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Listeria monocytogenes is a pathogenic bacterium causing potentially fatal foodborne infections in humans and animals. While the mechanisms used by Listeria to manipulate its host have been thoroughly characterized, how the host controls bacterial virulence factors remains to be extensively deciphered. Here, we found that the secreted Listeria virulence protein InlC is monoubiquitinated by the host cell machinery on K224, restricting infection. We show that the ubiquitinated form of InlC interacts with the intracellular alarmin S100A9, resulting in its stabilization and in increased reactive oxygen species production by neutrophils in infected mice. Collectively, our results suggest that posttranslational modification of InlC exacerbates the host response upon Listeria infection. IMPORTANCE The pathogenic potential of Listeria monocytogenes relies on the production of an arsenal of virulence determinants that have been extensively characterized, including surface and secreted proteins of the internalin family. We have previously shown that the Listeria secreted internalin InlC interacts with IκB kinase α to interfere with the host immune response (E. Gouin, M. Adib-Conquy, D. Balestrino, M.-A. Nahori, et al., Proc Natl Acad Sci USA, 107:17333-17338, 2010, https://doi.org/10.1073/pnas.1007765107). In the present work, we report that InlC is monoubiquitinated on K 224 upon infection of cells and provide evidence that ubiquitinated InlC interacts with and stabilizes the alarmin S100A9, which is a critical regulator of the immune response and inflammatory processes. Additionally, we show that ubiquitination of InlC causes an increase in reactive oxygen species production by neutrophils in mice and restricts Listeria infection. These findings are the first to identify a posttranscriptional modification of an internalin contributing to host defense., (Copyright © 2019 Gouin et al.)

Manipulation of host protein post-translational modifications (PTMs) is used by various pathogens to interfere with host cell functions. Among these modifications, ubiquitin (UBI) and ubiquitin-like proteins (UBLs) constitute key targets because they are regulators of pathways essential for the host cell. In particular, these PTM modifiers control pathways that have been described as crucial for infection such as pathogen entry, replication, propagation, or detection by the host. Although bacterial pathogens lack eucaryotic-like UBI or UBL systems, many of them produce proteins that specifically interfere with these host PTMs during infection. In this review we discuss the different mechanisms used by bacteria to interfere with host UBI and the two UBLs, SUMO and NEDD8., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.

The Gram-positive pathogen Listeria monocytogenes is able to promote its entry into a diverse range of mammalian host cells by triggering plasma membrane remodeling, leading to bacterial engulfment. Upon cell invasion, L. monocytogenes disrupts its internalization vacuole and translocates to the cytoplasm, where bacterial replication takes place. Subsequently, L. monocytogenes uses an actin-based motility system that allows bacterial cytoplasmic movement and cell-to-cell spread. L. monocytogenes therefore subverts host cell receptors, organelles and the cytoskeleton at different infection steps, manipulating diverse cellular functions that include ion transport, membrane trafficking, post-translational modifications, phosphoinositide production, innate immune responses as well as gene expression and DNA stability.

Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria modulate host gene transcription, thereby modifying the set of proteins synthetized by the infected cell. Bacteria can also target pre-existing host proteins and modulate their post-translational modifications or trigger their degradation. Analysis of protein levels variations in host cells during infection allows to integrate both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the toxin Listeriolysin O (LLO), secreted by the bacterial pathogen Listeria monocytogenes. We showed that a short-term treatment with LLO remodels the host cell proteome by specifically decreasing the abundance of 149 proteins. The same decrease in host protein levels was observed in different epithelial cell lines but not in macrophages. We show in particular that this proteome remodeling affects several ubiquitin and ubiquitin-like ligases and that LLO leads to major changes in the host ubiquitylome. Strikingly, this toxin-induced proteome remodeling involves only post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data reveal that different bacterial pore-forming toxins induce important host proteome remodeling, that may impair epithelial cell functions., (© 2018 Karim Malet et al.)

Listeria monocytogenes ( Lm ) is a facultative intracellular bacterial pathogen and the causative agent of listeriosis, a rare but fatal disease. During infection, Lm can traverse several physiological barriers; it can cross the intestine and placenta barrier and, in immunocompromised individuals, the blood-brain barrier. With the recent plethora of sequenced genomes available for Lm , it is clear that the complete repertoire of genes used by Lm to interact with its host remains to be fully explored. Recently, we focused on secreted Lm proteins because they are likely to interact with host cell components. Here, we investigated a putatively secreted protein of Lm , Lmo1656, that is present in most sequenced strains of Lm but absent in the nonpathogenic species Listeria innocua. lmo1656 gene is predicted to encode a small, positively charged protein. We show that Lmo1656 is secreted by Lm Furthermore, deletion of the lmo1656 gene (Δ lmo1656 ) attenuates virulence in mice infected orally but not intravenously, suggesting that Lmo1656 plays a role during oral listeriosis. We identified sorting nexin 6 (SNX6), an endosomal sorting component and BAR domain-containing protein, as a host cell interactor of Lmol656. SNX6 colocalizes with WT Lm during the early steps of infection. This colocalization depends on Lmo1656, and RNAi of SNX6 impairs infection in infected tissue culture cells, suggesting that SNX6 is utilized by Lm during infection. Our results reveal that Lmo1656 is a novel secreted virulence factor of Lm that facilitates recruitment of a specific member of the sorting nexin family in the mammalian host., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Listeria monocytogenes is a food-borne pathogen responsible for a disease called listeriosis, which is potentially lethal in immunocompromised individuals. This bacterium, first used as a model to study cell-mediated immunity, has emerged over the past 20 years as a paradigm in infection biology, cell biology and fundamental microbiology. In this Review, we highlight recent advances in the understanding of human listeriosis and L. monocytogenes biology. We describe unsuspected modes of hijacking host cell biology, ranging from changes in organelle morphology to direct effects on host transcription via a new class of bacterial effectors called nucleomodulins. We then discuss advances in understanding infection in vivo, including the discovery of tissue-specific virulence factors and the 'arms race' among bacteria competing for a niche in the microbiota. Finally, we describe the complexity of bacterial regulation and physiology, incorporating new insights into the mechanisms of action of a series of riboregulators that are critical for efficient metabolic regulation, antibiotic resistance and interspecies competition.

The opportunistic intracellular bacterial pathogen Listeria monocytogenes has in 30 years emerged as an exceptional bacterial model system in infection biology. Research on this bacterium has provided considerable insight into how pathogenic bacteria adapt to mammalian hosts, invade eukaryotic cells, move intracellularly, interfere with host cell functions and disseminate within tissues. It also contributed to unveil features of normal host cell pathways and unsuspected functions of previously known cellular proteins. This review provides an updated overview of our knowledge on this pathogen. In many examples, findings on L. monocytogenes provided the basis for new concepts in bacterial regulation, cell biology and infection processes.

Gene expression regulation is a critical question in host-pathogen interactions, and RNAs act as key players in this process. In this review, we focus on the mammalian RNA response to bacterial infection, with a special interest on microRNAs and long non-coding RNAs. We discuss the role of cellular miRNAs in immunity, the implication of circulating miRNAs as well as the influence of the microbiome on the miRNA response. We also review how pathogens counteract the host miRNA expression. Interestingly, bacterial non-coding RNAs regulate host gene expression and conversely eukaryotic miRNAs may regulate bacterial gene expression. Overall, the characterization of RNA regulatory networks represents an emerging theme in the field of host pathogen interactions., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Streptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present among Listeria monocytogenes strains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers resistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal system, and only in this environment LLS expression is detected in vivo Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections. IMPORTANCE The most severe human listeriosis outbreaks are caused by L. monocytogenes strains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulence in vivo once the intestinal barrier has been crossed. Importantly, we show that intravenous L. monocytogenes inoculation leads to bacterial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttranslational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity., (Copyright © 2017 Quereda et al.)

The promyelocytic leukemia protein (PML) is the main organizer of stress-responsive subnuclear structures called PML nuclear bodies. These structures recruit multiple interactors and modulate their abundance or their posttranslational modifications, notably by the SUMO ubiquitin-like modifiers. The involvement of PML in antiviral responses is well established. In contrast, the role of PML in bacterial infection remains poorly characterized. Here, we show that PML restricts infection by the pathogenic bacterium Listeria monocytogenes but not by Salmonella enterica serovar Typhimurium. During infection, PML undergoes oxidation-mediated multimerization, associates with the nuclear matrix, and becomes de-SUMOylated due to the pore-forming activity of the Listeria toxin listeriolysin O (LLO). These events trigger an antibacterial response that is not observed during in vitro infection by an LLO-defective Listeria mutant, but which can be phenocopied by specific induction of PML de-SUMOylation. Using transcriptomic and proteomic microarrays, we also characterized a network of immunity genes and cytokines, which are regulated by PML in response to Listeria infection but independently from the listeriolysin O toxin. Our study thus highlights two mechanistically distinct complementary roles of PML in host responses against bacterial infection., Importance: The promyelocytic leukemia protein (PML) is a eukaryotic protein that can polymerize in discrete nuclear assemblies known as PML nuclear bodies (NBs) and plays essential roles in many different cellular processes. Key to its function, PML can be posttranslationally modified by SUMO, a ubiquitin-like modifier. Identification of the role of PML in antiviral defenses has been deeply documented. In contrast, the role of PML in antibacterial defenses remains elusive. Here, we identify two mechanistically distinct complementary roles of PML in antibacterial responses against pathogens such as Listeria: (i) we show that PML regulates the expression of immunity genes in response to bacterial infection, and (ii) we unveil the fact that modification of PML SUMOylation by bacterial pore-forming toxins is sensed as a danger signal, leading to a restriction of bacterial intracellular multiplication. Taken together, our data reinforce the concept that intranuclear bodies can dynamically regulate important processes, such as defense against invaders., (Copyright © 2017 Ribet et al.)

Bacterial pathogens display an impressive arsenal of molecular mechanisms that allow survival in diverse host niches. Subversion of plasma membrane and cytoskeletal functions are common themes associated to infection by both extracellular and intracellular pathogens. Moreover, intracellular pathogens modify the structure/stability of their membrane-bound compartments and escape degradation from phagocytic or autophagic pathways. Here, we review the manipulation of host membranes by Listeria monocytogenes, Francisella tularensis, Shigella flexneri and Yersinia spp. These four bacterial model pathogens exemplify generalized strategies as well as specific features observed during bacterial infection processes., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Phosphoinositides control key cellular processes including vesicular trafficking and actin polymerization. Intracellular bacterial pathogens manipulate phosphoinositide metabolism in order to promote their uptake by target cells and to direct in some cases the biogenesis of their replication compartments. In this chapter, we review the molecular strategies that major pathogens including Listeria, Mycobacterium, Shigella, Salmonella, Legionella and Yersinia use to hijack phosphoinositides during infection. This article is part of a Special Issue entitled Phosphoinositides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function., Importance: Listeria is a bacterial pathogen that induces its internalization within the cytoplasm of human cells and has been used for decades as a major molecular tool to manipulate cells in order to explore and discover cellular functions. We have inactivated individually, for the first time in epithelial cells, all the genes of the human genome to investigate whether each gene modifies positively or negatively the Listeria infectious process. We identified novel signaling cascades that have never been associated with Listeria infection. We have also revisited the role of the molecular complex Arp2/3 involved in the polymerization of the actin cytoskeleton, which was shown previously to be required for Listeria entry and movement inside host cells, and we demonstrate that contrary to the general dogma, some subunits of the complex are dispensable for both Listeria entry and bacterial movement., (Copyright © 2015 Kühbacher et al.)

Bacterial pathogens have evolved a wide range of strategies to colonize and invade human organs, despite the presence of multiple host defense mechanisms. In this review, we will describe how pathogenic bacteria can adhere and multiply at the surface of host cells, how some bacteria can enter and proliferate inside these cells, and finally how pathogens may cross epithelial or endothelial host barriers and get access to internal tissues, leading to severe diseases in humans., (Copyright © 2015 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Invasion of nonphagocytic cells, a critical property of Listeria monocytogenes (Lm) that enables it to cross host barriers, is mediated by the interaction of two bacterial surface proteins, InlA and InlB, with their respective receptors E-cadherin and c-Met. Although InlA-E-cadherin interaction is necessary and sufficient for Lm crossing of the intestinal barrier, both InlA and InlB are required for Lm crossing of the placental barrier. The mechanisms underlying these differences are unknown. Phosphoinositide 3-kinase (PI3-K) is involved in both InlA- and InlB-dependent pathways. Indeed, InlA-dependent entry requires PI3-K activity but does not activate it, whereas InlB-c-Met interaction activates PI3-K. We show that Lm intestinal target cells exhibit a constitutive PI3-K activity, rendering InlB dispensable for InlA-dependent Lm intestinal barrier crossing. In contrast, the placental barrier does not exhibit constitutive PI3-K activity, making InlB necessary for InlA-dependent Lm placental invasion. Here, we provide the molecular explanation for the respective contributions of InlA and InlB to Lm host barrier invasion, and reveal the critical role of InlB in rendering cells permissive to InlA-mediated invasion. This study shows that PI3-K activity is critical to host barrier permissiveness to microbes, and that pathogens exploit both similarities and differences of host barriers to disseminate., (© 2015 Gessain et al.)

Interferons (IFNs) are secreted proteins of the cytokine family that regulate innate and adaptive immune responses to infection. Although the importance of IFNs in the antiviral response has long been appreciated, their role in bacterial infections is more complex and is currently a major focus of investigation. This review summarizes our current knowledge of the role of these cytokines in host defense against the bacterial pathogen Listeria monocytogenes and highlights recent discoveries on the molecular mechanisms evolved by this intracellular bacterium to subvert IFN responses.

Listeria monocytogenes is a model intracellular pathogen that can invade the cytoplasm of host mammalian cells. Cellular invasion can be measured using standard techniques such as the classical gentamicin protection assay, based on the quantification of colony-forming units from lysates of infected cells. In addition, there are methods based on immunofluorescence microscopy which allow for assaying invasion in a medium- to high-throughput manner. In the following sections we detail two different assays that can be used alone or in combination to quantify the internalization of L. monocytogenes in host cells.

Unlabelled: The intestinal tract is the largest reservoir of microbes in the human body. The intestinal microbiota is thought to be able to modulate alterations of the gut induced by enteropathogens, thereby maintaining homeostasis. Listeria monocytogenes is the agent of listeriosis, an infection transmitted to humans upon ingestion of contaminated food. Crossing of the intestinal barrier is a critical step of the infection before dissemination into deeper organs. Here, we investigated the role of the intestinal microbiota in the regulation of host protein-coding genes and microRNA (miRNA or miR) expression during Listeria infection. We first established the intestinal miRNA signatures corresponding to the 10 most highly expressed miRNAs in the murine ileum of conventional and germfree mice, noninfected and infected with Listeria. Next, we identified 6 miRNAs whose expression decreased upon Listeria infection in conventional mice. Strikingly, five of these miRNA expression variations (in miR-143, miR-148a, miR-200b, miR-200c, and miR-378) were dependent on the presence of the microbiota. In addition, as is already known, protein-coding genes were highly affected by infection in both conventional and germfree mice. By crossing bioinformatically the predicted targets of the miRNAs to our whole-genome transcriptomic data, we revealed an miRNA-mRNA network that suggested miRNA-mediated global regulation during intestinal infection. Other recent studies have revealed an miRNA response to either bacterial pathogens or commensal bacteria. In contrast, our work provides an unprecedented insight into the impact of the intestinal microbiota on host transcriptional reprogramming during infection by a human pathogen., Importance: While the crucial role of miRNAs in regulating the host response to bacterial infection is increasingly recognized, the involvement of the intestinal microbiota in the regulation of miRNA expression has not been explored in detail. Here, we investigated the impact of the intestinal microbiota on the regulation of protein-coding genes and miRNA expression in a host infected by L. monocytogenes, a food-borne pathogen. We show that the microbiota interferes with the microRNA response upon oral Listeria infection and identify several protein-coding target genes whose expression correlates inversely with that of the miRNA. Further investigations of the regulatory networks involving miR-143, miR-148a, miR-200b, miR-200c, and miR-378 will provide new insights into the impact of the intestinal microbiota on the host upon bacterial infection.

Listeria monocytogenes is a bacterial pathogen that promotes its internalization into host epithelial cells. Interaction between the bacterial surface molecules InlA and InlB and their cellular receptors E-cadherin and Met, respectively, triggers the recruitment of endocytic effectors, the subversion of the phosphoinositide metabolism, and the remodeling of the actin cytoskeleton that lead to bacterial engulfment. Additional bacterial surface and secreted virulence factors also contribute to entry, albeit to a lesser extent. Here we review the increasing number of signaling effectors that are reported as being subverted by L. monocytogenes during invasion of cultured cell lines. We also update the current knowledge of the early steps of in vivo cellular infection, which, as shown recently, challenges previous concepts generated from in vitro data.

The nucleus, at the heart of the eukaryotic cell, hosts and protects the genetic material, governs gene expression and regulates the whole cell physiology, including cell division. A growing number of studies indicate that various animal and plant pathogenic bacteria can deliver factors to this central organelle to subvert host defences by directly interfering with transcription, chromatin-remodelling, RNA splicing or DNA replication and repair. Such bacterial molecules entering the nucleus, which we propose to term 'nucleomodulins', use diverse strategies to hijack nuclear processes by targeting host DNA or an array of nuclear proteins. In some cases, bacteria can even enter the nucleus. These bacterial 'nuclear attacks' might have permanent genetic or long-term epigenetic effects on the host. Studying nucleomodulins and endonuclear bacteria can thus generate new insights into long-term impacts of infectious diseases and create novel tools for biotechnological applications and for deciphering the regulation of nuclear dynamics., (© 2012 Blackwell Publishing Ltd.)

Epithelia are highly organised structures protecting underlying tissues against microbial pathogens. Epithelial morphogenesis and maintenance is mediated by cell-cell adhesion molecules organised in junctional complexes, such as the adherens junctions. The tight organisation of these complexes and their interactions with cellular factors render the epithelia impermeable to potential invaders. Nevertheless, pathogens have developed strategies to target, interact and manipulate junctional complexes, in order to disrupt or cross the epithelial barriers and cause infection. Bacteria, viruses and parasites access the junctional molecular components either directly, often taking advantage of physiological alterations in epithelial polarity, or indirectly, by delivering into cells molecular factors that destabilise junctional integrity. Importantly, microbial interactions with junctional components are instrumental not only to elucidate mechanisms of invasion, but also to unravel fundamental physiological properties of the epithelial barriers, at the cellular and tissular level.

The Gram-positive bacterial pathogen Listeria monocytogenes has become one of the best studied models in infection biology. This review will update our knowledge of Listeria virulence factors and highlight their role during the Listeria infection process., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cell-cell adhesion plays a fundamental role in cell polarity and organogenesis. It also contributes to the formation and establishment of physical barriers against microbial infections. However, a large number of pathogens, from viruses to bacteria and parasites, have developed countless strategies to specifically target cell adhesion molecules in order to adhere to and invade epithelial cells, disrupt epithelial integrity, and access deeper tissues for dissemination. The study of all these processes has contributed to the characterization of molecular machineries at the junctions of eukaryotic cells that have been better understood by using pathogens as probes.

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The L. monocytogenes surface protein InlB interacts with c-Met, the hepatocyte growth factor (HGF) receptor, inducing bacterial internalization in numerous non-phagocytic cells. As InlB and HGF are known to trigger similar signaling pathways upon c-Met activation, we investigated the role of CD44, and more specifically its isoform CD44v6, in bacterial internalization in non-phagocytic cells. Indeed, CD44, the hyaluronic acid transmembrane receptor, and more specifically its isoform CD44v6 have been reported as necessary for the activation of c-Met upon the interaction with either the endogenous ligand HGF or the L. monocytogenes surface protein InlB. Our results demonstrate that, in the cell lines that we used, CD44 receptors play no role in the activation of c-Met, neither during L. monocytogenes entry, nor upon HGF activation. Furthermore, none of the CD44 isoforms was recruited at the L. monocytogenes entry site, and depletion by siRNA of total CD44 or of CD44v6 isoform did not reduce bacterial infections. Conversely, the overexpression of CD44 or CD44v6 had no significant effect on L. monocytogenes internalization. Together our results reveal that the activation of c-Met can be largely CD44-independent., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.

Subversion of host membrane machinery is important for the uptake, survival, and replication of bacterial pathogens. Understanding how pathogens manipulate host membrane transport pathways provides mechanistic insight into how infection occurs and is also revealing new information on biochemical processes involved in the functioning of eukaryotic cells. In this review we discuss several of the canonical host pathways targeted by bacterial pathogens and emerging areas of investigation in this exciting field., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes. InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans. Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans, we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans, like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.

The study of how microbes cause disease has initiated unique research avenues in both infection biology and cell biology. As our knowledge of the infected cell improves and innovative technologies become available, new domains of investigation hold promise for better understanding pathogenesis and fundamental cellular processes.

Listeria monocytogenes is an intracellular bacterial pathogen that promotes its internalization within nonprofessional phagocytes by interacting with specific host cell receptors. L. monocytogenes resides transiently in a membrane-bound compartment before escaping into the host cell cytosol where bacterial proliferation takes place. Actin-based motility then promotes cell-to-cell pathogen spread. Extensive studies on cytoskeleton rearrangements, membrane trafficking, and other events have established this microorganism as an archetype of cellular function subversion for intracellular parasitism. Here we discuss the most significant membrane trafficking pathways hijacked by L. monocytogenes during the host cell infection process and compare them to those of other intracellular pathogens, in particular Shigella flexneri, Salmonella enterica, and Mycobacterium tuberculosis.

The link between bacteria and host chromatin remodeling is an emerging topic. The exciting recent discoveries on bacterial impact on host epigenetics, as discussed in this Review, highlight yet another strategy used by bacterial pathogens to interfere with key cellular processes. The study of how pathogens provoke host chromatin changes will also provide new insights into host epigenetic regulation mechanisms.

Bacterial pathogens recruit clathrin upon interaction with host surface receptors during infection. Here, using three different infection models, we observed that host-pathogen interactions induce tyrosine phosphorylation of clathrin heavy chain. This modification was critical for recruitment of actin at bacteria-host adhesion sites during bacterial internalization or pedestal formation. At the bacterial interface, clathrin assembled to form coated pits of conventional size. Because such structures cannot internalize large particles such as bacteria, we propose that during infection, clathrin-coated pits serve as platforms to initiate actin rearrangements at bacteria-host adhesion sites. We then showed that the clathrin-actin interdependency is initiated by Dab2 and depends on the presence of clathrin light chain and its actin-binding partner Hip1R, and that the fully assembled machinery can recruit Myosin VI. Together, our study highlights a physiological role for clathrin heavy chain phosphorylation and reinforces the increasingly recognized function of clathrin in actin cytoskeletal organization in mammalian cells.