Human microbiota-secreted factors inhibit shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx(2) mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx(2) repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

Presence of activatable Shiga toxin genotype (stx2d) in Shiga toxigenic Escherichia coli from livestock sources

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c) (vha), stx(2c) (vhb), or stx(2d) (EH250). Subsequently, the stx(2c) (vha) and stx(2c) (vhb) operons were screened for the absence of a PstI site in the stx(2a) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable stx(2d) Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

Virulence factors of Escherichia coli isolated from diarrheic calves

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ocorrência de cepas de Escherichia coli que apresentam o gene de Shiga toxina em queijo mussarela produzido artesanalmente

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins

Escherichia coli O157:H7 causes Shiga toxin (Stx)-mediated vascular damage, resulting in hemorrhagic colitis and the hemolytic uremic syndrome in humans. These infections are often foodborne, and healthy carrier cattle are a major reservoir of E. coli O157:H7. We were interested in knowing why cattle are tolerant to infection with E. coli O157:H7. Cattle tissues were examined for the Stx receptor globotriaosylceramide (Gb3), for receptivity to Stx binding in vitro, and for susceptibility to the enterotoxic effects of Stx in vivo. TLC was used to detect Gb3 in tissues from a newborn calf. Gb3 was detected by TLC in kidney and brain, but not in the gastrointestinal tract. Immunohistochemistry was used to define binding of Stx1 and Stx2 overlaid onto sections from cattle tissues. Stx1 and Stx2 bound to selected tubules in the cortex of the kidney of both newborn calves (n = 3) and adult cattle (n = 3). Stx did not bind to blood vessels in any of the six gastrointestinal and five extraintestinal organs examined. The lack of Gb3 and of Stx receptivity in the gastrointestinal tract raised questions about the toxicity of Stx in bovine intestine. We found that neither viable E. coli O157:H7 nor Stx-containing bacterial extracts were enterotoxic (caused fluid accumulation) in ligated ileal loops in newborn calves. The lack of vascular receptors for Stx provides insight into why cattle are tolerant reservoir hosts for E. coli O157:H7.

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

Do Human Extraintestinal Escherichia coli Infections Resistant to Expanded-Spectrum Cephalosporins Originate From Food-Producing Animals? A Systematic Review

To find out whether food-producing animals (FPAs) are a source of extraintestinal expanded-spectrum cephalosporin-resistant Escherichia coli (ESCR-EC) infections in humans, Medline, Embase, and the Cochrane Database of Systematic Reviews were systematically reviewed. Thirty-four original, peer-reviewed publications were identified for inclusion. Six molecular epidemiology studies supported the transfer of resistance via whole bacterium transmission (WBT), which was best characterized among poultry in the Netherlands. Thirteen molecular epidemiology studies supported transmission of resistance via mobile genetic elements, which demonstrated greater diversity of geography and host FPA. Seventeen molecular epidemiology studies did not support WBT and two did not support mobile genetic element-mediated transmission. Four observational epidemiology studies were consistent with zoonotic transmission. Overall, there is evidence that a proportion of human extraintestinal ESCR-EC infections originate from FPAs. Poultry, in particular, is probably a source, but the quantitative and geographical extent of the problem is unclear and requires further investigation.

First detection of extended-spectrum cephalosporin- and fluoroquinolone-resistant Escherichia coli in Australian food-producing animals

This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n = 324) from Australian food-producing animals [cattle (n = 169), porcine (n = 114), poultry (n = 32) and sheep (n = 9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0–88.6%), ampicillin (9.4–71.1%), trimethoprim/sulfamethoxazole (11.1–67.5%) and streptomycin (21.9–69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.

Molecular characterisation of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

The vacuolating autotransporter (AT) toxin (Vat) contributes to Uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here we characterised Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, 73 and 95) and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator, which we termed vatX. The vat-vatX genes were present in the UPEC reference strain CFT073 and RT-PCR revealed both genes are co-transcribed. Over-expression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator H-NS; thus the hns gene was mutated in CFT073 (to generate CFT073hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hns compared to wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic SPATE secreted by UPEC during infection.

Production of F4 Fimbrial Adhesin in Plants: a Model for Oral Porcine Vaccine against Enterotoxigenic Escherichia coli

F4 fimbriae of enterotoxigenic Escherichia coli (ETEC) are highly stable multimeric structures with a capacity to evoke mucosal immune responses. With these characters F4 offer a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea. Postweaning diarrhea is a major problem in piggeries worldwide and results in significant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health. In this study, the capacity of transgenic plants to produce FaeG protein, the major structural subunit and adhesin of F4 fimbria, was evaluated. Using the model plant tobacco, the optimal subcellular location for FaeG accumulation was examined. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins (TSP) over the other investigated subcellular locations, namely, the endoplasmic reticulum and the apoplast. Moreover, we determined whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. The chloroplast-derived FaeG protein did show resistance against low pH and proteolysis in the simulated gastrointestinal conditions and was able to bind to the F4R, subsequently inhibiting the F4+ ETEC binding in a dose-dependent manner. To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the chloroplast-targeting construct and equally to tobacco plants, a high-yield FaeG accumulation of 1% of TSP was obtained. A similar yield was also obtained in the seeds of barley, a valuable crop plant, when the FaeG-encoding gene was expressed under an endosperm-specific promoter and subcellularly targeted into the endoplasmic reticulum. Furthermore, desiccated alfalfa plants and barley grains were shown to have a capacity to store FaeG protein in a stable form for years. When the transgenic alfalfa plants were administred orally to weaned piglets, slight F4-specific systemic and mucosal immune responses were induced. Co-administration of the transgenic alfalfa and the mucosal adjuvant cholera toxin enhanced the F4-specific immune response; the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were significantly reduced as compared with pigs that had received nontransgenic plant material. In conclusion, the results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine F4+ ETEC infections. The findings here thus present new approaches to develop the vaccination strategy against porcine postweaning diarrhea.

Suolistopatogeenisten Escherichia coli -bakteerien esiintyvyys ripulipotilailla ja raa’oissa lihatuotteissa Burkina Fasossa sekä enterohemorraagisten E. coli -kantojen karakterisointi

Suolistopatogeeniset Escherichia coli -bakteerit eli ripulikolit aiheuttavat ihmisellä suolistoinfektioita. Kuten normaalimikrobiston E. coli -bakteerit, ne esiintyvät ihmisen lisäksi muiden nisäkkäiden, etenkin märehtijöiden, ja lintujen suolistossa. Lisäksi ne voivat esiintyä maaperässä ja vesistöissä. Ihminen voi saada tartunnan eläinperäisten elintarvikkeiden välityksellä tai juomalla eläinten tai ihmisen ulosteilla saastunutta vettä. Ripulikolit voidaan jakaa ainakin viiteen ryhmään perustuen niiden erilaisiin virulenssiominaisuuksiin: enteropatogeeninen E. coli (EPEC), enterotoksigeeninen E. coli (ETEC), enterohemorraaginen E. coli (EHEC), enteroinvasiivinen E. coli (EIEC) ja enteroaggregatiivinen E. coli (EAEC). EPEC aiheuttaa etenkin kehitysmaissa pikkulapsille ripulia. ETEC aiheuttaa turistiripulia ja vastasyntyneiden ripulia kehitysmaissa. EHEC aiheuttaa ripulia tai veriripulia, joka voi varsinkin pienillä lapsilla johtaa hemolyyttis-ureemiseen oireyhtymään (HUS) ja munuaisten vaurioitumiseen. EIEC aiheuttaa Shigellan kaltaista ripulia, joka voi olla veristä. EAEC on yhdistetty lähinnä pitkittyneisiin ripuleihin. Tutkimuksessa selvitettiin suolistopatogeenisten E. coli -bakteerien esiintyvyyttä Burkina Fasossa, josta ei ole saatavilla aikaisempaa tietoa ripulikolien esiintymisestä ihmisissä ja elintarvikkeissa. Ulostenäytteitä otettiin ripulia sairastavilta alle viisivuotiailta lapsilta maaseudulta kahdesta kylästä, Boromosta ja Gourcysta, ja maan pääkaupungista Ouagadougousta (110 näytettä). Lihanäytteitä (kanaa, nautaa, lammasta ja naudan suolta, jota käytetään ihmisravinnoksi) otettiin Ouagadougoun toreilla myytävistä kypsentämättömistä lihoista (120 näytettä). Näytteistä saadut bakteerisekaviljelmät tutkittiin monialukkeisella PCR-menetelmällä, joka tunnistaa viiden ripulikoliryhmän virulenssigeenejä. Lisäksi lihanäytteistä eristettiin 20 EHEC-kantaa shigatoksiinin stx-geenin havaitsemiseen perustuvalla pesäkehybridisaatiolla ja PCR-seulonnalla, ja karakterisoitiin mahdollisten virulenssiominaisuuksien selvittämiseksi. Tutkimus osoitti, että ripulikolien aiheuttamat suolistoinfektiot ovat yleisiä ripulia sairastavilla pikkulapsilla Burkina Fasossa. Ulostenäytteistä 59 % oli positiivisia. Useimmiten lapsilla esiintyi EAEC- (32 %) ETEC- (31 %) ja EPEC-patoryhmiä (20 %). EIEC- (2 %) ja EHEC-patoryhmiä (1 %) esiintyi vähän. Myös useamman patoryhmän sekainfektiot olivat yleisiä (24 %). Eri paikkakuntien välillä oli tilastollisesti merkitseviä eroja ripulikolien esiintymisessä. Gourcyssa ripulikoleja esiintyi useammin kuin Ouagadougoussa ja Boromossa. Tutkimuksessa kävi ilmi, että Ouagadougoun toreilla myytävissä lihoissa on paljon ripulikoleja. Lihanäytteistä 43 % oli positiivisia. Yleisimmin lihoissa esiintyi EHEC (28 %), EPEC (20 %), ETEC (8 %) ja EAEC (5 %). EIEC-ryhmää ei havaittu lihoissa. Myös useamman patoryhmän sekakontaminaatioita löytyi (17 %) lihoista. Ripulikolien esiintyvyydessä eri lihojen välillä ei ollut tilastollisesti merkitseviä eroja, kun tarkasteltiin kaikkia patoryhmiä yhdessä. Eri patoryhmien esiintyvyyttä tarkasteltaessa EHEC-patoryhmää ei esiintynyt ollenkaan kanassa ja ero oli tilastollisesti merkitsevä muihin lihoihin verrattuna. Lihoista eristetyt 20 EHEC-kantaa kuuluivat 14 eri serotyyppiin, joista osa on aikaisemmin eristetty suolistoinfektioihin ja HUSoireyhtymään sairastuneilta ihmisiltä. Kaikki kannat olivat stx1-positiivisia ja puolella oli lisäksi stx2-geeni, jota pidetään shigatoksiinin virulentimpana muotona. Kahdelta EHEC-kannalta löytyi myös ETECpatoryhmän lämpöstabiilin enterotoksiini Ia:n geeni eli kannat olivat kahden patoryhmän välimuotoa ja osoitus geenien siirtymisestä eri patoryhmien välillä. Vaikka nuorimmat näytteen antaneet lapsipotilaat tuskin söivät lihaa, sen voidaan ajatella silti olevan edustava näyte lasten elinympäristöstä, sillä lasten ruoka valmistetaan usein samoissa oloissa, joissa raakaa lihaa käsitellään. Saastunut liha voi siten olla pikkulasten ripulikoli-infektioiden aiheuttaja.

Avaliação da interação de Escherichia coli enterohemorrágica (EHEC) pertencente ao sorotipo O157: H7 isoladas de bovinos assintomáticos e de doença humana com células enterocíticas humanas (linhagem Caco-2)

Reconhecida como agente de doença humana em 1982, E.coli enterohemorrágica (EHEC) pode causar diarréia sanguinolenta, colite hemorrágica e síndrome hemolítica urêmica (SHU). EHEC constitui um subgrupo especialmente virulento das E.coli produtoras de toxina de Shiga (Stx). O fator crítico da sua virulência é a toxina Shiga, capaz de interromper a síntese proteica da célula eucariótica. São conhecidos dois subgrupos de Stx, Stx1 e Stx2. Stx1 possui duas variantes Stx1c e Stx1d. Stx2 possui muitas variantes. Estudos epidemiológicos sugerem que cepas com os perfis toxigênicos Stx2 ou Stx2/Stx2c seriam mais frequentemente associadas a pacientes com SHU. Além da expressão de Stx, EHEC do sorotipo O157:H7 colonizam a mucosa intestinal induzindo a formação de lesões denominadas attaching/effacing (A/E). Para a produção da lesão A/E, é necessária a presença de uma ilha de patogenicidade cromossômica denominada LEE, composta por cinco operons, LEE 1 a LEE5. Em LEE 5 são codificadas a adesina intimina e o seu receptor Tir, o qual é translocado por um sistema de secreção tipo III (SSTT) e em LEE 4 são codificadas as proteínas secretadas EspA,B e D. Em EHEC O157:H7 são descritos muitos fatores de virulência, codificados em ilhas de patogenicidade, no cromossomo e no megaplasmídio pO157. Bovinos são o principal reservatório deste patógeno e alimentos de origem bovina e produtos contaminados com fezes de bovinos são causadores de surtos epidêmicos. Em nosso país EHEC O157:H7 é isolada do reservatório animal mas é muito rara a sua ocorrência em doença humana. Notamos que nas cepas bovinas predomina Stx2c, enquanto nas cepas humanas predomina o perfil toxigenico Stx2/Stx2c. Quanto a interação com enterocitos humanos cultivados in vitro (linhagem Caco-2), verificamos que tanto cepas bovinas quanto humanas mostram idêntica capacidade de invadir e persistir no compartimento intracelular das células Caco-2. No entanto, em comparação com as cepas humanas, as cepas bovinas mostram uma reduzida capacidade de produzir lesões A/E. Empregamos qPCR para aferir a transcrição de três diferentes locus (eae, espA e tir) situados nos operons LEE4 e LEE5 de cepas bovinas e humanas, durante a infecção de células Caco-2. Verificamos diferenças na expressão dos genes, especialmente espA, entre cepas bovinas e humanas com maior expressão para estas ultimas, em linha com os achados dos testes FAS. Através de clonagem e expressão de proteínas recombinantes, purificamos as proteínas Eae, EspA e Tir e obtivemos anticorpos específicos, empregados para acompanhar a sua expressão ao longo da infecção de células Caco-2, por imunofluorescencia. Verificamos que as três proteínas são detectadas tanto em cepas bovinas quanto humanas, mas nestas ultimas, a marcação é precoce e torna-se mais intensa com o avanço da infecção. Nossos resultados indicam que cepas EHEC O157:H7 isoladas do reservatório bovino em nosso país apresentam diferenças importantes em relação ao perfil toxigenico e a capacidade de indução de lesões A/E, características apontadas na literatura como relevantes para a virulência do micro-organismo. Por outro lado, nossos achados quanto a capacidade de invadir e multiplicar-se no interior de enterócitos pode explicar a persistência do patógeno no reservatório animal e a sua capacidade de transmissão horizontal.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

La toxine stable à la chaleur de type b (STb) est une des toxines produites par les souches Enterotoxigenic Escherichia coli (ETEC) impliquée dans le développement de la diarrhée. Une étude antérieure par Goncalves et al. (2009) a démontré que les cellules ayant internalisé la toxine STb démontraient une morphologie qui rappelle l’apoptose. Le changement du potentiel membranaire observé par Goncalves et al. (2009) nous a incité à vérifier la capacité de la toxine STb à induire l’apoptose des cellules HRT-18 et IEC-18 par la voie intrinsèque. Les cellules HRT-18 et IEC-18 ont été traitées avec de la toxine purifiée pour une durée de 24 heures puis ells ont été récoltées et examinées pour des caratéristiques de l’apoptose. L’activation des caspases-9 et -3, mais pas de la caspase-8, a été observée dans les deux lignées cellulaires à l’aide des substrats fluorescents spécifiques pour chaque caspase. L’ADN extrait des cellules HRT-18 et IEC-18 a révélé une fragmentation lorsque migré sur gel d’agarose. La condensation et la fragmentation des noyaux ont été observées en microscopie à fluorescence suite à une coloration de l’ADN au Hoechst 33342. Les indices apoptotiques des cellules HRT-18 et IEC-18 traitées avec des quantités croissantes de STb montrent une dose-réponse pour les deux lignées. L’activation de la caspase-9 est une indication que la voie intrinsèque de l’apoptose est activée dans les cellules HRT-18 et IEC-18. L’absence de l’activation de la caspase-8 démontre que la voie extrinsèque n’est pas impliquée dans la mort cellulaire médiée par STb.

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).

Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid bacteriophages

P>To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 1031 tailed-bacteriophages are estimated to populate the globe. To address this discrepancy a culture-independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 x 104 to 6.5 x 106 ml-1, with one in 103 free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation-dependent techniques, which grossly underestimate the abundance of viral entities.