Interaction d'Escherichia coli entérohémorragique (EHEC) avec Acanthamoeba castellanii et rôle du régulon Pho chez les EHEC

Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine).EHEC O157:H7 are an emerging zoonotic food- and water-borne hazard highly pathogenic to humans and associated with diseases ranging from acute gastroenteritis to hemolytic uremic syndrome. From their commensal carriage by farm animals to human targets, EHEC pass through a crucial step of persistence in the open environment. Microbial endosymbiosis is one strategy used by pathogenic bacteria to survive in aquatic environments. Amoebae species are free-living protozoa found in diverse environmental habitats and known to host several water-borne pathogens. Thus amoebae could contribute to transmission of EHEC to humans. The first part of my PhD project was focused on interaction of the free-living amoebae Acanthamoeba castellanii with EHEC. The results showed that the presence of amoeba extends the persistence of EHEC that survived phagocytosis by amoebae. This demonstrates the real potential of amoebae to harbourd EHEC that may contribute to their transmission. However, absence of shiga toxins enhanced the intra-amoeba survival. Moreover, EHEC had a toxic and lethal effect on amoebae partially due to shiga toxins. The involvement of shiga toxins in the intracellular survival and mortality of amoebae suggests the value of using amoebae as a model of host/pathogen interactions to study the pathogenicity of EHEC. During their transmission cycle, EHEC encounter limitation inorganic phosphate (Pi) in the environment. Using jointly the PhoB-R two-component system (TCS) and the Pst (Pi specific transport) system, EHEC detect and respond to this Pi limitation by activating the Pho regulon. The interplay between the EHEC virulence, the Pho-Pst and/or the environmental Pi remains unknown. The second part of my project explored the role of Pho regulon (responding to Pi-limitation stress) in the virulence of EHEC. Transcriptomic analysis showed that EHEC has evolved a sophisticated response to Pi deficiency involving not only biochemical strategies that are likely critical to its survival, but also coordinating its virulence response. In these conditions, the regulator PhoB regulates directly the expression of LEE and Stx2 genes. This is confirmed by an increase in EspB secretion and Stx2 production and secretion in low Pi conditions. Moreover, the activation of Pho regulon increases biofilm formation and reduces motility in EHEC. This correlated with the induction of genes regulating curli production and repression of indole production pathway and the flagellum and PGA biosynthesis

Interplay between genetic regulation of phosphate homeostasis and bacterial virulence.

International audienceBacterial pathogens, including those of humans, animals, and plants, encounter phosphate (Pi)-limiting or Pi-rich environments in the host, depending on the site of infection. The environmental Pi-concentration results in modulation of expression of the Pho regulon that allows bacteria to regulate phosphate assimilation pathways accordingly. In many cases, modulation of Pho regulon expression also results in concomitant changes in virulence phenotypes. Under Pi-limiting conditions, bacteria use the transcriptional-response regulator PhoB to translate the Pi starvation signal sensed by the bacterium into gene activation or repression. This regulator is employed not only for the maintenance of bacterial Pi homeostasis but also to differentially regulate virulence. The Pho regulon is therefore not only a regulatory circuit of phosphate homeostasis but also plays an important adaptive role in stress response and bacterial virulence. Here we focus on recent findings regarding the mechanisms of gene regulation that underlie the virulence responses to Pi stress in Vibrio cholerae, Pseudomonas spp., and pathogenic E. coli

PhoB activates Escherichia coli O157:H7 virulence factors in response to inorganic phosphate limitation.

International audienceEnterohemorrhagic 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

The ecological habitat and transmission of Escherichia coli O157:H7.

International audienceSince its first description in 1982, the zoonotic life-threatening Shiga toxin-producing Escherichia coli O157:H7 has emerged as an important food- and water-borne pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. In the last decade, increases in E. coli O157:H7 outbreaks were associated with environmental contamination in water and through fresh produce such as green leaves or vegetables. Both intrinsic (genetic adaptation) and extrinsic factors may contribute and help E. coli O157:H7 to survive in adverse environments. This makes it even more difficult to detect and monitor food and water safety for public health surveillance. E. coli O157:H7 has evolved in behaviors and strategies to persist in the environment

Bio-informatic analyses of PhoB-binding sites.

<p>Sequence logos determined from 12 putative PhoB-binding sites in EDL933 are indicated (upper panel). Potential consensus sequences identified in the promoter regions of LEE 1, LEE 2 operons and upstream <i>stx2AB</i> genes are shown with their statistical scores and genomic positions (lower panel). Pho box prediction probabilities were determined using the matrix frequencies of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094285#pone.0094285.s006" target="_blank">Table S4</a> that were uploaded into the Gibbs software algorithm. <a href="http://ccmbweb.ccv.brown.edu/gibbs/gibbs.html" target="_blank">http://ccmbweb.ccv.brown.edu/gibbs/gibbs.html</a>.</p

Effect of Pi and PhoB on <i>stx2</i> gene expression and toxin production.

<p>Low Pi and PhoB increased transcription of <i>stx2</i> and its toxin production and of BP933W genes except for repressor gene <i>cI</i> that was repressed. <b>A</b>. Heat map of the expression levels of BP933W genes between wild-type EDL933 strain grown in low or high Pi conditions and between wild-type and Δ<i>phoB</i> strain grown in low Pi conditions. <b>B.</b> The expression levels of the BP933W genes <i>cI, cro</i> and <i>stx2AB</i> were analyzed by RT-qPCR in the wild-type strain and the Δ<i>phoB</i> mutant grown in low or high Pi media as indicated. The wild-type strain induced by mitomycin C (WT+MitC) was used as a positive control <b>C.</b> Fluorescence of the wild-type EDL933 strain carrying a chromosomal fusion reporting <i>stx2</i> transcription level grown in Pi+ or Pi- conditions. <b>D.</b> The production of Stx2 measured by ELISA in extra-cellular protein (ECP) and whole cell protein (WCP) fractions of the EDL933 wild-type strain grown in Pi+ or Pi- conditions and in the Δ<i>phoB</i> mutant and its complemented derivative. Asterisks represent the significant ANOVA <i>P value</i> (*<0.05, **<0.01, ***<0.001).</p

Global analysis of differentially expressed genes in response to Pi starvation or phoB inactivation in EDL933 strain.

<p><b>A.</b> Classification of genes whose expression levels were altered in Pi-dependent and in PhoB-dependent manners. Left and right circles indicate the differentially expressed genes of wild-type and Δ<i>phoB</i>-mutant strains with expression levels that were altered over 2-fold under Pi limitation. Venn diagram: Group 1 includes 936 PhoB-independent genes that are differentially expressed under Pi limitation in the wild-type strain, but that did not change in the Δ<i>phoB</i> mutant. Group 3 includes 472 PhoB-dependent genes differentially expressed in the Δ<i>phoB</i> mutant but did not change under Pi-limitation in the wild-type strain. Group 2 included 131 PhoB-dependent Pi response genes that are differentially expressed under Pi limitation in the wild-type strain and between the wild-type and the Δ<i>phoB</i> strains. <b>B.</b> Functional classification of genes with altered expression in strain EDL933 grown in Pi-limited conditions compared to cells grown in Pi-rich conditions (Pi-dependent (white bars)) and EDL933 incubated in Pi-limited conditions compared to Δ<i>phoB</i> mutant cells grown in the Pi-limited conditions (PhoB-dependent (gray bars)).</p

Genes among upregulated and downregulated PhoB-dependent Pi response genes.

<p>Genes among upregulated and downregulated PhoB-dependent Pi response genes.</p

PhoB binds in vitro to LEE1, LEE2 and <i>stx2</i> promoter regions.

<p>A. Schematic representation of LEE and lambdoid prophage BP933W DNA regions. Arrows indicate the orientation of transcription. The blue lines/arrows indicate the probes used for EMSA assays. The square symbol indicates the predicted Pho box. B. Increasing amounts of GST-purified recombinant PhoB^CA were used in the EMSA assay to shift the 6-FAM labeled DNA probes amplified from LEE1 (−253 to +64 bp), LEE2 (−228 to +81 bp), LEE3 (−109 to −259 bp) and <i>stx2AB</i> (−402 to −3 bp) promoter regions.</p

<i>E. coli</i> strains and plasmids used in this study.

a<p>Km^r, kanamycin-resistant; Cm^r, chloramphenicol-resistant; Gm^r, gentamicin-resistant; Ap^r; ampicillin-resistant</p