Search

My scientific career has resulted from key decisions and reorientations, sometimes taken rapidly but not always, guided by discussions or collaborations with amazing individuals from whom I learnt a lot scientifically and humanly. I had never anticipated that I would accomplish so much in what appeared as terra incognita when I started to interrogate the mechanisms underlying the virulence of the bacterium Listeria monocytogenes . All this has been possible thanks to a number of talented team members who ultimately became friends.

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.)

Background: Small regulatory RNAs (sRNAs) are widely found in bacteria and play key roles in many important physiological and adaptation processes. Studying their evolution and screening for events of coevolution with other genomic features is a powerful way to better understand their origin and assess a common functional or adaptive relationship between them. However, evolution and coevolution of sRNAs with coding genes have been sparsely investigated in bacterial pathogens., Results: We designed a robust and generic phylogenomics approach that detects correlated evolution between sRNAs and protein-coding genes using their observed and inferred patterns of presence-absence in a set of annotated genomes. We applied this approach on 79 complete genomes of the Listeria genus and identified fifty-two accessory sRNAs, of which most were present in the Listeria common ancestor and lost during Listeria evolution. We detected significant coevolution between 23 sRNA and 52 coding genes and inferred the Listeria sRNA-coding genes coevolution network. We characterized a main hub of 12 sRNAs that coevolved with genes encoding cell wall proteins and virulence factors. Among them, an sRNA specific to L. monocytogenes species, rli133, coevolved with genes involved either in pathogenicity or in interaction with host cells, possibly acting as a direct negative post-transcriptional regulation., Conclusions: Our approach allowed the identification of candidate sRNAs potentially involved in pathogenicity and host interaction, consistent with recent findings on known pathogenicity actors. We highlight four sRNAs coevolving with seven internalin genes, some of which being important virulence factors in Listeria.

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, a facultative intracellular bacterium responsible for severe foodborne infections, is now recognized as a multifaceted model in infection biology. Comprehensive studies of the molecular and cellular basis of the infection have unraveled how the bacterium crosses the intestinal and feto-placental barriers, invades several cell types in which it multiplies and moves, and spreads from cell to cell. Interestingly, although Listeria does not actively invade host cell organelles, it can interfere with their function. We discuss the effect of Listeria on the endoplasmic reticulum (ER) and the mechanisms leading to the fragmentation of the mitochondrial network and its consequences, and review the strategies used by Listeria to subvert nuclear functions, more precisely to control host gene expression at the chromatin level., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Listeria monocytogenes is a human, food-borne pathogen. Genomic comparisons between L. monocytogenes and Listeria innocua, a closely related non-pathogenic species, were pivotal in the identification of protein-coding genes essential for virulence. However, no comprehensive comparison has focused on the non-coding genome. We used strand-specific cDNA sequencing to produce genome-wide transcription start site maps for both organisms, and developed a publicly available integrative browser to visualize and analyze both transcriptomes in different growth conditions and genetic backgrounds. Our data revealed conservation across most transcripts, but significant divergence between the species in a subset of non-coding RNAs. In L. monocytogenes, we identified 113 small RNAs (33 novel) and 70 antisense RNAs (53 novel), significantly increasing the repertoire of ncRNAs in this species. Remarkably, we identified a class of long antisense transcripts (lasRNAs) that overlap one gene while also serving as the 5' UTR of the adjacent divergent gene. Experimental evidence suggests that lasRNAs transcription inhibits expression of one operon while activating the expression of another. Such a lasRNA/operon structure, that we named 'excludon', might represent a novel form of regulation in bacteria.

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.)

We report the complete and annotated genome sequence of the animal pathogen Listeria ivanovii subsp. ivanovii strain PAM 55 (serotype 5), isolated in 1997 in Spain from an outbreak of abortion in sheep. The sequence and its analysis are available at an interactive genome browser at the Institut Pasteur (http://genolist.pasteur.fr/LivaList/).

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.

Actin-based motility is used by various pathogens such as Listeria and Shigella for dissemination within cells: and tissues, yet host factors counteracting this process have not been identified. We have recently discovered that infected host cells can prevent actin-based motility of Shigella by compartmentalizing bacteria inside 'septin cages,' revealing a novel mechanism of host defense that restricts dissemination. Because bacterial proteins controlling actin-based motility also regulate the autophagy process, we hypothesized and then established a link between septin caging and autophagy. Together, these results unveiled the first cellular mechanism that counteracts pathogen dissemination. Understanding the role of septins, a so far poorly characterized component of the cytoskeleton, will thus provide new insights into bacterial infection and autophagy.

This review provides an overview of recent progress in the exploration of genomic, transcriptomic, and proteomic data in Listeria spp. to understand genome evolution and diversity, as well as physiological aspects of metabolism utilized by the bacteria when growing in diverse and varied environments.

Listeria monocytogenes is an ubiquitous gram positive bacterium responsible for a severe food borne disease in human and animals which has become a multifaceted model. Indeed, it is a facultative intracellular bacterium that induces an amazingly rapid and sterilizing T-cell response and has been--and still is--widely used by immunologists. Listeria's strategies to invade non-phagocytic cells and spread from cell to cell have been and still are outstandingly instrumental to address key questions in cell biology. Orally acquired listeriosis can now be studied in relevant animal models. Finally, Listeria is among the few bacteria which have so rapidly benefited from comparative genomics. It now ranks among the most documented pathogens.

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-beta 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.

Genome analyses have revealed that the Gram-positive bacterial species Listeria monocytogenes and L. innocua contain a large number of genes encoding surface proteins predicted to be covalently bound to the cell wall (41 and 34, respectively). The function of most of these proteins is unknown and they have not even been identified biochemically. Here, we report the first characterization of the Listeria cell wall proteome using a nonelectrophoretic approach. The material analyzed consisted of a peptide mixture obtained from a cell wall extract insoluble in boiling 4% SDS. This extract, containing peptidoglycan (intrinsically resistant to proteases) and strongly associated proteins, was digested with trypsin in a solution with 0.01% SDS, used to favor protein digestion throughout the peptidoglycan. The resulting complex peptide mixture was fractionated and analyzed by two-dimensional nanoliquid chromatography coupled to ion-trap mass spectrometry. A total of 30 protein species were unequivocally identified in cell wall extracts of the genome strains L. monocytogenes EGD-e (19 proteins) and L. innocua CLIP11262 (11 proteins). Among them, 20 proteins bearing an LPXTG motif recognized for covalent anchoring to the peptidoglycan were identified. Other proteins detected included peptidoglycan-lytic enzymes, a penicillin-binding protein, and proteins bearing an NXZTN motif recently proposed to direct protein anchoring to the peptidoglycan. The marked sensitivity of the method makes it highly attractive in the post-genome era for defining the cell wall proteome in any bacterial species. This information will be useful to study novel protein-peptidoglycan associations and to rapidly identify new targets in the surface of important bacterial pathogens.

Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Interaction of internalin with E-cadherin promotes entry of Listeria monocytogenes into human epithelial cells. This process requires actin cytoskeleton rearrangements. Here we show, by using a series of stably transfected cell lines expressing E-cadherin variants, that the ectodomain of E-cadherin is sufficient for bacterial adherence and that the intracytoplasmic domain is required for entry. The critical cytoplasmic region was further mapped to the beta-catenin binding domain. Because beta-catenin is known to interact with alpha-catenin, which binds to actin, we generated a fusion molecule consisting of the ectodomain of E-cadherin and the actin binding site of alpha-catenin. Cells expressing this chimera were as permissive as E-cadherin-expressing cells. In agreement with these data, alpha- and beta-catenins as well as E-cadherin clustered and colocalized at the entry site, where F-actin then accumulated. Taken together, these results reveal that E-cadherin, via beta- and alpha-catenins, can trigger dynamic events of actin polymerization and membrane extensions culminating in bacterial uptake.

The surface protein InlB of the bacterial pathogen Listeria monocytogenes is required for inducing phagocytosis in various nonphagocytic mammalian cell types in vitro. InlB causes tyrosine phosphorylation of host cell adaptor proteins, activation of phosphoinositide 3-kinase, and rearrangements of the actin cytoskeleton. These events lead to phagocytic uptake of the bacterium by the host cell. InlB belongs to the internalin family of Listeria proteins, which also includes InlA, another surface protein involved in host cell invasion. The internalins are the largest class of bacterial proteins containing leucine-rich repeats (LRR), a motif associated with protein-protein interactions. The LRR motif is found in a functionally diverse array of proteins, including those involved in the plant immune system and in the mammalian innate immune response. Structural and functional interpretations of the sequences of internalin family members are presented in light of the recently determined x-ray crystal structure of the InlB LRR domain.

Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the IcsA (VirG) surface protein in S. flexneri. ActA and IcsA share no sequence homology. To assess the role of the two proteins in the generation of actin-based movement, we expressed them in the genetic context of two non-actin polymerizing, non-pathogenic bacterial species, Listeria innocua and Escherichia coli. In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing IcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half-lives) were similar in IcsA- and ActA-induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.

A gene homologous to the actA gene of Listeria monocytogenes was cloned from Listeria ivanovii (strain CLIP257) by chromosome walking starting from the ilo gene that encodes the pore-forming toxin ivanolysin. The nucleotide sequence revealed that this gene, named iactA, encodes a protein of 1,044 amino acids (IactA) comprising a central region with seven highly conserved tandem proline-rich repeats of 47 amino acids. Although IactA and ActA share an overall similar structure, these two proteins are only distantly related. Like ActA, IactA migrates aberrantly on sodium dodecyl sulfate gels. When expressed in an L. monocytogenes actA deletion mutant strain, iactA restored actin polymerization.

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp-2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic-like activity. CNF1-treated cells acquired the ability to ingest latex beads as well as non-invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen-induced phagocytosis, since L. innocua transformed with DNA coding for the pore-forming toxin listeriolysin O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1-producing E. coli may invade epithelia by this novel induced phagocytic-like mechanism.

Two DNA fragments encoding part of the listeriolysin O of Listeria monocytogenes have been used as probes in Southern blot experiments to detect the presence of the gene in the genus Listeria. Under stringent conditions, and using a fragment internal to the gene, only L. monocytogenes reacted with the probe.

Abstract Background Small regulatory RNAs (sRNAs) are widely found in bacteria and play key roles in many important physiological and adaptation processes. Studying their evolution and screening for events of coevolution with other genomic features is a powerful way to better understand their origin and assess a common functional or adaptive relationship between them. However, evolution and coevolution of sRNAs with coding genes have been sparsely investigated in bacterial pathogens. Results We designed a robust and generic phylogenomics approach that detects correlated evolution between sRNAs and protein-coding genes using their observed and inferred patterns of presence-absence in a set of annotated genomes. We applied this approach on 79 complete genomes of the Listeria genus and identified fifty-two accessory sRNAs, of which most were present in the Listeria common ancestor and lost during Listeria evolution. We detected significant coevolution between 23 sRNA and 52 coding genes and inferred the Listeria sRNA-coding genes coevolution network. We characterized a main hub of 12 sRNAs that coevolved with genes encoding cell wall proteins and virulence factors. Among them, an sRNA specific to L. monocytogenes species, rli133, coevolved with genes involved either in pathogenicity or in interaction with host cells, possibly acting as a direct negative post-transcriptional regulation. Conclusions Our approach allowed the identification of candidate sRNAs potentially involved in pathogenicity and host interaction, consistent with recent findings on known pathogenicity actors. We highlight four sRNAs coevolving with seven internalin genes, some of which being important virulence factors in Listeria.

During infection, the foodborne bacterial pathogen Listeria monocytogenes dynamically influences the gene expression profile of host cells. Infection-induced transcriptional changes are a typical feature of the host-response to bacteria and contribute to the activation of protective genes such as inflammatory cytokines. However, by using specialized virulence factors, bacterial pathogens can target signaling pathways, transcription factors, and epigenetic mechanisms to alter host gene expression, thereby reprogramming the response to infection. Therefore, the transcriptional profile that is established in the host is delicately balanced between antibacterial responses and pathogenesis, where any change in host gene expression might significantly influence the outcome of infection. In this review, we discuss the known transcriptional and epigenetic processes that are engaged during Listeria monocytogenes infection, the virulence factors that can remodel them, and the impact these processes have on the outcome of infection.

ABSTRACT 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.

ABSTRACT As for many model organisms, the amount of Listeria omics data produced has recently increased exponentially. There are now >80 published complete Listeria genomes, around 350 different transcriptomic data sets, and 25 proteomic data sets available. The analysis of these data sets through a systems biology approach and the generation of tools for biologists to browse these various data are a challenge for bioinformaticians. We have developed a web-based platform, named Listeriomics, that integrates different tools for omics data analyses, i.e., (i) an interactive genome viewer to display gene expression arrays, tiling arrays, and sequencing data sets along with proteomics and genomics data sets; (ii) an expression and protein atlas that connects every gene, small RNA, antisense RNA, or protein with the most relevant omics data; (iii) a specific tool for exploring protein conservation through the Listeria phylogenomic tree; and (iv) a coexpression network tool for the discovery of potential new regulations. Our platform integrates all the complete Listeria species genomes, transcriptomes, and proteomes published to date. This website allows navigation among all these data sets with enriched metadata in a user-friendly format and can be used as a central database for systems biology analysis. IMPORTANCE In the last decades, Listeria has become a key model organism for the study of host-pathogen interactions, noncoding RNA regulation, and bacterial adaptation to stress. To study these mechanisms, several genomics, transcriptomics, and proteomics data sets have been produced. We have developed Listeriomics, an interactive web platform to browse and correlate these heterogeneous sources of information. Our website will allow listeriologists and microbiologists to decipher key regulation mechanism by using a systems biology approach.

How invasive bacteria exploit mammalian host cell components to induce their entry into cells has received a lot of attention in the last two decades. Model organisms have emerged and helped understanding the various mechanisms that are used. Among those, Listeria monocytogenes is one of the most documented organisms. It enters into cells via two bacterial proteins, internalin (also called InlA) and InlB, which interact with cell surface receptors, E-cadherin and the hepatocyte growth factor receptor, Met, respectively. These interactions initiate a series of events that leads to actin polymerization, membrane invagination and bacterial internalization. Investigations on internalin- and InlB-mediated entries have repeatedly shown that Listeria fully usurps the host cell machinery. Moreover, they have also shown that previously unknown components discovered during the study of Listeria invasion play a role either in E-cadherin-mediated cell–cell adhesion or Met signalling. Unexpectedly, recent studies have highlighted a role for clathrin in Listeria InlB-mediated actin polymerization and entry, revealing a new role for this endocytic protein, i.e. in bacterial-induced internalization. Furthermore, comparative studies have demonstrated that the clathrin-mediated endocytosis machinery is also used in the internalin–E-cadherin pathway, and for the entry of other bacteria that enter by a ‘zipper’ mechanism. By contrast, the clathrin-mediated endocytic machinery is not used by bacteria that inject effectors into mammalian cells via the type III secretion system and enter by the so-called trigger mechanism, characterized by enormous membrane ruflles that result in the macropinocytosis of the corresponding bacteria. Finally, adherent bacteria, for example enteropathogenic Escherichia coli (EPEC), also co-opt clathrin to induce the formation of actin-rich pedestals. Together, these new data illuminate our view on how actin rearrangements may be coupled to clathrin recruitment during bacterial infection. They also shed light on a new function for clathrin in mammalian cells, i.e. internalization of objects much larger than previously accepted. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Evidence for pleiotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA-deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl-inositol-specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo-protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase, in addition, two prf4-specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co-transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA-prfA transcript, in late-exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis. [ABSTRACT FROM AUTHOR]

Abstract: The Listeria monocytogenes genome includes a large family of proteins harbouring leucine-rich repeats known as internalins (Inl). The generation of novel mutants and comparative analysis of Inl variability among Listeria and other bacterial genomes suggest that beyond the extensively-studied invasins, InlA and InlB, additional internalins also play important functions in the infectious process. [Copyright &y& Elsevier]

The article analyzes the effects of the modifications induced by the K220T substitution on the activity of PrfA and on the protein conformation. It shows that a naturally occurring mutation in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity.