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Functional diversity within isogenic spatially organized bacterial populations has been shown to trigger emergent community properties such as stress tolerance. Taking advantage of confocal laser scanning microscopy combined with a transcriptional fluorescent fusion reporting at single cell scale the expression of the glutamic acid decarboxylasegadBinE. coliO157:H7, it was possible to visualize for the first-time spatial patterns of bacterial gene expression in microcolonies grown in a gelled matrix. ThegadBgene is involved inE. colitolerance to acidic conditions and its strong over-expression was observed locally on the periphery of embedded microcolonies grown in acidic hydrogels. This spatialization ofgadBexpression did not correlate with live/dead populations that appeared randomly distributed in the colonies. While the planktonic population of the pathogens was eradicated by an exposition to a pH of 2 (HCl) for 4h, mimicking a stomachal acidic stress, bacteria grown in gel-microcolonies were poorly affected by this treatment, in particular in conditions wheregadBwas spatially overexpressed. Consequences of these results for food safety are further discussed.

International audience; In complex food systems, bacteria live in heterogeneous microstructures, and the population displays phenotypic heterogeneities at the single-cell level. This review provides an overview of spatiotemporal drivers of phenotypic heterogeneity of bacterial pathogens in food matrices at three levels. The first level is the genotypic heterogeneity due to the possibility for various strains of a given species to contaminate food, each of them having specific genetic features. Then, physiological heterogeneities are induced within the same strain, due to specific microenvironments and heterogeneous adaptative responses to the food microstructure. The third level of phenotypic heterogeneity is related to cellular heterogeneity of the same strain in a specific microenvironment. Finally, we consider how these phenotypic heterogeneities at the single-cell level could be implemented in mathematical models to predict bacterial behavior and help ensure microbiological food safety.

International audience; Emergence and dissemination of multidrug resistance among pathogenic Escherichia coli have posed a serious threat to public health across developing and developed countries. In combination with a flexible repertoire of virulence mechanisms, E. coli can cause a vast range of intestinal (InPEC) and extraintestinal (ExPEC) diseases but only a very limited number of antibiotics still remains effective against this pathogen. Hence, a broad spectrum E. coli vaccine could be a promising alternative to prevent the burden of such diseases, while offering the potential for covering against several InPEC and ExPEC at once. SslE, the Secreted and Surface-associated Lipoprotein of E. coli, is a widely distributed protein among InPEC and ExPEC. SslE functions ex vivo as a mucinase capable of degrading mucins and reaching the surface of mucus-producing epithelial cells. SslE was identified by reverse vaccinology as a protective vaccine candidate against an ExPEC murine model of sepsis, and further shown to be cross-effective against other ExPEC and InPEC models of infection. In this study, we aimed to gain insight into the immune response to antigen SslE and identify an immunization strategy suited to generate robust mucosal and systemic immune responses. We showed, by analyzing T cell and antibody responses, that mice immunized with SslE via an intranasal prime followed by two intramuscular boosts developed an enhanced overall immune response compared to either intranasal-only or intramuscular-only protocols. Importantly, we also report that this regimen of immunization did not impact the richness of the murine gut microbiota, and mice had a comparable cecal microbial composition, whether immunized with SslE or PBS. Collectively, our findings further support the use of SslE in future vaccination strategies to effectively target both InPEC and ExPEC while not perturbing the resident gut microbiota.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans, such as hemolytic and uremic syndrome. It has been previously demonstrated that the interplay between EHEC and nitric oxide (NO), a mediator of the host immune innate response, is critical for infection outcome, since NO affects both Shiga toxin (Stx) production and adhesion to enterocytes. In this study, we investigated the role of the NO reductase NorVW in the virulence and fitness of two EHEC strains in a murine model of infection. We determined that the deletion of norVW in the strain O91:H21 B2F1 has no impact on its virulence, whereas it reduces the ability of the strain O157:H7 620 to persist in the mouse gut and to produce Stx. We also revealed that the fitness defect of strain 620 &Delta, norVW is strongly attenuated when mice are treated with an NO synthase inhibitor. Altogether, these results demonstrate that the NO reductase NorVW participates in EHEC resistance against NO produced by the host and promotes virulence through the modulation of Stx synthesis. The contribution of NorVW in the EHEC infectious process is, however, strain-dependent and suggests that the EHEC response to nitrosative stress is complex and multifactorial.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Epub 2019 Feb 26; International audience; Enterohaemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for bloody diarrhoea and renal failure in humans. While Shiga toxin (Stx) is the cardinal virulence factor of EHEC, its production by E. coli is not sufficient to cause disease and many Shiga-toxin producing E. coli (STEC) strains have never been implicated in human infection. So far, the pathophysiology of EHEC infection is not fully understood and more knowledge is needed to characterize the "auxiliary" factors that enable a STEC strain to cause disease in humans. In this study, we applied a recombinase-based in vivo expression technology (RIVET) to the EHEC reference strain EDL933 in order to identify genes specifically induced during the infectious process, using mouse as an infection model. We identified 31 in vivo-induced (ivi) genes having functions related to metabolism, stress adaptive response and bacterial virulence or fitness. Eight of the 31 ivi genes were found to be heterogeneously distributed in EHEC strains circulating in France these last years. In addition, they are more prevalent in strains from the TOP 7 priority serotypes and particularly strains carrying significant virulence determinants such as Stx2 and intimin adhesin. This work sheds further light on bacterial determinants over-expressed in vivo during infection that may contribute to the potential of STEC strains to cause disease in humans.

Enterohemorrhagic E. coli (EHEC) is a major cause of large outbreaks worldwide associated with hemorrhagic colitis and hemolytic uremic syndrome. While vaccine development is warranted, a licensed vaccine, specific for human use, against EHEC is not yet available. In this study, the reverse vaccinology approach combined with genomic, transcriptional and molecular epidemiology data was applied on the EHEC O157:H7 genome to select new potential vaccine candidates. Twenty-four potential protein antigens were identified and one of them (MC001) was successfully expressed onto Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing this vaccine candidate was immunogenic, raising a specific antibody response. Immunization with the MC001 candidate was able to reduce the bacterial load of EHEC O157:H7 strain in feces, colon and caecum tissues after murine infection. MC001 is homologue to lipid A deacylase enzyme (LpxR), and to our knowledge, this is the first study describing it as a potential vaccine candidate. Gene distribution and sequence variability analysis showed that MC001 is present and conserved in EHEC and in enteropathogenic E. coli (EPEC) strains. Given the high genetic variability among and within E. coli pathotypes, the identification of such conserved antigen suggests that its inclusion in a vaccine might represent a solution against major intestinal pathogenic strains.

Part of special issue:14ème Congrès Francophone d’Allergologie - 16-19 avril 2019 - Paris, Palais des Congrès; Le microbiote comme outil thérapeutique dans l’allergie alimentaire

Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen responsible for human diseases ranging from diarrhoea to life-threatening complications. Survival of the pathogen and modulation of virulence gene expression along the human gastrointestinal tract (GIT) are key features in bacterial pathogenesis, but remain poorly described, due to a paucity of relevant model systems. This review will provide an overview of the in vitro and in vivo studies investigating the effect of abiotic (e.g., gastric acid, bile, low oxygen concentration or fluid shear) and biotic (e.g., gut microbiota, short chain fatty acids or host hormones) parameters of the human gut on EHEC survival and/or virulence (especially in relation with motility, adhesion and toxin production). Despite their relevance, these studies display important limitations considering the complexity of the human digestive environment. These include the evaluation of only one single digestive parameter at a time, lack of dynamic flux and compartmentalization, and the absence of a complex human gut microbiota. In a last part of the review, we will discuss how dynamic multi-compartmental in vitro models of the human gut represent a novel platform for elucidating spatial and temporal modulation of EHEC survival and virulence along the GIT, and provide new insights into EHEC pathogenesis.

International audience; Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E coil responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coil O157:H7 outside the bovine gut. The E. coil O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coil MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra intestinal environments was analyzed. E. coil MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 151 degrees C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HOP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E coil MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coil strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15 degrees C. E coil MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coil to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coil MC2 in the farm environment.

Probiotiques et allergie. Part of special issue: 13eme Congres Francophone d'Allergologie - 17-20 avril 2018 - Paris, Palais des Congres

International audience; The gastrointestinal tract (GIT) of healthy cattle is the main reservoir of enterohaemorrhagic Escherichia coli (EHEC). Therefore, it is crucial to better understand the physiology of EHEC in the bovine GIT. In this study, we demonstrate that aspartate present in bovine small intestine content (BSIC), was exhausted after incubation of the reference EHEC strain EDL933 but was poorly assimilated by the endogenous microbiota. Furthermore, the bovine commensal E. coli strain BG1 appeared less efficient than EDL933 in aspartate assimilation suggesting a competitive ability of EHEC to assimilate this amino acid. Our results strongly suggest that aspartate, internalized via the DcuA aspartate:succinate antiporting system, is then converted to fumarate and carbamoyl-aspartate, the precursor for UMP biosynthesis. Aspartate assimilation by these two pathways conferred a competitive growth advantage to EHEC in BSIC. In summary, supply of intracellular fumarate due to aspartate deamination and used as an electron acceptor for anaerobic fumarate respiration, as well as de novo synthesis of pyrimidine from aspartate appear to be important pathways favouring EHEC persistence in the bovine gut. Aspartate probably represents an ecological niche for EHEC in the bovine small intestine.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are human pathogens responsible for bloody diarrhea and renal failures. EHEC employ a type 3 secretion system to attach directly to the human colonic epithelium. This structure is encoded by the locus of enterocyte effacement (LEE) whose expression is regulated in response to specific nutrients. In this study, we show that the mucin-derived sugars N-acetylglucosamine (NAG) and N-acetylneuraminic acid (NANA) inhibit EHEC adhesion to epithelial cells through down-regulation of LEE expression. The effect of NAG and NANA is dependent on NagC, a transcriptional repressor of the NAG catabolism in E. coli. We show that NagC is an activator of the LEE1 operon and a critical regulator for the colonization of mice intestine by EHEC. Finally, we demonstrate that NAG and NANA as well as the metabolic activity of Bacteroides thetaiotaomicron affect the in vivo fitness of EHEC in a NagC-dependent manner. This study highlights the role of NagC in coordinating metabolism and LEE expression in EHEC and in promoting EHEC colonization in vivo.

Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.

Erratum to: Unraveling the pectinolytic function of Bacteroides xylanisolvens using a RNA-seq approach and mutagenesis BMC Genomics 2016 17:426 DOI: 10.1186/s12864-016-2758-3 After publication of the original article [1], it came to the authors’ attention that a funding source received by B. Henrissat had been accidently omitted from the Acknowledgements. The following sentence should have been included in the original article; International audience; BACKGROUND: Diet and particularly dietary fibres have an impact on the gut microbiome and play an important role in human health and disease. Pectin is a highly consumed dietary fibre found in fruits and vegetables and is also a widely used additive in the food industry. Yet there is no information on the effect of pectin on the human gut microbiome. Likewise, little is known on gut pectinolytic bacteria and their enzyme systems. This study was undertaken to investigate the mechanisms of pectin degradation by the prominent human gut symbiont Bacteroides xylanisolvens.RESULTS: Transcriptomic analyses of B. xylanisolvens XB1A grown on citrus and apple pectins at mid- and late-log phases highlighted six polysaccharide utilization loci (PUL) that were overexpressed on pectin relative to glucose. The PUL numbers used in this report are those given by Terrapon et al. (Bioinformatics 31(5):647-55, 2015) and found in the PUL database: http://www.cazy.org/PULDB/. Based on their CAZyme composition, we propose that PUL 49 and 50, the most overexpressed PULs on both pectins and at both growth phases, are involved in homogalacturonan (HG) and type I rhamnogalacturonan (RGI) degradation, respectively. PUL 13 and PUL 2 could be involved in the degradation of arabinose-containing side chains and of type II rhamnogalacturonan (RGII), respectively. Considering that HG is the most abundant moiety (>70%) within pectin, the importance of PUL 49 was further investigated by insertion mutagenesis into the susC-like gene. The insertion blocked transcription of the susC-like and the two downstream genes (susD-like/FnIII). The mutant showed strong growth reduction, thus confirming that PUL 49 plays a major role in pectin degradation.CONCLUSION: This study shows the existence of six PULs devoted to pectin degradation by B. xylanisolvens, one of them being particularly important in this function. Hence, this species deploys a very complex enzymatic machinery that probably reflects the structural complexity of pectin. Our findings also highlight the metabolic plasticity of B. xylanisolvens towards dietary fibres that contributes to its competitive fitness within the human gut ecosystem. Wider functional and ecological studies are needed to understand how dietary fibers and especially plant cell wall polysaccharides drive the composition and metabolism of the fibrolytic and non-fibrolytic community within the gut microbial ecosystem.

International audience; Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2), the main virulence factor of EHEC. Among them, soluble factors produced by human gut microbiota and in particular, by the predominant species Bacteroides thetaiotaomicron (B. thetaiotaomicron), inhibit Stx2 gene expression. In this study, we investigated the molecular mechanisms underlying the B. thetaiotaomicron-dependent inhibition of Stx2 production by EHEC. We determined that Stx2-regulating molecules are resistant to heat treatment but do not correspond to propionate and acetate, two short-chain fatty acids produced by B. thetaiotaomicron. Moreover, screening of a B. thetaiotaomicron mutant library identified seven mutants that do not inhibit Stx2 synthesis by EHEC. One mutant has impaired production of BtuB, an outer membrane receptor for vitamin B12. Together with restoration of Stx2 level after vitamin B12 supplementation, these data highlight vitamin B12 as a molecule produced by gut microbiota that modulates production of a key virulence factor of EHEC and consequently may affect the outcome of an infection.

Background Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1AT. Results Transcriptomic analyses of B. xylanisolvens XB1AT grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. Conclusion This study shows that xylan degradation by B. xylanisolvens XB1AT is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1AT has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2680-8) contains supplementary material, which is available to authorized users.

Xenorhabdus nematophila engages in complex interactions with invertebrates, through its symbiosis with soil nematodes and its pathogenicity to a broad range of insect larvae. Among the regulatory proteins of Xenorhabdus involved in host interactions, the sigma factor FliA and the regulator FliZ, expressed from the fliAZ operon, play a key role in mediating the production of exoenzymes, motility and full virulence in insects (Lanois et al., 2008). In this study, we investigated the dynamics of the FliA-dependent flagellin gene fliC and FliZ-dependent haemolysin genes xaxAB during insect infection and nematode association by carrying out real-time expression analysis using an unstable GFP monitoring system. We showed that expression of the FliAZ-dependent genes in infected insects is not restricted to a specific tissue but increases significantly just prior to host death and reaches a maximal level in larvae cadaver. Using an iron availability reporter construct, we also showed that iron starvation conditions inhibit expression of FliAZ-dependent genes in vitro, as well as during the first steps of the infectious process. These findings shed further light on the role of the FliAZ regulon in the Xenorhabdus life cycle and suggest that iron may constitute a signal governing Xenorhabdus adaptation to shifting host environments.

There is a complex interplay between the regulation of flagellar motility and the expression of virulence factors in many bacterial pathogens. We investigated the role of FliZ in the regulation of flagellar and virulence genes in Xenorhabdus nematophila, an insect pathogen. The fliZ gene is the second gene in the fliAZ operon in X. nematophila. In vivo transcription analysis revealed a positive feedback loop of fliAZ transcription in which FliZ activates flhDC, the master operon of flagellar regulon in X. nematophila, leading to an increased transcription of the FlhDC-dependent promoter of fliAZ. We also showed that fliAZ and flhDC mutants lacked motility, had no haemolysin or Tween lipase activity and displayed an attenuated virulence phenotype in insects. Lipase activity is controlled by FliA, whereas haemolysin production and full virulence phenotype have been reported to be FliZ-dependent. Transcriptional analysis revealed that FliZ directly controlled expression of the xhlBA and xaxAB operons, which encode haemolysins from the two-partner secretion system and the binary XaxAB toxin family respectively. We suggest that this regulatory pathway may also occur in other pathogenic enterobacteria with genes encoding members of these two growing families of haemolysins.

This study was aimed at characterizing a cell-surface 25 kDa glycoprotein (GP25) that was previously shown to be underproduced by a spontaneous adhesion-defective mutant D5 of Ruminococcus albus 20. An antiserum against wild-type strain 20 was adsorbed with the mutant D5 to enrich it in antibodies 'specific' to adhesion structures of R. albus 20. The resulting antiserum, called anti-Adh serum, blocked adhesion of R. albus 20 and reacted mainly with GP25 in bacterial and extracellular protein fractions of R. albus 20. The N-terminal sequence of purified GP25 was identical to that of CbpC, a 21 kDa cellulose-binding protein (CBP) of R. albus 8. The nucleotide sequence of the gp25 gene was determined by PCR and genomic walking procedures. The gp25 gene encoded a protein of 165 aa with a calculated molecular mass of 16940 Da that showed 72.9% identity with CbpC and presented homologies with type IV pilins of Gram-negative pathogenic bacteria. Negative-staining electron microscopy revealed fine and flexible pili surrounding R. albus 20 cells while mutant cells were not piliated. In addition, immunoelectron microscopy showed that the anti-Adh serum probing mainly GP25, completely decorated the pili surrounding R. albus 20, thereby showing that GP25 was a major pilus subunit. This study shows for the first time the presence of pili at the surface of R. albus and identifies GP25 as their major protein subunit. Though GP25 was not identified as a CBP, isolated pili were shown to bind cellulose. In conclusion, these pili, which belong to the family of type IV pili, mediate adhesion of R. albus 20 to cellulose.

International audience; In developed countries, enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a leading cause of bloody diarrhea and renal failures in human. Understanding strategies employed by EHEC to colonize the intestine is of major importance since to date no cure exists to eradicate the pathogen. In this study, the adaptive response of EHEC to the intestinal milieu conditioned by a human microbiota was examined. A transcriptomic analysis was performed on the EHEC strain EDL933 incubated in vitro in the sterile-filtrated cecal content of human microbiota-associated rats (HMC) compared with EDL933 incubated in the sterile-filtrated cecal content of germ-free rat (GFC). EDL933 switches from a glycolytic metabolic profile in the GFC to an anaplerotic metabolic profile in HMC. The expression of several catabolism genes was strongly affected such as those involved in the utilization of sugars, glycerol, N-acetylneuraminic acid, amino acids and secondary metabolites. Interestingly, expression level of critical EHEC O157:H7 virulence genes including genes from the locus of enterocyte effacement was reduced in HMC. Altogether, these results contribute to the understanding of EHEC adaptive response to a digestive content and highlight the ability of the microbiota to repress EHEC virulence gene expression.

International audience; Enterohemorrhagic Escherichia coli (EHEC), an emerging food- and water-borne hazard, is highly pathogenic to humans. In the environment, EHEC must survive phosphate (Pi) limitation. The response to such Pi starvation is an induction of the Pho regulon including the Pst system that senses Pi variation. The interplay between the virulence of EHEC, Pho-Pst system and environmental Pi remains unknown. To understand the effects of Pi deprivation on the molecular mechanisms involved in EHEC survival and virulence under Pho regulon control, we undertook transcriptome profiling of the EDL933 wild-type strain grown under high Pi and low Pi conditions and its isogenic ΔphoB mutant grown in low Pi conditions. The differentially expressed genes included 1067 Pi-dependent genes and 603 PhoB-dependent genes. Of these 131 genes were both Pi and PhoB-dependent. Differentially expressed genes that were selected included those involved in Pi homeostasis, cellular metabolism, acid stress, oxidative stress and RpoS-dependent stress responses. Differentially expressed virulence systems included the locus of enterocyte effacement (LEE) encoding the type-3 secretion system (T3SS) and its effectors, as well as BP-933W prophage encoded Shiga toxin 2 genes. Moreover, PhoB directly regulated LEE and stx2 gene expression through binding to specific Pho boxes. However, in Pi-rich medium, constitutive activation of the Pho regulon decreased LEE gene expression and reduced adherence to HeLa cells. Together, these findings reveal that EHEC has evolved a sophisticated response to Pi limitation involving multiple biochemical strategies that contribute to its ability to respond to variations in environmental Pi and to coordinating the virulence response.

International audience; Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC.

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria., Author Summary Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.

International audience; Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G2/M phase transition in human cell lines. We report here the first direct functional analysis of CifPl, from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cifPl gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cifPl was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. CifPl inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G2/M phase transition. It also triggered host cell death by apoptosis. The integrity of the CifPl catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.

Correspondance auteur: R. Zumbihl E-mail: zumbihl@univ-montp2.fr; International audience; Photorhabdus asymbiotica is unique among the entomopathogenic bacteria of this genus in also being able to infect humans, leading to its isolation from some clinical samples. Recent comparative genomics data and the results of studies of interactions between bacteria and cells provide insight into the adaptation of this bacterium to its new niche, the human body

Correspondance auteur: E. Oswald E-mail: e.oswald@envt.fr Numéro article: e4855; International audience; The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G(1) and G(2) cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21(waf1/cip1) and p27(kip1) and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans

The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated.

Bacterial pathogens have evolved a sophisticated arsenal of virulence factors to modulate host cell biology. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) use a type III protein secretion system (T3SS) to inject microbial proteins into host cells. The T3SS effector cycle inhibiting factor (Cif) produced by EPEC and EHEC is able to block host eukaryotic cell-cycle progression. We present here a crystal structure of Cif, revealing it to be a divergent member of the superfamily of enzymes including cysteine proteases and acetyltransferases that share a common catalytic triad. Mutation of these conserved active site residues abolishes the ability of Cif to block cell-cycle progression. Finally, we demonstrate that irreversible cysteine protease inhibitors do not abolish the Cif cytopathic effect, suggesting that another enzymatic activity may underlie the biological activity of this virulence factor.

The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Cif-induced cyclin-dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome-mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.

There is a complex interplay between the regulation of flagellar motility and the expression of virulence factors in many bacterial pathogens. We investigated the role of FliZ in the regulation of flagellar and virulence genes in Xenorhabdus nematophila, an insect pathogen. The fliZ gene is the second gene in the fliAZ operon in X. nematophila. In vivo transcription analysis revealed a positive feedback loop of fliAZ transcription in which FliZ activates flhDC, the master operon of flagellar regulon in X. nematophila, leading to an increased transcription of the FlhDC-dependent promoter of fliAZ. We also showed that fliAZ and flhDC mutants lacked motility, had no haemolysin or Tween lipase activity and displayed an attenuated virulence phenotype in insects. Lipase activity is controlled by FliA, whereas haemolysin production and full virulence phenotype have been reported to be FliZ-dependent. Transcriptional analysis revealed that FliZ directly controlled expression of the xhlBA and xaxAB operons, which encode haemolysins from the two-partner secretion system and the binary XaxAB toxin family respectively. We suggest that this regulatory pathway may also occur in other pathogenic enterobacteria with genes encoding members of these two growing families of haemolysins.

Summary The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. Cif-induced cyclin-dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome-mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.

International audience; Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehelin, M. (2003) J. Mal. Chem. 278, 3030-3039). We show here that this toxin, Xenorhabdus alpha-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C I. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins.