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Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation and cancer, however its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane.We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection.

Scavenger receptors are part of a complex surveillance system expressed by host cells to efficiently orchestrate innate immune response against bacterial infections. Stabilin-1 (STAB-1) is a scavenger receptor involved in cell trafficking, inflammation, and cancer; however, its role in infection remains to be elucidated. Listeria monocytogenes (Lm) is a major intracellular human food-borne pathogen causing severe infections in susceptible hosts. Using a mouse model of infection, we demonstrate here that STAB-1 controls Lm-induced cytokine and chemokine production and immune cell accumulation in Lm-infected organs. We show that STAB-1 also regulates the recruitment of myeloid cells in response to Lm infection and contributes to clear circulating bacteria. In addition, whereas STAB-1 appears to promote bacterial uptake by macrophages, infection by pathogenic Listeria induces the down regulation of STAB-1 expression and its delocalization from the host cell membrane. We propose STAB-1 as a new SR involved in the control of Lm infection through the regulation of host defense mechanisms, a process that would be targeted by bacterial virulence factors to promote infection.

Listeria monocytogenes is among the best-characterized intracellular pathogens. Its virulence factors, and thewaythey interfere with host cells to hijack host functions and promote the establishment and dissemination of the infection, have been the focus of multiple studies over the last 30 years. During cellular infection, L. monocytogenes was shown to induce host DNA damage and delay the host cell cycle to its own benefit. However, whether the cell cycle stage would interfere with the capacity of Listeria to infect human cultured cell lines was never assessed. We found here that L. monocytogenes preferentially infects cultured cells in G2/M phases. Inside G2/M cells, the bacteria lead to an increase in the overall mitosis duration by delaying the mitotic exit. We showed that L. monocytogenes infection causes a sustained activation of the spindle assembly checkpoint, which we correlated with the increase in the percentage of misaligned chromosomes detected in infected cells. Moreover, we demonstrated that chromosome misalignment in Listeria-infected cells required the function of two Listeria virulence factors, ActA and InlC. Our findings show the pleiotropic role of Listeria virulence factors and their cooperative action in successfully establishing the cellular infection. This research was funded by the Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Programme (COMPETE) and by National funds through FCT (Fundação para a Ciência e Tecnologia) under the projects (PTDC/BIA-BCM/111215/2009FCOMP-01-0124-FEDER-014178). A.C.C., J.P. were supported by FCT grants (SFRH/BPD/88769/2012 and SFRH/BD/86871/2012, respectively). S.S. was supported by the FCT in the framework of the CEEC-Institutional 2017 program. This research was funded by the Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Programme (COMPETE) and by National funds through FCT (Funda??o para a Ci?ncia e Tecnologia) under the projects (PTDC/BIA-BCM/111215/2009FCOMP-01-0124-FEDER-014178). A.C.C., J.P. were supported by FCT grants (SFRH/BPD/88769/2012 and SFRH/BD/86871/2012, respectively). S.S. was supported by the FCT in the framework of the CEEC-Institutional 2017 program.

The capacity of bacterial pathogens to infect their hosts depends on the tight spatiotemporal regulation of virulence genes. The Listeria monocytogenes (Lm) metal efflux pump repressor CadC is highly expressed during late infection stages, modulating lipoprotein processing and host immune response. Here we investigate the potential of CadC as broad repressor of virulence genes. We show that CadC represses the expression of the bile salt hydrolase impairing Lm resistance to bile. During late infection, in absence of CadC-dependent repression, the constitutive bile salt hydrolase expression induces the overexpression of the cholic acid efflux pump MdrT that is unfavorable to Lm virulence. We establish the CadC regulon and show that CadC represses additional virulence factors activated by sB during colonization of the intestinal lumen. CadC is thus a general repressor that promotes Lm virulence by down-regulating, at late infection stages, genes required for survival in the gastrointestinal tract. This demonstrates for the first time how bacterial pathogens can repurpose regulators to spatiotemporally repress virulence genes and optimize their infectious capacity. This work was supported by grants to DC (FEDER - Fundo Europeu de Desenvolvimento Regional funds through the NORTE 2020 - Norte Portugal Regional Operational Programme, Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project NORTE-01-0145-FEDER-030020 PTDC/SAU-INF/30020/2017); to PC (European Research Council - ERC - Advanced Grant BacCellEpi 670823). R.P. and A.C. received an FCT Doctoral Fellowship (SFRH/BD/89542/2012, SFRH/BD/29314/2006) through FCT/MEC co-funded by QREN and POPH (Programa Operacional Potencial Humano). SS was supported by FCT Investigator program (COMPETE, POPH and FCT).

Innate immunity is the most broadly effective host defense, being essential to clear the majority of microbial infections. Scavenger Receptors comprise a family of sensors expressed in a multitude of host cells, whose dual role during microbial pathogenesis gained importance over recent years. SRs regulate the recruitment of immune cells and control both host inflammatory response and bacterial load. In turn, pathogens have evolved different strategies to overcome immune response, avoid recognition by SRs and exploit them to favor infection. Here, we discuss the most relevant findings regarding the interplay between SRs and pathogens, discussing how these multifunctional proteins recognize a panoply of ligands and act as bacterial phagocytic receptors. This work received funding from Norte-01–0145-FEDER-000012– Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL Partnership Agreement, through the European Regional Development Fund (FEDER). R.P received an FCT Doctoral Fellowship [SFRH/BD/89542/2012] through FCT/MEC co-funded by QREN and POPH (Programa Operacional Potencial Humano). SS was supported by FCT Investigator program (COMPETE, POPH, and FCT).

Wall teichoic acids (WTAs) are important surface glycopolymers involved in various physiological processes occurring in the Gram-positive cell envelope. We previously showed that the decoration of Listeria monocytogenes (Lm) WTAs with l-rhamnose conferred resistance against antimicrobial peptides. Here, we show that WTA l-rhamnosylation also contributes to physiological levels of autolysis in Lm through a mechanism that requires efficient association of Ami, a virulence-promoting autolysin belonging to the GW protein family, to the bacterial cell surface. Importantly, WTA l-rhamnosylation also controls the surface association of another GW protein, the invasin internalin B (InlB), that promotes Lm invasion of host cells. Whereas WTA N-acetylglucosaminylation is not a prerequisite for GW protein surface association, lipoteichoic acids appear to also play a role in the surface anchoring of InlB. Strikingly, while the GW domains of Ami, InlB and Auto (another autolysin contributing to cell invasion and virulence) are sufficient to mediate surface association, this is not the case for the GW domains of the remaining six uncharacterized Lm GW proteins. Overall, we reveal WTA l-rhamnosylation as a bacterial surface modification mechanism that contributes to Lm physiology and pathogenesis by controlling the surface association of GW proteins involved in autolysis and infection.

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible animals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence factors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively controls the expression of the Agr quorum sensing system (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcription factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infections the role of MouR in Lm virulence. Norte-01-0145-FEDER-000012 – Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme [NORTE 2020], under the PORTUGAL Partnership Agreement, through the European Regional Development Fund (FEDER); FCT Doctoral Fellowship [SFRH/BD/86871/2012, SFRH/BD/89542/2012, SFRH/BD/61825/2009 and SFRH/BD/112217/2015 to J.P., R.P., F.C. and C.B.] through FCT/MEC co-funded by QREN and POPH (Programa Operacional Potencial Humano); FCT Investigator program (COMPETE, POPH and FCT) (to S.S.). Funding for open access charge: Norte-01-0145-FEDER-000012 – Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme [NORTE 2020].

The cell wall of Listeria monocytogenes (Lm), a major intracellular foodborne bacterial pathogen, comprises a thick peptidoglycan layer that serves as a scaffold for glycopolymers such as wall teichoic acids (WTAs). WTAs contain non-essential sugar substituents whose absence prevents bacteriophage binding and impacts antigenicity, sensitivity to antimicrobials, and virulence. Here, we demonstrated, for the first time, the triple function of Lm WTA glycosylations in the following: (1) supporting the correct anchoring of major Lm virulence factors at the bacterial surface, namely Ami and InlB, (2) promoting Lm resistance to antimicrobial peptides (AMPs), and (3) decreasing Lm sensitivity to some antibiotics. We showed that while the decoration of WTAs by rhamnose in Lm serovar 1/2a and by galactose in serovar 4b are important for the surface anchoring of Ami and InlB, N-acetylglucosamine in serovar 1/2a and glucose in serovar 4b are dispensable for the surface association of InlB or InlB/Ami. We found that the absence of a single glycosylation only had a slight impact on the sensibility of Lm to AMPs and antibiotics, however the concomitant deficiency of both glycosylations (rhamnose and N-acetylglucosamine in serovar 1/2a, and galactose and glucose in serovar 4b) significantly impaired the Lm capacity to overcome the action of antimicrobials. We propose WTA glycosylation as a broad mechanism used by Lm, not only to properly anchor surface virulence factors, but also to resist AMPs and antibiotics. WTA glycosyltransferases thus emerge as promising drug targets to attenuate the virulence of bacterial pathogens, while increasing their susceptibility to host immune defenses and potentiating the action of antibiotics.

We report here the draft genome sequence of Listeria monocytogenes 2542, a serotype 4b clinical strain recovered from a placental sample during a cheese-related listeriosis outbreak in Portugal. Work done by T.C. and T.H. was supported by LOEWE Medical RNomics (B3) and theGermanCentre for Infection Research, Justus-Liebig University Giessen. Financial sup-port for R.M. and V.F. was provided by Fundação para a Ciência e a Tecnologia(FCT)through Ph.D. (SFRH/BD/71704/2010) and postdoctoral (SFRH/BPD/72617/2010) fellow-ships, respectively. Open-access publication was cofinanced by the NEWFOOD NORTE-01-0246-FEDER-000043 project supported by the Norte Portugal Regional OperationalProgramme (NORTE 2020), under the Portugal 2020 Partnership Agreement throughthe European Regional Development Fund (ERDF). We also acknowledge the scientificcollaboration under the FCT project UID/Multi/50016/2013.

Listeria monocytogenes is a major intracellular human foodborne bacterial pathogen. We previously revealed L. monocytogenes cadC as highly expressed during mouse infection. Here we show that L. monocytogenes CadC is a sequence-specific, DNA-binding and cadmium-dependent regulator of CadA, an efflux pump conferring cadmium resistance. CadC but not CadA is required for L. monocytogenes infection in vivo. Interestingly, CadC also directly represses lspB, a gene encoding a lipoprotein signal peptidase whose expression appears detrimental for infection. lspB overexpression promotes the release of the LpeA lipoprotein to the extracellular medium, inducing tumor necrosis factor α and interleukin 6 expression, thus impairing L. monocytogenes survival in macrophages. We propose that L. monocytogenes uses CadC to repress lspB expression during infection to avoid LpeA exposure to the host immune system, diminishing inflammatory cytokine expression and promoting intramacrophagic survival and virulence. CadC appears as the first metal efflux pump regulator repurposed during infection to fine-tune lipoprotein processing and host responses.

Bacterial pathogenicity deeply depends on the ability to secrete virulence factors that bind specific targets on host cells and manipulate host responses. The Gram-positive bacterium Listeria monocytogenes is a human foodborne pathogen that remains a serious public health concern. To transport proteins across its cell envelope, this facultative intracellular pathogen engages a set of specialized secretion systems. Here we show that L. monocytogenes EGDe uses a specialized secretion system, named ESX-1, to secrete EsxA, a homolog of the virulence determinants ESAT-6 and EsxA of Mycobacterium tuberculosis and Staphylococcus aureus, respectively. Our data show that the L. monocytogenes ESX-1 secretion system and its substrates are dispensable for bacterial invasion and intracellular multiplication in eukaryotic cell lines. Surprisingly, we found that the EssC-dependent secretion of EsxA has a detrimental effect on L. monocytogenes in vivo infection.

Listeria monocytogenes, a foodborne pathogenic bacterium, remains a serious public health concern due to its frequent occurrence in food products coupled with a high mortality rate. Bacterial pathogenicity depends greatly on the ability to secrete virulence factors to or beyond the bacterial cell surface. The Tat pathway, one of the secretion systems present in L. monocytogenes, was until now only investigated in silico. In L. monocytogenes strain EGDe two genes constitute this pathway, tatC(lmo0361) and tatA(lmo0362). Here we show that tatC and tatA are cotranscribed in a bicistronic- and growth-phase-dependent manner, being downregulated in the stationary phase. An EGDe tatAC mutant strain (EGDe ΔtatAC) was constructed, confirming that the Tat pathway is not essential for L.monocytogenes survival or biofilm-forming ability. When compared to the wild-type EGDe, deletion of tatAC did not decrease the virulence potential of EGDe ΔtatAC in HT-29 human epithelial cell line and even increased (p < 0.05) the virulence potential for mice. Moreover, we show that tat genes are prevalent in L. monocytogenes strains belonging to genetic lineage II and are generally absent from lineage I, which is more associated with human cases, thus excluding the possibility of using the Tat system as a target for novel antimicrobial compounds targeting L.monocytogenes.

Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favor microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signaling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed. This work was supported by FEDER funds through Programa Operational Factores de Competitividade - COMPETE and by national funds through FCT - Fundacao para a Ciencia e a Tecnologia (project PTDC/BIA-MIC/3463/2012 FCOMP-01-0124-FEDER-028364; to AV). Research in the groups of Molecular Microbiology and Fish Immunology and Vaccinology is supported by national funds through FCT Fundacao para Ciencia e a Tecnologia/MEC - Ministerio da Educacao e Ciencia and co-funded by FEDER within the partnership agreement: PT2020 related with the research unit number 4293. The Group of Molecular Microbiology also receives support from a Research Grant 2014 by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (to SS) and the PT2020 research project Infect-ERA/0001/2013 PROANTILIS. AdV received the FCT fellowship SFRH/BPD/95777/2013 by national funds through FCT - Fundacao para a Ciencia e a Tecnologia/MEC - Ministerio da Educacao e Ciencia and co-funded by QREN and POPH (Programa Operational Potential Humano). SS is supported by FCT-Investigator program.

Listeria monocytogenes is an intracellular food-borne pathogen causing listeriosis in humans. This bacterium deploys an arsenal of virulence factors that act in concert to promote cellular infection. Bacterial surface proteins are of primary importance in the process of host cell invasion. They interact with host cellular receptors, inducing/modulating specific cellular responses. We previously identified Vip, a Listeria surface protein covalently attached to the bacterial cell wall acting as a key virulence factor. We have shown that Vip interacts with Gp96 localized at the surface of host cells during invasion and that this interaction is critical for a successful infection in vivo. To better understand the importance of Vip-Gp96 interaction during infection, we aimed to characterize this interaction at the molecular level. Here we demonstrate that, during infection, L. monocytogenes triggers the cellular redistribution of Gp96, inducing its exposure at the cell surface. Upon infection, Gp96 N-terminal domain is exposed to the extracellular milieu in L2071 fibroblasts and interacts with Vip expressed by Listeria. We identified Gp96 (Asp(1)-Leu(170)) as sufficient to interact with Vip; however, we also showed that the region Tyr(179)-Leu(390) of Gp96 is important for the interaction. Our findings unravel the Listeria-induced surface expression of Gp96 and the topology of its insertion on the plasma membrane and improve our knowledge on the Vip-Gp96 interaction during Listeria infection.

Understanding the strategies used by pathogens to infect, survive, and proliferate in their hosts requires the identification of virulence factors. We developed PCR-based screening of targeted mutants to facilitate quick, simultaneous detection of multiple novel bacterial virulence genes. Based on direct PCR screening of pooled targeted mutants, this approach provides a fast and sensitive measure of virulence attenuation while significantly reducing the number of animals and time required. We demonstrate that the careful design of gene-specific primers allows the direct relative quantification of mixed mutants in infected mouse organs. Indeed, we show that the band intensity of the PCR product is directly related to the quantity of the corresponding strain in a pool of mutants. We applied the PCR-based screening of targeted mutants to the murine model of listeriosis and revealed new genes required for full pathogenicity of Listeria monocytogenes, a facultative human intracellular pathogen. PCR-based screening is a simple, useful, and fast technique to test pools of targeted bacterial mutants in vivo, without the requirements for a rigorous purification step, complicated PCR set-up, or special equipment. This approach can be adapted to other bacterial systems, constituting a significant advance in the field of infection biology.

International audience; Listeria monocytogenes is a food-borne pathogen able to invade non-phagocytic cells. InlA, a L. monocytogenes surface protein, interacts with human E-cadherin to promote bacterial entry. L. monocytogenes internalization is a dynamic process involving co-ordinated actin cytoskeleton rearrangements and host cell membrane remodelling at the site of bacterial attachment. Interaction between E-cadherin and catenins is required to promote Listeria entry, and for the establishment of adherens junctions in epithelial cells. Although several molecular factors promoting E-cadherin-mediated Listeria internalization have been identified, the proteins regulating the transient actin polymerization required at the bacterial entry site are unknown. Here we show that the Arp2/3 complex acts as an actin nucleator during the InlA/E-cadherin-dependent internalization. Using a variety of approaches including siRNA, expression of dominant negative derivatives and pharmacological inhibitors, we demonstrate the crucial role of cortactin in the activation of the Arp2/3 complex during InlA-mediated entry. We also show the requirement of the small GTPase Rac1 and that of Src-tyrosine kinase activity to promote Listeria internalization. Together, these data suggest a model in which Src tyrosine kinase and Rac1 promote recruitment of cortactin and activation of Arp2/3 at Listeria entry site, mimicking events that occur during adherens junction formation.

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection., Author Summary Listeria monocytogenes is a foodborne bacterial pathogen that preferentially infects immunocompromised hosts, eliciting a severe and often lethal disease. In humans, clinical manifestations range from asymptomatic intestinal carriage and gastroenteritis to harsher systemic states of the disease such as sepsis, meningitis or encephalitis, and fetal infections. The surface of L. monocytogenes is decorated with wall teichoic acids (WTAs), a class of carbohydrate-based polymers that contributes to cell surface-related events with implications in physiological processes, such as bacterial division or resistance to antimicrobial peptides (AMPs). The addition of other molecules to the backbone of WTAs modulates their chemical properties and consequently their functionality. In this context, we studied the role of WTA tailoring mechanisms in L. monocytogenes, whose WTAs are strictly decorated with monosaccharides. For the first time, we link WTA glycosylation with AMP resistance by showing that the decoration of L. monocytogenes WTAs with l-rhamnose confers resistance to host defense peptides. We suggest that this resistance is based on changes in the permeability of the cell wall that delay its crossing by AMPs and therefore promote the protection of the bacterial membrane integrity. Importantly, we also demonstrate the significance of this WTA modification in L. monocytogenes virulence.

Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src. This work was supported by the Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Programme (COMPETE), by National Funds through Fundação para a Ciência e Tecnologia (FCT) under Project PTDC/BIA-BCM/100088/2008FCOMP-01-0124- FEDER-008860 and ERANet-Pathogenomics LISTRESS ERA-PTG/0003/ 2010, Project NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions, co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referência Estratégico Nacional, through FEDER, by FCT, and by a Research Grant 2014 by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (to S. S.). M. T. A. received a Travel Grant from Boehringer Ingelheim Fonds. Recipients of Fundação para a Ciência e Tecnologia Doctoral Fellowships BD/43352/2008 and BD/90607/2012. Recipient of EMBO Long Term Post-doctoral Fellowship EMBO ALTF 864-2012. Supported by Ciência 2008 and FCT Investigator Programs COMPETE, POPH, and FCT.

International audience; By comparative genomics, we have identified a gene of the intracellular pathogen Listeria monocytogenes that encodes an LPXTG surface protein absent from nonpathogenic Listeria species. This gene, vip, is positively regulated by PrfA, the transcriptional activator of the major Listeria virulence factors. Vip is anchored to the Listeria cell wall by sortase A and is required for entry into some mammalian cells. Using a ligand overlay approach, we identified a cellular receptor for Vip, the endoplasmic reticulum (ER) resident chaperone Gp96 recently shown to interact with TLRs. The Vip-Gp96 interaction is critical for bacterial entry into some cells. Comparative infection studies using oral and intravenous inoculation of nontransgenic and transgenic mice expressing human E-cadherin demonstrated a role for Vip in Listeria virulence, not only at the intestine level but also in late stages of the infectious process. Vip thus appears as a new virulence factor exploiting Gp96 as a receptor for cell invasion and/or signalling events that may interfere with the host immune response in the course of the infection.

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

Summary Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work "behind the frontline", either supporting virulence effectors or ensuring the survival of the bacterium within its host. We apologize to authors whose relevant work could not be cited owing to space limitations. Research in the group of Molecular Microbiology is funded by the project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions" co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referencia Estrategico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER), the Operational Competitiveness Programme (COMPETE) and FCT (Fundacdo para a Ciencia e Tecnologia), and by projects ERANet Pathogenomics LISTRESS ERA-PTG/0003/2010, PTDC/SAU-MIC/111581/2009FCOMP-FEDER, PTDC/BIA-BCM/100088/2008FCOMP-01-0124-FEDER-008860 and PTDC/BIA-BCM/111215/2009FCOMP-01-0124-FEDER-014178. Filipe Carvalho was supported by FCT doctoral grant SFRH1BD16182512009, and Sandra Sousa by the Ciencia 2008 and FCT-Investigator programs (COMPETE, POPH, and FCT).

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation. We thank AFCU and ALM facilities (IBMC), T Duarte (IBMC), G Almeida, and R Guimarães (ESB) for technical support, M Moroso for the LmΔinlB strain and members of Maiato’s and Sunkel’s lab (IBMC) for fruitful discussions. This work was funded by the Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Programme (COMPETE) and by National funds through FCT (Fundação para a Ciência e Tecnologia) under the projects (PTDC/BIA-BCM/111215/2009FCOMP-01-0124- FEDER-014178, ERANet-Pathogenomics LISTRESS ERAPTG/0003/2010); Project “NORTE-07-0124-FEDER-000002- Host-Pathogen Interactions” co-funded by Programa Operacional Regional do Norte (ON.2, O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through the FEDER and by FCT. E.L., A.C.C., and R.P. were supported by FCT grants (SFRH/BPD/62926/2009, SFRH/BPD/88769/2012 and SFRH/BD/89542/2012, respectively), L.C. by ERASMUS program and S.S. by Ciência 2008 program (COMPETE, POPH, and FCT).

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent at which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we analysed by proteomics mutants of the intracellular pathogen L. monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in large series of new inlG or vip mutants constructed for this purpose were however unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using widely used genetic procedures or during sub-culturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components. Fil: Quereda, Juan J.. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España Fil: Graciela Pucciarelli, M.. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España. Universidad Autónoma de Madrid. Departamento de Biología Molecular. Centro de Biología Molecular "Severo Ochoa"; España Fil: Botello Morte, Laura. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España Fil: Calvo, Enrique. Centro Nacional Investigaciones Cardiovasculares; España Fil: Carvalho, Filipe. Universidade do Porto. Instituto de Biologia Molecular e Celular. Group of Molecular Microbiology; Portugal Fil: Bouchier, Christiane. Institut Pasteur. Département Génomes et Génétique. Plate-forme PF1 Génomique; Francia Fil: Vieira, Ana. Universidade do Porto. Instituto de Biologia Molecular e Celular. Group of Molecular Microbiology; Portugal Fil: Mariscotti, Javier Fernando. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Chakraborty, Trinad. Justus-Liebig-University. Institute of Medical Microbiology; Alemania Fil: Cossart, Pascale. Institut National de la Santé et de la Recherche Médicale. Unité des Interactions Bactéries-Cellules; Francia. Instituto Pasteur; Francia. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia Fil: Hain, Torsten. Justus-Liebig-University. Institute of Medical Microbiology; Alemania Fil: Cabanes, Didier. Universidade do Porto. Instituto de Biologia Molecular e Celular. Group of Molecular Microbiology; España Fil: Garcia del Portillo, Francisco. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España

Gram-positive bacteria contain a cell wall consisting of a thick peptidoglycan layer decorated with surface proteins and polysaccharide-based polymers. The latter include the wall teichoic acids (WTAs), which are anionic glycopolymers covalently linked to the peptidoglycan matrix. They are constituted by a long backbone containing sugars of various sizes (trioses to hexoses) which can be reduced (polyols as in Listeria) or oxidized (uronic acids) and can undergo a variety of species- or often strain-specific modifications and substitutions. These confer unique biochemical properties to WTAs and any defect in the modification or substitution process can potentially affect their biological role in the overall cell wall physiology. Surface proteins can be associated to the cell wall by covalent bonds that anchor the protein to the peptidoglycan lattice. Due to the chemical nature of this bond, covalently bound proteins "co-purify" with peptidoglycan sacculi and are intrinsically insoluble at high temperatures and/or in the presence of ionic detergents. Analysis of this type of proteins therefore requires enzymatic digestion of peptidoglycan for the subsequent release of associated proteins. In contrast, proteins associated to the cell wall by non-covalent interactions are easier to isolate using ionic detergents. In this chapter, we describe methods for the extraction and analysis of (i) WTAs, (ii) covalently, and (iii) non-covalently cell wall-bound surface proteins from the Gram-positive pathogen Listeria monocytogenes.

Listeria monocytogenes is an intracellular Gram-positive pathogen and the etiological agent of listeriosis, a human food-borne disease potentially fatal for certain risk groups. The virulence of L. monocytogenes is supported by a highly complex and coordinated intracellular life cycle that comprises several crucial steps: host cell adhesion and invasion, intracellular multiplication and motility, and intercellular spread. The completion of each stage is dependent on the orchestrated activity of specialized bacterial factors, in turn tightly controlled by a specific set of regulators. Some virulence factors and modulators also assume an important role in bacterial resistance and evasion to host defense mechanisms. In the last years, the advent of genomics promoted an increasingly prolific identification and functional characterization of new Listeria virulence factors. In this review, we summarize the current knowledge on nearly 50 molecules deployed by L. monocytogenes to promote its cell infection cycle.

International audience; Intracellular pathogens such as $Listeria\ monocytogenes$ subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon IFN-stimulated genes (ISGs). IFN-$\lambda$ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with $lntA$-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1$^{+/-}$ mice or when $lntA$ was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-$\lambda$-mediated immune response to control bacterial colonization of the host.

Attachment to mucosal surfaces is the initial event in the pathogenesis of the human foodborne pathogen Listeria monocytogenes. By use of comparative genomics, we identified a L. monocytogenes-specific gene, lapB, that encodes an LPXTG surface protein that is absent from nonpathogenic Listeria species. We showed that lapB expression is positively regulated by PrfA, the major transcriptional activator of the virulence genes of Listeria species, and is up-regulated in mouse spleens during infection. We demonstrated that LapB is an SrtA-anchored surface protein required for adhesion to and entry into mammalian cells and for virulence following intravenous or oral inoculation in mice. Our results highlight LapB as a new L. monocytogenes virulence adhesin with a function that is supported by its unique N-terminal domain through the probable interaction with a cellular receptor.

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence., Author Summary The facultative intracellular bacterial pathogen Listeria monocytogenes is the etiological agent of a severe foodborne disease. In humans it causes a variety of manifestations ranging from asymptomatic intestinal carriage and gastroenteritis to invasive and disseminated severe diseases. Septicemia, meningoencephalitis, and infection of the foetus in pregnant women are the most serious clinical features of listeriosis. Virulence is a trait that only manifests in a susceptible host, involving a highly coordinated interaction between bacterial factors and host components. This article reports the use of in vivo genome expression profiling as a powerful approach to gain a detailed understanding of the Listeria responses and the molecular cross-talk taking place in infected mice. We showed that, during infection, L. monocytogenes shifts the expression of its entire genome to promote virulence, subverting host defenses and adapting to host conditions. This first analysis of L. monocytogenes gene expression in vivo significantly enhances our understanding of the means by which intracellular pathogens promote infection.

Listeria monocytogenes is an intracellular foodborne pathogen that causes listeriosis, an infection characterized by gastroenteritis, meningitis, encephalitis, and maternofetal infections in humans. L. monocytogenes enters the host via contaminated foods, invades the small intestine, translocates to mesenteric lymph nodes, and spreads to the liver, spleen, brain and, in pregnant women, the fetoplacental unit. Many pathogenicity tests for studying L. monocytogenes have been developed, including tests using laboratory animals. A number of small animal species can be experimentally infected with Listeria. Mice and guinea pigs can be infected either intragastrically or intravenously, and virulence evaluated either by enumerating bacteria within infected target organs or by evaluating the 50% lethal dose (LD50). Although mice and guinea pigs can be infected with Listeria by a variety of routes, the intragastric route is the most relevant to the human foodborne listeriosis.

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N -acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA , in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-β response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.

Listeria monocytogenes expresses surface proteins covalently anchored to the peptidoglycan by sortase enzymes. Inactivation of srtA attenuates Listeria virulence in mice (H. Bierne, S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garcia-del Portillo, O. Schneewind, and P. Cossart, Mol. Microbiol. 43: 869-881, 2002). We show here that an srtA mutant is more attenuated than an internalin mutant in orally infected guinea pigs and transgenic mice expressing human E-cadherin (hEcad mice), indicating the involvement of other SrtA substrates, LPXTG proteins, in food-borne listeriosis. Data recently generated with a listerial DNA macroarray identified two LPXTG protein-encoding genes present in the genomes of L. monocytogenes strains and absent from all other Listeria species, inlI (lmo0333) and inlJ (lmo2821). They also revealed two other LPXTG protein-encoding genes, ORF29 and ORF2568, present only in a subclass of L. monocytogenes serovars, including the epidemic serovar 4b. We report here that an inlJ deletion mutant, in contrast to inlI and ORF29 mutants, is significantly attenuated in virulence after intravenous infection of mice or oral inoculation of hEcad mice. Interestingly, a ΔORF2568 strain showed a slight increase in virulence. inlJ encodes a leucine-rich repeat (LRR) protein that is structurally related to the listerial invasion factor internalin. However, the consensus sequence of the InlJ LRR defines a novel subfamily of cysteine-containing LRRs in bacteria. In conclusion, this postgenomic approach identified InlJ as a new virulence factor among the proteins belonging to the internalin family in L. monocytogenes .

International audience; The use of the food-grade bacterium Lactococcus lactis as antigen delivery vehicle at the mucosal level is an attractive vaccination strategy intensively explored during the last decade. In this study, we developed L. lactis strains which could be used as a DNA delivery vector to combine both advantages of mucosal delivery and of DNA vaccination. To render lactococci capable of invading epithelial cells, the Listeria monocytogenes inlA gene was cloned and expressed in L. lactis under transcriptional control of the native promoter. Western blot and immunofluorescence assays revealed that recombinant lactococci efficiently displayed the cell wall anchored form of InlA. We demonstrated that this expression promotes internalization of L. lactis inlA+ into the human epithelial cell line Caco-2. Gentamicin assay showed that invasiveness of L. lactis in these cells is approximately 100-fold higher for L. lactis inlA+ than for wild type (wt) L. lactis strains. Moreover, we showed that L. lactis inlA+ is able to enter intestinal cells in vivo, after oral inoculation of guinea pigs. After internalization, L. lactis inlA+ was able to deliver a functional eukaryotic gfp gene into epithelial Caco-2 cells; GFP was detected in 1% of internalized cells. The L. lactis inlA+ strain will be a useful bacterial vector for the development of new live oral DNA vaccines. It also constitutes an interesting new model to study the role of internalin in bacterial localization in the animal host.

International audience; On the basis of the recently determined genome sequence of Listeria monocytogenes, we performed a global analysis of the surface-protein-encoding genes. Only proteins displaying a signal peptide were taken into account. Forty-one genes encoding LPXTG proteins, including the previously known internalin gene family, were detected. Several genes encoding proteins that, like InlB and Ami, possess GW modules that attach them to lipoteichoic acids were also identified. Additionally, the completed genome sequence revealed genes encoding proteins potentially anchored in the cell membrane by a hydrophobic tail as well as genes encoding P60-like proteins and lipoproteins. We describe these families and discuss their putative implications for host-pathogen interactions.

RNA fingerprinting by arbitrarily primed PCR was used to isolate Sinorhizobium meliloti genes regulated during the symbiotic interaction with alfalfa ( Medicago sativa ). Sixteen partial cDNAs were isolated whose corresponding genes were differentially expressed between symbiotic and free-living conditions. Thirteen sequences corresponded to genes up-regulated during symbiosis, whereas three were instead repressed during establishment of the symbiotic interaction. Seven cDNAs corresponded to known or predicted nif and fix genes. Four presented high sequence similarity with genes not yet identified in S. meliloti , including genes encoding a component of the pyruvate dehydrogenase complex, a cell surface protein component, a copper transporter, and an argininosuccinate lyase. Finally, five cDNAs did not exhibit any similarity with sequences present in databases. A detailed expression analysis of the nine non- nif-fix genes provided evidence for an unexpected variety of regulatory patterns, most of which have not been described so far.

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment., Author Summary Listeria monocytogenes (Lm) is a ubiquitous bacterium that survives and thrives within the environment, and a facultative intracellular pathogen that induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we identify ActA, which is a major Lm virulence determinant mediating actin-based motility, as critical for bacterial aggregation and biofilm formation. ActA promotes Lm aggregation via direct ActA-ActA interaction and ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation. Whereas ActA-mediated Lm aggregation is not observed in infected tissues, it occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the gut lumen, and are shed in the feces three order of magnitude more and for twice as long than bacteria unable to aggregate. This study identifies a novel function for ActA, which plays a key role in Lm persistence within the host and transmission.