocytogenes, listeriolysin O (LLO), can also be obtained with other pore-forming toxins of the same family. Listeria-induced mitochondrial fragmentation is transient and impairs the energy-producing function of the organelle. Functional mitochondrial dynamics is required for efficient infection; indeed decreased infection is observed in cells with impaired mitochondrial fusion, while cells with impaired mitochondrial fission display improved infection. Interestingly, LLO-dependent fission of mitochondria is independent of the dynamin-like protein Drp1. However, similarly to the canonical Drp1-dependent fission, it requires transient contacts with the endoplasmic reticulum, as well as actin polymerisation [START_REF] Stavru | Atypical mitochondrial fission upon bacterial infection[END_REF]. The involvement of other cellular components in this atypical fission mechanism is currently being addressed.

Post-translational modifications of host proteins: A role for SUMO in the infection.

The above mentioned toxin, listeriolysin O, which was originally described for its role in the disruption of the phagocytic vacuole after Listeria entry, turns out to be a highly versatile virulence factor with multiple effects on host cell functions [reviewed in 64]. One of them is the proteasomeindependent degradation of various cellular proteins, such as Ubc9, telomerase and Mitochondrial fission factor (Mff) [START_REF] Stavru | Atypical mitochondrial fission upon bacterial infection[END_REF][START_REF] Ribet | Listeria monocytogenes impairs SUMOylation for efficient infection[END_REF][START_REF] Samba-Louaka | Role for telomerase in Listeria monocytogenes infection[END_REF] (Fig. 3D). Ubc9 is the unique E2-ligase responsible for SUMO conjugation pathways; the modification of proteins by this small ubiquitin-like polypeptide controls their function in key cellular pathways such as transcription, intracellular transport, or stress responses. Ubc9 degradation results in the global de-SUMOylation of cellular proteins [START_REF] Ribet | Listeria monocytogenes impairs SUMOylation for efficient infection[END_REF]. Given that hyper-SUMOylation impairs infection efficiency, we have proposed that some SUMOylated proteins participate in controlling infection, and that LLO-dependent de-SUMOylation prevents their function. Whether the effect on infection depends on the modulation of one or several protein functions or localisations when they are SUMOylated, or on a role of the conjugated SUMO group per se, is the object of current investigations.

Novel insights into the host response to Listeria infection.

Host response to infection revealed by transcriptomics.

As described above, the transcriptome of L. monocytogenes has been investigated in depth at various phases of the bacterial life cycle. In contrast, studies dedicated to the transcriptional response of the infected host remain fragmentary. Nevertheless, several transcriptome analyses of Listeria-infected immune [START_REF] Frande-Cabanes | Dissociation of innate immune responses in microglia infected with Listeria monocytogenes[END_REF][START_REF] Mccaffrey | A specific gene expression program triggered by Gram-positive bacteria in the cytosol[END_REF] or non-immune [START_REF] Baldwin | A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes[END_REF][START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF] cell lines have been reported. Typically, the host transcriptional response corresponds mostly to innate immune signalling pathways, driven by NF-κB and interferons (IFN), with cell-type-specific variations around this core. Insights into more complex signalling events occurring in host infected tissues have been provided by transcriptome studies performed in the intestine of gnotobiotic humanized mice [START_REF] Lecuit | Functional genomic studies of the intestinal response to a foodborne enteropathogen in a humanized gnotobiotic mouse model[END_REF]. Another interesting approach allowing the analysis of a homogenous cell population rather than a tissular response has been adopted by Best et al.: The authors explored the differentiation process of memory T-cells, by analysing the transcriptome of CD8 + T cells sorted from infected mice [START_REF] Best | Transcriptional insights into the CD8(+) T cell response to infection and memory T cell formation[END_REF].

To address the role of commensal and probiotic bacteria in listeriosis, a transcriptome of the host and of Listeria has been undertaken, in the germ free "humanized" mouse model of listeriosis after colonisation of the intestine with lactobacilli [START_REF] Archambaud | Impact of lactobacilli on orally acquired listeriosis[END_REF]. It has highlighted that lactobacilli have a protective role against the in vivo dissemination of Listeria. They influence the transcriptome of the

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bacterium, essentially by modulating expression of genes involved in the utilisation of intestinal available nitrogen and carbon sources such as propanediol and ethanolamine. On the host side, lactobacilli not only affect the host immune defences by dampening IFN response; they also strikingly modulate the expression of several microRNAs.

The influence of the microbiota itself has been further investigated using deep-sequencing approaches on RNA extracted from intestinal tissue of infected conventional or germ free mice [START_REF] Archambaud | The Intestinal Microbiota Interferes with the microRNA Response upon Oral Listeria Infection[END_REF]. This has shown the role of the gut flora in modulating the host microRNA response to infection, and provided a regulatory network defined by host microRNAs and their target genes.

Set-up of host innate immune responses.

An important achievement of the past five years in the understanding of host innate immune responses to infection has been the unravelling by several groups of a number of bacterial pathogenassociated molecular patterns (PAMPs), cellular sensors and signalling pathways resulting in the onset of interferon responses [reviewed in 75]. Among these, secretion of cyclic-di-AMP by Listeria appears as a major agonist of the pathway, and signalling occurs via DDX41, STING, TBK1 and IRF3 [START_REF] Parvatiyar | The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response[END_REF][START_REF] Woodward | c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response[END_REF]. Bacterial nucleic acids can also be sensed via RIG-I, and then induce STING-dependent IFN responses [START_REF] Abdullah | RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids[END_REF][START_REF] Hagmann | RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells[END_REF]. In murine macrophages, we have uncovered a nonconventional intracellular pathway resulting in the induction of IFNβ, which depends on the tolllike receptor TLR2, the adaptor TRIF, IRF3 and IRF7 [START_REF] Aubry | Both TLR2 and TRIF contribute to interferon-beta production during Listeria infection[END_REF].

To date, whether the type I IFN response of the host is beneficial or detrimental to infection is still unclear. Indeed, mice invalidated for the type I IFN receptor (IFNAR1 -/-) are more resistant to Listeria infection when inoculated via intravenous route, but more sensitive to an oral challenge [START_REF] Kernbauer | Route of Infection Determines the Impact of Type I Interferons on Innate Immunity to Listeria monocytogenes[END_REF][START_REF] O'connell | Type I interferon production enhances susceptibility to Listeria monocytogenes infection[END_REF]. In line with this, recent studies suggest that the outcome of the IFN response also depends on its timing: a delayed onset of type I IFN induction proves detrimental to the host, while IFNβ treatment in the early phase of infection shows anti-bacterial properties [START_REF] Pontiroli | The timing of IFNbeta production affects early innate responses to Listeria monocytogenes and determines the overall outcome of lethal infection[END_REF]. This suggests that type I IFN antibacterial effects can be efficient against Listeria, while its properties to down-regulate host defences have opposite effects at later time points [START_REF] Carrero | Mechanisms and immunological effects of apoptosis caused by Listeria monocytogenes[END_REF][START_REF] Rayamajhi | Antagonistic crosstalk between type I and II interferons and increased host susceptibility to bacterial infections[END_REF].

The analysis of host gene expression reprogramming in response to infection has recently revealed that Listeria also induces type-III IFN (IFN-λ) in infected intestinal epithelial cells, an event so far only known for viral infections [START_REF] Lebreton | A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response[END_REF]. Detailed analysis has shown that production of IFN-λ requires bacterial entry into host cells, and increases with bacterial replication [START_REF] Bierne | Activation of Type III Interferon Genes by Pathogenic Bacteria in Infected Epithelial Cells and Mouse Placenta[END_REF]. These findings were extended to other gram-positive bacterial species during the infection of epithelial cells and tissues [START_REF] Bierne | Activation of Type III Interferon Genes by Pathogenic Bacteria in Infected Epithelial Cells and Mouse Placenta[END_REF]. So far, it is still unknown whether IFN-λ plays a positive or a negative role on the outcome of infection.

Strategies to escape or counteract innate immune responses.

L. monocytogenes has evolved ingenious mechanisms to evade the innate immune defences of its host. One such mechanism is the O-acetylation of muramic residues contained in the peptidoglycan, performed by the O-acetyltransferase OatA [START_REF] Aubry | OatA, a peptidoglycan Oacetyltransferase involved in Listeria monocytogenes immune escape, is critical for virulence[END_REF]. In a way reminiscent of the previously characterised peptidoglycan N-deacetylase PgdA mode of action [START_REF] Boneca | A critical role for peptidoglycan Ndeacetylation in Listeria evasion from the host innate immune system[END_REF], OatA increases the bacterial cell wall resistance to antimicrobial compounds such as lysozyme, thereby favouring bacterial persistence in macrophages and virulence in vivo. In addition, the stronger resistance of the bacterial cell wall limits the triggering of host pro-inflammatory response.

Listeria also dampens its host pro-inflammatory response through the secretion of InlC, a virulence factor of the internalin family [START_REF] Gouin | The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the IκB kinase subunit IKKα[END_REF] (Fig. 4A). In the cytosol, InlC directly interacts with the IKKα kinase and prevents the phosphorylation of IκB, the regulatory subunit in the NF-κB complex. As a consequence, IκB is no longer degraded in response to pro-inflammatory signals

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Trends in Cell Biology 2014 10 such as TNF-α, blocking the translocation of activated NF-κB to the nucleus, as well as the downstream production of cytokines and the chemo-attraction of neutrophils to the infection site.

Epigenetics and infection: Chromatin-based regulations of host transcription

In eukaryotic cells, DNA is condensed into chromatin, which can be either in an open, transcriptionally active, or in a closed, transcriptionally silent state. An important component in the control of the host transcriptional response to infection is the open or closed status of chromatin. Recent research aiming at exploring this level of regulation in the context of Listeria infection broadly contributed to the emerging field of bacterial infection and epigenetics [START_REF] Bierne | Epigenetics and bacterial infections[END_REF].

It had previously been shown that LLO as well as other bacterial pore forming toxins of the same family trigger the dephosphorylation of histone H3 (H3deP) and deacetylation of histone H4 (H4deAc) [START_REF] Hamon | Histone modifications induced by a family of bacterial toxins[END_REF], which correlate with the repression of a subset of host genes (Fig. 4B). Further mechanistic investigations have highlighted that H3deP is induced by potassium efflux when the host cell plasma membrane is permeabilized [START_REF] Hamon | K+ efflux is required for histone H3 dephosphorylation by Listeria monocytogenes listeriolysin O and other pore-forming toxins[END_REF]. In an independent pathway, this K + efflux also activates caspase-1 and thereby the inflammasome.

Infection by L. monocytogenes additionally stimulates the deacetylation of histone H3 on lysine 18 (H3K18) [START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF] (Fig. 4C). H3K18 is deacetylated by the NAD-dependent deacetylase sirtuin 2 (SIRT2), which upon binding of InlB to the Met receptor translocates to the nucleus and associates with the promoter of a number of genes. SIRT2 activity has a major influence on the downregulation host genes in infected cells, including a significant number of immune-related genes. Impaired SIRT2 function is detrimental to infection in cultured cells or in a murine listeriosis model, suggesting that the newly described role of SIRT2 in gene expression reprogramming is actively exploited by the pathogen to its own benefit. Control of the host chromatin by L. monocytogenes was also illustrated in another report. LntA is a recently discovered virulence factor from Listeria monocytogenes, belonging to the emerging family of nucleomodulins [START_REF] Bierne | When bacteria target the nucleus: the emerging family of nucleomodulins[END_REF]. After secretion in the host cell cytosol, LntA enters the infected cell nucleus, interacts directly with a chromatin component, BAHD1, and inhibits its activity [START_REF] Lebreton | A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response[END_REF][START_REF] Lebreton | Structural basis for the inhibition of the chromatin repressor BAHD1 by the bacterial nucleomodulin LntA[END_REF] (Fig. 4D). In parallel to the study of the bacterial effector LntA, the function of BAHD1 in noninfected cells has been explored, reinforcing the notion that bacterial pathogens may help unravelling unexpected aspects of cell physiology; BAHD1 participates in the nucleation and spreading of a novel repressive chromatin complex, which induces gene silencing when recruited at promoters [START_REF] Bierne | Human BAHD1 promotes heterochromatic gene silencing[END_REF]. During Listeria infection, the BAHD1-associated chromatin complex represses the expression of interferon-stimulated genes (ISG) by a so-far unknown mechanism [START_REF] Lebreton | Structural basis for the inhibition of the chromatin repressor BAHD1 by the bacterial nucleomodulin LntA[END_REF]. Upon interaction with LntA, BAHD1 dissociates from the promoters of ISGs, resulting in a reactivation of these genes, and affecting the outcome of in vivo infections in mice. Strikingly, the expression of lntA is tightly regulated, thereby avoiding uncontrolled activation of host innate immune responses. Altogether, the LntA-BAHD1 interplay fine-tunes the expression of ISGs during the progression of infection.

The three latter examples significantly participated in the rise of a new field of research named patho-epigenetics, a promising area for future investigations. One of the discussed issues is whether the targeting of chromatin by Listeria and other pathogenic bacteria could leave a permanent imprint, so that host cells -including, perhaps, non-immune stem cells -might keep a memory of past infections. Ongoing studies should soon provide answers to this hypothesis.

Conclusions

A true Renaissance is occurring in Microbiology [START_REF] Radoshevich | The New Microbiology: a conference at the Institut de France[END_REF]. By using new and cutting edge

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approaches such as transcriptomics, in vivo imaging, single cell analysis etc., this "New Microbiology" is generating novel concepts, including the discovery of several unsuspected RNAbased mechanisms to regulate gene expression. It is also highlighting the importance of the microbial social life and that of cell communication in microbial assemblies. The notions of persistence and dormancy are increasingly influencing the field of infection biology, which itself has been revolutionized since nearly three decades by live cell imaging. In this "New Microbiology", Listeria has been one of the champions and we anticipate that it will continue to be. A. The in vivo infection process. Following ingestion of contaminated food (1), bacteria colonize the digestive track. They can cross the intestinal barrier (2) and, after reaching the mesenteric lymph nodes (MLN), gain access to the systemic circulation (3). The primary target organs of the infection are the liver and spleen (4), which appear to constitute reservoirs of bacterial persistence if the infection is not controlled by immune defences. Release of bacteria into the blood stream can give rise to septicaemia. In some cases, L. monocytogenes cross the blood-brain barrier and reach the brain (5), resulting in meningitis or encephalitis. In pregnant women, crossing of the placental barrier ( 6 3) Bacteria can replicate in the cytosol, using cytosolic resources to their own benefit. The bacterial surface protein ActA stimulates the polymerisation of cellular actin via the recruitment of the Arp2/3 complex. This gives rise to actin comet tails, which allow intracellular motility (4) and cell-to-cell spread (5) of the bacteria. ( 6) Rupture of the two-membrane vacuole is mainly mediated by the action of LLO and phosphatidylcholine-specific phospholipase C (PC-PLC).

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A trip with Listeria When it is produced, LntA is targeted to the nucleus and binds directly with BAHD1. This interaction releases BAHD1 from the promoter of ISGs and allows their induction.

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FiguresFig. 1 .

 1 Figures

  ) can lead to abortion, or generalized neonatal infection. B. Intracellular life cycle of Listeria monocytogenes. (1) Listeria enters into host cells via a zipper mechanism, which requires the interaction of surface internalins InlA and InlB with their respective cell surface receptors E-cadherin and Met. (2) The endocytic vacuole is ruptured via the action of secreted effectors, the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositide phospholipase C (PI-PLC). (

Fig. 2 .

 2 Fig. 2. Novel regulatory mechanisms in prokaryotic gene expression. A. Model for the B 12 -dependent regulation of pocR via AspocR. (Left) In absence of B 12 , AspocR is transcribed and inhibits pocR expression by base-pairing with PocR mRNA. (Right) In presence of B 12 , binding of this ligand to the riboswitch (RS) triggers the premature ranscription termination of AspocR; as a consequence, PocR mRNA can be produced. Note that transcription of pocR is also dependent on propanediol (PDO). B. Regulation of prfA by cis-and trans-acting RNA elements. The 5'-UTR of PrfA mRNA forms a closed stem loop structure at low temperatures, masking the Shine-Dalgarno (SD) sequence and thereby inhibiting ribosome binding. At 37 °C, the stem loop structure melts, desequestering the SD sequence and allowing ribosomes to initiate translation. In presence of high concentration of methionine, a S-adenosyl-methionine (SAM)-dependent riboswitch terminates transcription upstream of lmo2419, thus generating the 227-nucleotide sRNA SreA. By binding to the 5′ UTR of PrfA mRNA, terminated or un-terminated transcripts containing the SreA sequence block the translation of PrfA, independently of SAM binding. C. Typical example of an excludon: the mogR-fli locus.An excludon consists of divergent transcriptional units overlapped by a long antisense RNA. Anti0677 can act as a negative regulator for the fli operon, but its distal part also harbours information as a mRNA, contributing to the expression of mogR. MogR is a transcriptional repressor of flagellum genes, while Fli proteins participate in the flagellum export apparatus; the two divergent arrays of genes thus have opposite functions.

Fig. 3 .

 3 Fig. 3. Subversion of host cell functions by Listeria. A. Proposed model for the recruitment of the endocytic machinery during Listeria entry. (Left) InlA-mediated entry. (Right) InlB-mediated entry. B. Localisation of various cell components at the site of Listeria entry. In each one of these independent immunofluorescence snapshots, host cell components are labelled in green; bacteria are in red, Actin, Clathrin, Septin, reprinted from [9] with permission of PNAS; OCRL, courtesy of J. Pizarro-Cerdá. C. Threedimensional architecture of actin filaments in L. monocytogenes comet tails. Filaments were projected into the XY plane. The cell wall of the bacterium is shown in grey. (Top) Colours of the filaments correspond to their angle with respect to the Y-axis: 0-15° (blue), 15-30° (green), 30-45° (red). (Bottom) Pairs of parallel filaments are highlighted in black. Reprinted from [60] with permission of PNAS. D. Listeriolysin O (LLO) affects protein levels and mitochondrial dynamics. Secretion of LLO induces the degradation of the E2-SUMO ligase Ubc9, and a global de-SUMOylation of host cell proteins. Via its pore-forming activity, LLO also leads to calcium influx, which promotes the proteasome-independent degradation of telomerase, and the fragmentation of the mitochondrial network. The endoplasmic reticulum (ER) and actin participate in the Drp1-independend mechanism of mitochondrial fission. E. The endoplasmic reticulum marks sites of mitochondrial fission. Arrowheads indicate mitochondrial fragmentation sites marked by ER-mitochondria contact sites. Mitochondria (DsRed2-mito; red) and ER (Sec61β-GFP; green) are shown, by confocal microscopy of Sec61β-GFP cells after addition of 2 nM LLO (Courtesy of F. Stavru).

Fig. 4 .

 4 Fig. 4. Control of host gene transcription by Listeria. A. InlC inhibits NF-κB dependent pro-inflammatory signalling. Secreted InlC interacts with the kinase IKKα, and prevents it to phosphorylate IκB. This impairs the ubiquitin (Ub)-proteasome dependent degradation of IκB, which sequesters NF-κB (p50-RelA) in the cytoplasm and dampens the induction of pro-inflammatory responses. B. Listeriolysin O (LLO) induces histone modifications. The pore-forming activity of LLO is responsible for an efflux of cellular potassium (K + ). This triggers a global dephosphorylation of histone 3 on serine 10 (H3deP) and deacetylation of H4 (H4deAc), and modulates the expression of host genes. C. InlB-Met signalling targets SIRT2 to the chromatin. Activation of Met-dependent signalling cascades by InlB triggers the translocation of the protein deacetylase SIRT2 to the chromatin, where it deacetylates histone 4 on lysine 18 (H3K18deAc). This leads to the repression of various host genes including transcriptional regulators and genes involved in innate immune responses, and promotes infection. D. The LntA-BAHD1 interplay fine-tunes the expression of interferon-stimulated genes (ISG). The chromatin-associated repressor BAHD1 controls the expression of ISGs during Listeria infection.When it is produced, LntA is targeted to the nucleus and binds directly with BAHD1. This interaction releases BAHD1 from the promoter of ISGs and allows their induction.

Acknowledgements

We are grateful to Fabrizia Stavru for critical reading of the manuscript and, together with Javier Pizarro-Cerdá, for sharing immunofluorescence images. We also thank Nina Sesto for helpful remarks about bacterial RNA processes. Work in the Cossart laboratory received financial support from Pasteur Institute, INSERM and INRA, from the European Research Council (ERC) advanced grant 233348, from Agence Nationale de la Recherche (ANR BacNet 10-BINF-0201 and EpiLis 11-BSV3-003-01), from Laboratoires d'Excellence (IBEID ANR-10-LABX-62-01), from Fondation Jeantet and Fondation le Roch Les Mousquetaires. P. Cossart is a Senior International Research Scholar of the Howard Hughes Medical Institute.

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Keywords: Listeria monocytogenes, cellular microbiology, organelle, mitochondria, nucleus, endomembrane system

Listeria monocytogenes, a facultative intracellular bacterium responsible for severe foodborne infections, is now recognized as a multifaceted model in infection biology. Comprehensive studies of the molecular and cellular basis of the infection have unravelled how the bacterium crosses the intestinal and feto-placental barriers, invades several cell types in which it multiplies and moves, and spreads from cell to cell. Interestingly, although Listeria does not actively invade host cell organelles, it can interfere with their function. We discuss the effect of Listeria on the ER and the mechanisms leading to the fragmentation of the mitochondrial network and its consequences, and review the strategies used by Listeria to subvert nuclear functions, more precisely to control host gene expression at the chromatin level.

Highlights

-Organelle manipulation by Listeria determines the outcome of infection.

-Disruption of mitochondrial dynamics affects the efficiency of Listeria infection.

-Bacteria secrete nucleomodulins to reprogram host cell transcription.

-Listeria-induced perturbations in ion homeostasis impact all organelles.

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Organelle targeting by Listeria 2

Bacterial life in the complex intracellular environment

Intracellular bacteria have developed various strategies to invade, survive, and multiply inside cells. Many bacterial pathogens including Legionella, Chlamydia, Salmonella and Brucella co-opt inner membranes to create a specific compartment adapted to their needs [reviewed in [START_REF] Salcedo | Bacterial interactions with the eukaryotic secretory pathway[END_REF]2]. Few bacteria, most of which are endosymbionts, reside inside pre-existing organelles [e.g. 3 -5]. By contrast, several pathogenic species such as Listeria monocytogenes, Shigella flexneri and members of the genus Rickettsia, escape from their internalization vacuoles and complete their life cycle in the cytosol [reviewed in 6]. The respective benefits and detriments of these diverse habitats concerning nutrient availability or sensitivity to host immune recognition are difficult to compare. In each case, bacteria have evolved survival strategies adapted to their specific lifestyles. Despite of their different residence sites, most intracellular bacteria directly or indirectly interact with organelles and perturb their function. We illustrate this issue with the example of Listeria monocytogenes.

Organelles are among the most prominent features that distinguish eukaryotic from prokaryotic cells (see Glossary). They form intracellular compartments delimited by at least one membrane. They are defined by a characteristic composition and specific metabolic pathways. Organelles can furthermore act as platforms for signalling pathways, allowing the coordination of activities such as metabolism, innate immune sensing, and the stress response [START_REF] Malhas | The nucleoplasmic reticulum: form and function[END_REF][START_REF] Tait | Mitochondria and cell signalling[END_REF]. Therefore, they represent attractive targets for bacterial pathogens to hijack host cell function and ensure their own survival.

L. monocytogenes (hereafter also Listeria) is a food-borne bacterial pathogen capable of invasion and replication in phagocytic and non-phagocytic cells. This capacity allows it to cross the protective epithelial barriers of the human body and cause severe disease in immunocompromised individuals [reviewed in [START_REF] Allerberger | Listeriosis: a resurgent foodborne infection[END_REF]10]. To invade host cells, Listeria expresses several dedicated proteins, notably internalin (InlA) and InlB. When the bacterium contacts the host cell membrane, InlA and InlB bind to E-Cadherin and to the hepatocyte growth factor (HGF) receptor Met respectively and induce bacterial internalization [reviewed in 11]. An internalization vacuole is formed from which the bacterium usually escapes; however, in vivo analysis of the crossing of the intestinal barrier has shown that Listeria can also transcytose across epithelial cells [12].

To evade membrane-bound structures, Listeria secretes two phospholipases (PlcA and PlcB), as well as a cholesterol-dependent cytolysin, listeriolysin O (LLO) [START_REF] Gaillard | In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2[END_REF][START_REF] Gründling | Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells[END_REF][15]. When the pH of the internalization vacuole acidifies, LLO forms pores in the vacuolar membrane and destabilizes it. In addition to its role in vacuole escape, LLO acts at several steps of the infection cycle and appears as a multifunctional toxin [see 16 for a recent review].

An alternative pathway implicating the intravacuolar replication of Listeria has been documented in macrophages of mice with severe combined immunodeficiency [17]. The formation of non-acidic, non-degradative compartments named spacious Listeria-containing phagosomes (SLAPS) is dependent on macroautophagy, and on moderate secretion of LLO. SLAPs have been proposed to provide a niche for persistent Listeria infection within macrophages, but do not represent a general rule for intracellular proliferation.

Listeria does not only escape the internalisation vacuole, but also avoids autophagic recognition, entrapment in autophagosomes and degradation. During cellular infection, the membrane damage induced by LLO leads to a transient amino-acid starvation, to which the host cell responds through inhibition of the mTOR pathway and induction of autophagy [START_REF] Meyer-Morse | Listeriolysin O is necessary and sufficient to induce autophagy during Listeria monocytogenes infection[END_REF]19]. However, PlcA and PlcB inhibit the cell autophagic flux; by stalling pre-

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autophagosome structures, they protect bacteria from clearance [19]. Another player in escape from autophagy is the Listeria surface protein ActA, which recruits the actin polymerisation machinery onto the bacterial surface to promote intracellular movement and spreading to neighbouring cells [START_REF] Tilney | Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes[END_REF]21]. The recruitment of actin by ActA [START_REF] Yoshikawa | Listeria monocytogenes ActA-mediated escape from autophagic recognition[END_REF] and that of the Major Vault Protein (MVP) by InlK [START_REF] Dortet | Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy[END_REF] cover the bacterial surface and prevent the sensing and targeting of bacteria to autophagy [reviewed in 24].

Apart from its ability to escape from internalisation compartments and autophagosomes, Listeria has developed a broad range of strategies allowing it to interfere with the function of bona fide cellular organelles, i.e. pre-existing the bacterial infection. These activities range from the perturbation of vesicle trafficking, or of the ER, to more elaborate mechanisms leading to fragmentation of the mitochondrial network, or to the modulation of host gene expression via direct chromatin-targeting mechanisms. We review here our current understanding of these processes and of the benefit they convey to the bacterium.

Perturbation of the endomembrane system during Listeria infection

The endomembrane system defines the subset of cellular compartments enclosed by membrane structures that are involved in transport. It includes the ER, the Golgi apparatus, lysosomes, endo-and exocytic compartments, as well as the nuclear envelope. The different components of these systems can exchange membrane and contents through vesicular transport. During infection, Listeria interacts successively with several of these compartments.

Inhibition of phagosomal killing

A primary strategy of the cell to counteract invading bacteria is maturation and then fusion of the phagocytic vacuole containing the bacteria (the phagosome) with lysosomes, a pathway that is particularly active in macrophages (Fig. 1). The small GTPase Rab5a is associated with the membrane of early endosomes and phagosomes and recruits effectors required for phagosome maturation, including enzymes leading to phosphoinositide conversion [START_REF] Wandinger-Ness | Rab proteins and the compartmentalization of the endosomal system[END_REF]26]. Rab5a has also been reported to play a role in the killing of Listeria after uptake by macrophages, by inducing the translocation of the small Rho GTPase Rac to the phagosome, which in turn promotes the assembly, at the phago-lysosomal membrane, of a NADPH oxidase complex producing reactive oxygen species (ROS) in the mature phagosome [START_REF] Prada-Delgado | Interferon-gamma listericidal action is mediated by novel Rab5a functions at the phagosomal environment[END_REF]. By targeting Rab5a, Listeria blocks the maturation of phagosomes and thus avoids killing. Towards this aim, Listeria secretes a 40-kDa protein annotated as a glyceraldehyde-3phosphate dehydrogenase (Lmo2459). Lmo2459 can mediate the ADP-ribosylation of Rab5a, which impedes phago-lysosomal maturation [START_REF] Alvarez-Domínguez | Characterization of a Listeria monocytogenes protein interfering with Rab5a[END_REF]; however, how Lmo2459 reaches its target Rab5a has not been defined. Whether a similar scenario occurs in epithelial cells is also unknown.

Inhibition of lysosome fusion

In addition to the mechanism described above, the pore-forming activity of LLO contributes to impede the fusion of lysosomes with the endocytic vacuole [START_REF] Henry | Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes[END_REF]30] (Fig. 1). Before bacterial escape into the cytosol, LLO-mediated perforation of the phagosomal membrane allows ion fluxes between compartments, with a notable drop in calcium concentration and an increase in pH inside the phagosome. This perturbation of the ion gradient across the phagosomal membrane inhibits fusion with LAMP1-positive lysosomes,

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as previously reported for the depletion of vacuolar calcium [31]. One should note that the activity of LLO itself is sensitive, in addition to pH variations [32], to other vacuolar ion concentrations, such as K + /Cl - [START_REF] Radtke | Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome[END_REF]. In line with this, the host chloride channel Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the phagosomal membrane promotes Listeria phagosomal escape by increasing intraphagosomal Cl -concentration and thereby potentiating LLO oligomerization [START_REF] Radtke | Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome[END_REF].

Altogether, by blocking the maturation of endocytic compartments, LLO and Lmo2459 allow the bacteria to avoid intraphagolysosomal degradation.

Perturbation of intracellular calcium storage and signalling

LLO triggers a massive increase in cytosolic calcium concentration, primarily due to an influx of extracellular Ca 2+ via pores in the plasma membrane [START_REF] Repp | Listeriolysin of Listeria monocytogenes forms Ca 2+ -permeable pores leading to intracellular Ca 2+ oscillations[END_REF]. Ca 2+ influx has been shown to promote bacterial entry, possibly through the fusion of subcortical vesicles that increase the membrane availability required for the uptake of a particle as large as a bacterium [START_REF] Dramsi | Listeriolysin O-mediated calcium influx potentiates entry of Listeria monocytogenes into the human Hep-2 epithelial cell line[END_REF]36]. In addition, compartments of the endomembrane system, such as the ER, release stored Ca 2+ into the cytosol upon infection. This release relies on two LLO-dependent mechanisms. First, InlB and LLO trigger signalling cascades including protein tyrosine kinases, which result in the activation of phospholipases C (PLC) and the generation of inositol triphosphate (IP 3 ), thereby stimulating the IP 3 receptor Ca 2+ channels [37,[START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF]. Second, LLO is responsible for reversible damages to the ER and lysosome membranes and the release of their content [30,[START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF], inducing a transient increase in cytosolic calcium concentration. In both epithelial and immune cells, LLO-induced Ca 2+ signals were shown to lead to a production of pro-inflammatory cytokines, which may contribute to pathology in different tissues, such as the intestine and the liver [START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF][START_REF] Dewamitta | Listeriolysin O-dependent bacterial entry into the cytoplasm is required for calpain activation and interleukin-1 alpha secretion in macrophages infected with Listeria monocytogenes[END_REF][40].

Activation of the unfolded protein response/ER stress

The ER damage mediated by LLO even prior to bacterial entry causes rapid swelling and loss of ER-tracker staining, which is typical of ER stress [START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF]. This stress is likely amplified by the concomitant increase in cytosolic calcium concentration [START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF][START_REF] Bentley | Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling[END_REF]. In response to this stress, the unfolded protein response (UPR) is activated, and ultimately leads to cell death if the infection is sustained [30,[START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF]42]. This cell response is considered to be antibacterial, as induction of the UPR prior to infection reduces the bacterial number [42].

Similar to the ER-based UPR, the mitochondrial UPR (mtUPR) is induced upon mitochondrial stress, where the protein ATFS1 accumulates in the nucleus instead of mitochondria and activates the transcription of proteins exerting protective functions in mitochondria [START_REF] Gekara | The multiple mechanisms of Ca 2+ signalling by listeriolysin O, the cholesteroldependent cytolysin of Listeria monocytogenes[END_REF]43]. The mtUPR pathway was recently found to activate an innate immunity program, suggesting that surveillance of organelle homeostasis is part of the active antimicrobial strategy in infected cells [44]. It remains to be uncovered whether this pathway is activated upon Listeria infection.

Mitochondrial dynamics and infection

Mitochondria are essential organelles that form a dynamic network within the cell. They play a crucial role in energy metabolism and cellular signalling pathways such as innate immunity, calcium signalling or apoptosis, which makes them an attractive target for pathogens to interfere with cellular function. Indeed, several bacteria and viruses modulate mitochondrial function to maintain their replicative niche by preventing host cell apoptosis or

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to favour dissemination by inducing apoptosis [reviewed in [START_REF] Galluzzi | Viral control of mitochondrial apoptosis[END_REF][46][47]. A variety of physiological and pathological conditions have been shown to induce either fusion or fission of the mitochondrial network and thereby affect mitochondrial function.

Infection with L. monocytogenes induces the host cell mitochondrial network to undergo a rapid, transient fragmentation already at early time points of infection (1-3h), along with a loss in the mitochondrial membrane potential, which results in decreased intracellular ATP levels [48] (Fig. 2A). This effect is specific to pathogenic Listeria, as it is not observed with the non-pathogenic species L. innocua or several other intracellular bacteria. The main bacterial factor responsible for mitochondrial fragmentation is the pore-forming toxin LLO, which causes calcium influx across the plasma membrane, although the exact signalling pathway leading to mitochondrial fragmentation is yet to be discovered. Other pore-forming toxins from the same family as LLO, such as pneumolysin, perfringolysin or streptolysin induce the same morphological phenotype as LLO, but it is largely unknown whether and how infection with the corresponding bacteria would affect mitochondria. In contrast to the bacterial pore-forming proteins Helicobacter pylori VacA, Neisseria gonorrhoeae PorB or Staphylococcus aureus Panton-Valentine leukocydin [reviewed in 46] (Fig 2B ), which reach and perturb mitochondrial membranes, LLO does not appear to localize to mitochondria, although it might do so if delivered by bacterial membrane vesicles, as has been recently shown for the enteropathogenic Escherichia coli Hly toxin [START_REF] Bielaszewska | Enterohemorrhagic Escherichia coli hemolysin employs outer membrane 11 vesicles to target mitochondria and cause endothelial and epithelial apoptosis[END_REF].

Strikingly, functional mitochondrial dynamics appears to be important for Listeria infection, because perturbing the fusion-fission balance by siRNA against key components of the fusion machinery causes a decrease in the infection efficiency, while interfering with fission results in its augmentation. Together, these findings led to the hypothesis that Listeria targets the host cell mitochondrial network to temporarily shut down cellular bioenergetics and to interfere with mitochondrial innate immune signalling [48]. Current efforts are aimed at obtaining a global view of the mitochondrial pathways that are reorganized upon Listeria infection (Box 1).

Mechanistically, the atypical mitochondrial fragmentation induced by Listeria differs from VacA-induced mitochondrial fragmentation [50], since it is independent of the key mitochondrial fission protein, the dynamin-related protein Drp1, which dissociates from mitochondria upon LLO treatment [START_REF] Stavru | Atypical mitochondrial fission upon bacterial infection[END_REF]. As in canonical fragmentation, the ER and actin were found to contribute significantly to the process; however, the fragmentation sites were marked by the ER also in the absence of Drp1, suggesting that although the initial constriction machinery is shared, molecules other than Drp1 can terminate mitochondrial abscission downstream of the ER. Uncovering such molecule(s) is a challenging issue, since to date LLO and Listeria are the only known exogenous stimuli of Drp1-independent mitochondrial fragmentation (Box 1).

Recently, mitochondria have been shown to play an important role in innate immunity through Mitochondrial AntiViral Signalling protein (MAVS), an adaptor protein located on the mitochondrial outer membrane that regulates signal transduction from cytosolic RNA sensors. MAVS activity has been shown to depend on mitochondrial dynamics and function [START_REF] Castanier | Mitochondrial localization of viral proteins as a means to subvert host defense[END_REF][START_REF] Onoguchi | Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1[END_REF][54]. Through the assembly of a large signalling complex on mitochondria, activated MAVS triggers nuclear translocation of the transcription factors NF-κB, IRF3 and IRF7, which results in the production of interferons, cytokines and interferon-stimulated genes (ISGs) [reviewed in 55]. Similar to other mitochondrial tail-anchored proteins, MAVS has additionally been found to localize at peroxisomes [START_REF] Dixit | Peroxisomes are signaling platforms for antiviral innate immunity[END_REF]. MAVS is one of the mediators of the innate immune response to Listeria in epithelial cells, leading to the production of type I and type III interferons [START_REF] Hagmann | RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells[END_REF]58]. Consistent with the disruption of mitochondrial morphology and

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Organelle targeting by Listeria 6 function by Listeria, it appears that MAVS-dependent stimulation of the interferon response is mainly carried out by peroxisomal rather than mitochondrial MAVS [58].

Targeting nuclear functions: strategies to reprogram the host

Upon infection by Listeria, the transcriptome of host cells is vastly reshaped. Different patterns of transcriptional response have been identified, depending on the nature of target cells in experiments performed in vitro [START_REF] Cohen | Monitoring cellular responses to Listeria monocytogenes with oligonucleotide arrays[END_REF][START_REF] Baldwin | A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes[END_REF][61][START_REF] Frande-Cabanes | Dissociation of innate immune responses in microglia infected with Listeria monocytogenes[END_REF], or on more complex regulatory circuits occurring in mouse models [START_REF] Lecuit | Functional genomic studies of the intestinal response to a foodborne enteropathogen in a humanized gnotobiotic mouse model[END_REF][START_REF] Archambaud | Impact of lactobacilli on orally acquired listeriosis[END_REF]. This reprogramming is largely the consequence of cellular signalling events occurring upon bacterial entry and/or subsequent recognition of Pathogen Associated Molecular Patterns (PAMPs) by a variety of host sensors named Pattern-Recognition Receptors (PRRs) [reviewed in 65]. In addition to these pathways, the notion recently emerged that pathogenic bacteria could manipulate host transcription by directly acting at the nuclear level and interfering with chromatin-based regulation [66,67], giving rise to the emerging field of patho-epigenetics (Box 1). Research on Listeria has been on the leading edge of this topic. As documented below, it has revealed that bacterial-triggered signalling events can impact the post-translational modification of histone tails [68], and that a virulence factor, belonging to a new class of bacterial secreted effectors named nucleomodulins [69] can localize to the cell nucleus and perturb chromatin silencing.

Perturbation of histone modifications

Eukaryotic DNA is organised in the nucleus into a structure named chromatin [reviewed in 70]. The primary unit of chromatin architecture is the nucleosome, which consists of a core octamer of histones H2A/B, H3 and H4, around which DNA wraps together with linker histones H1. Successive nucleosomes are packed more or less tightly to form chromatin fibres. The post-translational status of core histone tails plays a key function in the organization of this structure; by modulating the compaction of the chromatin assembly, and acting as specific docking sites for regulatory factors, these modifications constitute a histone code that adds another layer in the regulation of gene expression and cell fate decisions [START_REF] Jenuwein | Translating the histone code[END_REF].

LLO induces histone deacetylation and dephosphorylation

Listeria was one of the first examples of an invasive bacterium reported to have an effect on its host's histone modifications [START_REF] Hamon | Histone modifications induced by a family of bacterial toxins[END_REF]. Following the infection of HeLa cells, the total levels of serine 10 phosphorylation histone H3 (H3S10deP) and lysine acetylation of histone H3 (H3S10deP), as well as acetylation of histone H4 (H4KdeAc) are significantly decreased (Fig. 3, left). Both H3S10deP and H4KdeAc are induced by secreted LLO, and can be recapitulated by addition of the purified toxin. Transcriptional studies have highlighted that a specific subset of host genes is de-regulated within 20 minutes of LLO treatment. Interestingly, Chromatin Immunoprecipitation (ChIP) experiments have revealed that H3S10deP and H4KdeAc occur specifically at promoter genes that are differentially regulated upon LLO addition, suggesting that histone modifications induced by LLO are responsible for the reprogramming of host gene expression. In terms of mechanism, the intracellular signalling leading to H3S10deP is dependent on potassium efflux driven by increased membrane permeability upon LLO treatment, and can be recapitulated with other bacterial pore-forming toxins or potassium-specific ionophores [START_REF] Hamon | K + efflux is required for histone H3 dephosphorylation by Listeria monocytogenes listeriolysin O and other pore-forming toxins[END_REF]. Future investigations are needed to clarify how the decrease in cellular potassium concentration is sensed, and which signalling pathways result in histone modifications.

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Histone deacetylation by SIRT2

In contrast to H3S10deP and H4KdeAc, H3KdeAc has been shown to be a LLOindependent process [START_REF] Hamon | Histone modifications induced by a family of bacterial toxins[END_REF]. It is driven by another virulence factor, InlB, one of the two surface internalins involved in Listeria entry into its target cells [START_REF] Eskandarian | A role for SIRT2-dependent histone H3K18 deacetylation in bacterial infection[END_REF]. Upon binding to its cell membrane receptor Met (the receptor of HGF), InlB triggers the activation of a signalling cascade via the PI3K/Akt pathway, which results in the translocation of the tubulindeacetylase SIRT2 [START_REF] North | The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase[END_REF] from the cytoplasm to the chromatin fraction of the nucleus (Fig. 3, right). There, SIRT2 is responsible for the specific deacetylation of lysine 18 of H3 during infection, and for the transcriptional deregulation of hundreds of host genes. ChIP experiments showed that the promoter of genes that are repressed in a SIRT2-dependent manner during infection are enriched in deacetylated H3K18, suggesting that the activity of the histone deacetylase at these promoters is responsible for their repression during infection. The subversion of SIRT2 function by InlB proves to be pro-bacterial, as SIRT2 -/-mice can control listeriosis more efficiently than their wild type littermates. Further studies will be required to identify which SIRT2-regulated genes are most important for host defence against Listeria. Given that Listeria is the only known stimulus that induces SIRT2 translocation to the nucleus, it will provide an essential tool to elucidate the precise mechanisms mediating SIRT2 regulation at the chromatin.

Interaction of the nucleomodulin LntA with the heterochromatin protein BAHD1

Upon infection of epithelial cells, Listeria, like other intracellular bacteria, activates host innate immunes responses, which lead to the production and secretion of a variety of cytokines including type I and III interferons [reviewed in 76]. However, the expression of downstream ISGs is repressed by the heterochromatinization factor, BAHD1 [77] (Fig. 3, bottom). In non-infected cells, BAHD1 can nucleate the formation of hererochromatin at the promoter of a subset of host genes [START_REF] Bierne | Human BAHD1 promotes heterochromatic gene silencing[END_REF]; during infection, BAHD1 additionally targets ISGsvia a so-far unknown mechanism. Listeria can, under conditions that are still poorly defined, secrete the nucleomodulin LntA into the cell cytosol. This protein enters the host nucleus where it binds directly to BAHD1, causing it to dissociate from chromatin, and thus activating the expression of ISGs [77,[START_REF] Lebreton | Structural basis for the inhibition of the chromatin repressor BAHD1 by the bacterial nucleomodulin LntA[END_REF]. The lntA virulence gene, and a tight regulation of its expression, is required for an optimal infectious process in vivo, indicating that the deregulation of BAHD1 must be carefully timed and tuned. In line with this hypothesis, hypomorphic BAHD1 +/-mice are more resistant to Listeria infection than their wild type littermates. The precise effects of the host interferon responses on the patho-physiological outcomes of infection is still a matter of debate [80]; their study will certainly lead to surprising findings, which should clarify why their accurate modulation is essential to the host as well as to the bacterium.

Other secreted effectors of Listeria that can translocate to the host cell nucleus are currently under investigation. Their extensive functional characterisation should highlight novel mechanisms of subversion of nuclear functions by Listeria nucleomodulins.

Concluding remarks

The strategies of organelle subversion by Listeria that we have reviewed here reveal some of the ingenious tricks allowing the bacterium to adapt to its intracellular environment. Via a variety of molecular mechanisms, the bacterium is able to reorganize the host cell endomembrane dynamics, its energetics and gene expression program, most often for its own

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Organelle targeting by Listeria 8 profit. Depending on the organelle, strategies and consequences differ significantly. For instance, mitochondrial targeting results in a global reshaping of their structure, dynamics and functionality, and we are only beginning to understand the functional consequences of these events; they might represent the mammalian counterpart to the surveillance pathway described in Caenorhabditis elegans, which correlates mitochondrial dysfunction with pathogen attack [START_REF] Liu | Caenorhabditis elegans pathways that surveil and defend mitochondria[END_REF].

In contrast to the profound effect on mitochondria, the harnessing of chromatin by the Listeria protein LntA appears as a more focused process. It allows Listeria to selectively control the transcription of a subset of host genes, like ISGs, independently from other nuclear functions affected by infection, such as the DNA damage response described in other studies [82,83].

Organelle targeting during infection is not restricted to L. monocytogenes; a number of viruses, parasites and intracellular bacteria seem to have evolved alternative approaches to hijack the functions of metazoan organelles such as mitochondria or nuclei [see for instance 84-87], of which nuclear targeting has been previously reviewed [69]. As exemplified by H. pylori VacA [88] or the enteropathogenic E. coli proteins EspF and Map [START_REF] Kenny | Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria[END_REF]90], targeting of microbial proteins to mitochondria can directly or indirectly disrupt the mitochondrial membranes and modulate cell death. Recently, more subtle mechanisms have emerged: the V. cholerae secreted effector VopE acts as a mitochondria-localized GTPase-activating protein for the mitochondrial protein Miro, thereby disrupting its ability to mediate kinesin-dependent mitochondrial motility and resulting in mitochondrial fragmentation [91]. Similar strategies are used by plant pathogens. For instance, the type III secretion system (T3SS) of Pseudomonas syringae pv tomato DC3000 secretes Hop1N, a cysteine protease targeted to the host chloroplast, which interferes with photosynthesis by degrading a component of the photosystem, PsbQ [START_REF] Rodríguez-Herva | A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses[END_REF]. This suppresses ROS production involved in the plant innate immune response to bacterial infection, and counteracts host programmed cell death. Other pathogens, such as Chlamydia, Legionella or Toxoplasma recruit mitochondria to manipulate bioenergetic and innate immune pathways [93][94][START_REF] Matsumoto | Morphological studies of the association of mitochondria with chlamydial inclusions and the fusion of chlamydial inclusions[END_REF]. In the case of Toxoplasma, the molecular basis for this phenomenon has recently been uncovered with the identification of mitochondrial association factor 1 (MAF1), a Toxoplasma protein that directly binds host cell mitochondria [96].

Ultimately, the subversion of organelles can result in their colonisation, as mentioned in the introduction. This has so far been essentially documented for endosymbionts, such as the bacterial inhabitants of paramecium macro-and micronuclei [97] or the intra-mitochondrial bacterial species discovered in ticks, Candidatus Midichloria mitochondrii [3], and intranuclear growth has also been reported for a pathogen, the spotted fever agent Rickettsia rickettsii [98,99].

Together, these studies pave the way to a better understanding of how bacteria take advantage of their host at the nano-and microscopic levels. However, our perception of organelle dynamics during infection remains incomplete. Functional studies by high resolution and live-cell imaging should, in the coming years, allow us to better define the mechanisms that govern the morphological and functional changes in the ER and mitochondria. They should allow us to fully appreciate the impact of bacteria on vesicle trafficking, and the key role played by the cytoskeleton in the redistribution of intracellular compartments during infection. Whether Listeria infection also influences other key organelles such as peroxisomes, the nucleo-cytoplasmic transport pathways, or the architecture and function of sub-compartments not delineated by a membrane, such as Pbodies or stress granules, are still open questions.

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Box 1. Outstanding Questions 1. Does the organelle response to infection impact bacterial replication beyond innate immune signalling? In particular, how does mitochondrial bioenergetics affect bacterial replication?

2. What molecules perform mitochondrial abscission in the absence of the main fission protein Drp1? Is this pathway also at work in Drp1-containing cells? 3. Does Listeria infection cause peroxisome restructuring, given that the mitochondrial division machinery also acts in peroxisomal division [100]? 4. Can cells keep an epigenetic memory of previous infections? The discovery that bacterial pathogens can change the pattern of histone modification on chromatin has led to the tempting hypothesis that infected cells could be imprinted with a memory of infection, independently of the well-described commitment of memory B and T lymphocytes. Research in the patho-epigenetics field still has to provide evidence that the epigenetic marks deposited during infection are long-lasting and play a role in host cell behaviour during re-infection.

5. Are the infection-induced signals from different organelles integrated? If they are, how is this achieved, and at which cellular level?

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Fig. 3. Subversion of nuclear functions by Listeria. Left. By secreting the pore-forming toxin LLO, Listeria generates potassium efflux from the host-cell, which promotes a global deacetylation of histone H4 and dephosphorylation of Histone H3 on serine 10. This results in the down-regulation of a subset of host genes. Right. Binding of Listeria InlB to its receptor Met at the host cell membrane triggers, via a PI3K/Akt-dependent signalling cascade, the translocation of the lysine deacetylase SIRT2 to the nucleus. SIRT2 associates with chromatin at the promoter of various host genes, triggers the deacetylation of lysine 18 on H3, and thereby represses these genes. Bottom. In response to Listeria infection of epithelial cells, type I and III interferons are produced. However, the expression of ISGs is repressed downstream of JAK/STAT signalling, due to the condensation of a repressive complex at their promoter by the host chromatin component BAHD1. Under specific conditions, Listeria secretes the nucleomodulin LntA, which translocates to the nucleus, binds directly with BAHD1 and inhibits its function, resulting in an enhanced expression of ISGs.

FiguresFig. 1 .

 1 Figures

Fig. 2 .

 2 Fig. 2. Mitochondria and bacterial infections. (A) Listeria-induced mitochondrial fragmentation. LLO secreted by Listeria can form ion-permeable pores in the plasma membrane, thereby causing mitochondrial fragmentation. This fragmentation is facilitated by the ER and actin, but probably requires additional downstream factors, as it is independent of the main mitochondrial fission factor Drp1. Structural and functional disruption of the mitochondrial network causes a decrease in cellular bioenergetics and may modulate the mitochondria-based innate immune response. Although LLO has not been detected on the ER, it was shown to cause IP 3 R-independent calcium release. (B) Pathogen strategies to interfere with host cell mitochondria. Bacterial, viral or protozoan proteins can target mitochondria in different ways. Several pore-forming toxins, such as Listeriolysin O (LLO, Listeria monocytogenes), Streptolysin O (SLO, Streptococcus pyogenes), Perfringolysin (PFO, Clostridium perfringens), Pneumolysin (PLY, Streptococcus pneumoniae) and Panton-Valentine leukocydin (PVL, Staphylococcus aureus) insert into the plasma membrane, thereby causing ion imbalances and activating signalling pathways that affect mitochondrial morphology and function (1). Other toxins, such as VacA (Helicobacter pylori), Omp38 (Neisseria gonorrhoeae) and PVL insert into the outer or inner mitochondrial membrane, causing a loss in the mitochondrial membrane potential ΔΨ (2). The viral proteins vMia (cytomegalovirus) and NS3/4A (Hepatitis C) localize to the outer membrane and respectively induce Bax inhibition along with mitochondrial fragmentation, and MAVS cleavage. Vibrio cholerae VopE is a GTPase activating protein that inactivates Miro at mitochondria, inducing mitochondrial fragmentation. The secreted effectors EspF, Map (enteropathogenic Escherichia coli) and Ats1 (Anaplasma phagocytophilum) are imported into mitochondria and act "from within" to interfere with mitochondrial apoptosis pathways (3). Yet other effectors act on mitochondria while remaining pathogen-associated (4): Toxoplasma gondii MAF1 recruits mitochondria to the parasitophorous vacuole; Salmonella enterica SipB induces the formation of mitochondria-derived membrane structures; Neisseria gonorrhoeae type IV pili inhibit apoptosis. Other microbial effectors either remain unknown or act on mitochondria from unknown locations that include other organelles or the cytoplasm (5).

Acknowledgements

We are grateful to Mélanie Hamon and Javier Pizarro-Cerdá for critical reading of the manuscript.

Role of the funding sources

Work in the Cossart laboratory received financial support from Pasteur Institute, INSERM and INRA, from the European Research Council (ERC) advanced Grant 233348, from Agence Nationale de la Recherche (ANR blancs Mitopatho 12-BSV3-0017-03 and EpiLis 11-BSV3-003-01; ERA-NET Infect-ERA ProAntiLis), from Laboratoires d'Excellence (IBEID ANR-10-LABX-62-01), from Fondation Jeantet and Fondation le Roch Les Mousquetaires. P. Cossart is a Senior International Research Scholar of the Howard Hughes Medical Institute, F. Stavru is a CNRS research associate.

We declare no conflict of interest.

Glossary

Chromatin: Eukaryotic DNA is condensed into an organised ribonucleoproteic assembly named chromatin. The more or less dense structure of this arrangement has several functions. During metaphase, chromatin displays its most compact form, which participates in the partition of genetic material among daughter cells. In other phases, the tightness of chromatin varies locally, on distances spanning over a few nucleosomes to a whole chromosome. The ability of chromatin to switch from an open (euchromatin) to a closed (heretochromatin) state is a potent regulator of gene expression.

Intracellular bacteria: Bacteria that invade and replicate inside host cells. They are subdivided into facultative intracellular bacteria (for example Listeria monocytogenes, Salmonella Typhimurium or Shigella flexneri) and obligate intracellular bacteria (for example several Rickettsia).

Mitochondrial membrane potential: An electrochemical gradient formed when the electron transport chain located in the inner mitochondrial membrane pumps protons from the mitochondrial matrix to the intermembrane space. Proton backflux from the intermembrane space to the matrix through the FoF1 ATPase leads to ATP synthesis.

Nucleomodulins: An emerging class of secreted bacterial effectors that enter the nucleus of the host cell, where they can affect various functions including DNA replication and repair, transcription, chromatin remodelling or RNA splicing [69].

Organelle: A cellular compartment that is separated from the cytosol by one or several membranes. Note that the definition of organelles may vary, from a stricter version only encompassing DNA-containing compartments (namely, mitochondria and plastids), to a more relaxed terminology that would include non-membrane enclosed structures visible in microscopy such as nucleoli, centrioles, stress-or P-granules. For the purpose of clarity in this review, we opted for the most-conventional definition as a membrane-enclosed intracellular compartment.

Unfolded Protein Response:

A cellular quality control and defence mechanism, which is activated upon disruption of the proteostatic balance in the ER. To compensate the loss of ER function, this pathway aims at reducing the accumulation of abnormally-folded proteins, by (i) decreasing protein synthesis and translocation into the ER; (ii) stimulating the production of ER protein chaperones, which assist protein folding; (iii) promoting the degradation of unfolded proteins in the cytoplasm. If these mechanisms are ineffective, cell death is triggered [101].

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