Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarreia infantil tanto em países desenvolvidos como em países em desenvolvimento. Esta bactéria possui dois fatores de virulência comprovadamente envolvidos na patogênese, intimina e bundle-forming pilus (BFP). Este patotipo está dividido em EPEC típica e EPEC atípica, ambos apresentam uma ilha de patogenicidade cromossomal denominada locus of enterocyte effacement (região LEE) onde está localizado o gene eae (E. coli attachment effacement), que codifica a intimina, uma proteína de membrana externa que medeia a adesão íntima da bactéria ao enterócito. Diferente da EPEC típica, as cepas de EPEC atípica não possuem o plasmídeo EAF (EPEC adherence factor) no qual encontra-se o operon bfp (bundle forming pilus) constituído por 14 genes incluindo bfpA o qual codifica a bundlina (BfpA), principal subunidade da fímbria Bfp, que possibilita a agregação bacteriana. Na infecção por EPEC ocorre grave disfunção da barreira epitelial, e uma das conseqüências é a inflamação. Na literatura, é bem descrito a interação entre as proteínas efetoras de EPEC com as células epiteliais e os processos iniciais da interação bactéria à célula hospedeira. Entretanto, poucos são os estudos que analisam a produção de citocinas em infecções por EPEC ou suas moléculas efetoras com relação a ativação de macrófagos, fundamentais para o controle do processo inflamatório e geração da resposta imune durante esta infecção. A análise das citocinas produzidas constitui uma parte importante da resposta imune e representa a tentativa do hospedeiro em lidar com um determinado microrganismo. Em função disto analisou-se o papel da intimina e do BfpA na capacidade de ativar a resposta inata mediada por macrófagos in vitro, onde avaliou-se a produção de citocinas pró-inflamatórias (TNF-α, IL-1, IL-6 e IL-12), citocina antiinflamatória (IL-10) e quimiocina (MCP-1). Os resultados demonstraram que as proteínas recombinantes intimina e BfpA são potentes ativadores de macrófagos, de forma dose dependente, produzindo TNF-α, IL-12 e IL-6, IL-10 e MCP-1, mas não IL-1β. Neste estudo não foi observado efeito sinérgico na produção de citocinas pró-inflamatórias ao associar intimina e BfpA, entretanto em dose mais elevada potenciou a produção de IL-10, um mediador antiinflamatório. O efeito imune obtido foi atribuído majoritariamente a estas proteínas uma vez que o tratamento destas com polimixina B não alterou a produção de TNF-α. Conclui-se que intimina e BfpA são potentes ativadores de macrófagos durante a resposta inata podendo colaborar para o controle do processo inflamatório durante a infecção por EPEC. / Enteropathogenic Escherichia coli (EPEC) is a common cause of childhood diarrhea in developed countries as well as developing countries. This bacterium has two proven virulence factors involved in pathogenesis, intimin and bundle-forming pilus (BFP). This pathotype EPEC is divided into typical and atypical EPEC, both having a chromosomal pathogenicity island called locus of enterocyte effacernent (LEE region) which contains the gene eae (E. coli attachment effacement). eae encodes intimin, an outer membrane protein that mediates the intimate adherence of bacteria to the enterocyte. Unlike typical EPEC, atypical EPEC strains do not possess the plasmid EAF (EPEC adherence factor) which is in the operon bfp (bundle forming pilus) consisting of 14 genes including bfpA, which encodes bundlin (BfpA), the main subunit of BFP allowing bacterial aggregation. EPEC infection occurs in severe dysfunction of the epithelial barrier, and one consequence is inflammation. In the literature, the interaction between effector proteins of EPEC and epithelial cells and the initial processes of bacterial interaction with the host cell are well described. However, there are few studies that have examined cytokine production in EPEC infections or their effector molecules with respect to macrophage activation, essential for controlling inflammation and immune response during this infection. The production of cytokines is an important part of the immune response and represents the host\'s attempt to deal with a particular microorganism. Therefore, we examined in vitro the role of intimin and BfpA in the ability to activate the innate response mediated by macrophages, where we analyzed the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, and the antiinflammatory cytokine IL-10 and chemokine MCP-1. The results show that recombinant intimin and BfpA are potent activators of macrophages in a dose-dependent manner, where the stimulated cells produce TNF-α, IL-12 and IL-6, IL-10 and MCP-1, but not IL-1β. In this study, no synergistic effect was observed in the production of proinflammatory cytokines by combining BfpA and intimin, although production of IL-10, an antiinflammatory mediator, was potentiated at a higher dose. The effect obtained was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. We conclude that intimin and BfpA are potent activators of macrophages during the innate response and may contribute to the control of inflammation during infection with EPEC.

Escherichia coli Enteropatogênicas são um importante patógeno causador de diarréia. As EPEC podem ser classificadas em típica e atípica, baseado na presença do plasmídeo EAF. As amostras de EPEC apresentam em seu genoma uma ilha de patogenicidade denominada região LEE, na qual estão contidos os genes relacionados a formação da lesão (A/E). A regulação gênica da região LEE é multifatorial, sendo o principal regulador o gene ler. Até o momento não existem trabalhos sobre a participação de Hfq em EPEC, assim sendo, o presente estudo analisa o papel de Hfq na regulação dos fatores de virulência de EPEC típica (O127:H6) e atípica (O55:H7). A mutagênese do gene hfq foi obtida através do sistema l Red de recombinação alélica. As amostras mutantes apresentaram uma diminuição na capacidade aderir e formar a lesão A/E. Analise transcricional dos mutantes revelou uma significativa diminuição na transcrição do gene espA e do gene eae. Foi possível evidenciar uma diminuição da motilidade das amostras mutantes. A analise in silico revelou a possibilidade do dobramento natural do mRNA ler, ocultando o sitio de ligação do ribossomo. Aqui demonstramos a necessidade Hfq para a transcrição dos genes responsáveis pela lesão A/E. responsible for the A/E lesion. / Enteropathogenic Escherichia coli are an important pathogen responsible for causing diarrhea. EPEC can be classified as typical and atypical, based on the presence of the EAF plasmid. EPEC strains have in their genome a pathogenicity island known as LEE region, where it harbours genes related to the formation of a lesion A/E. LEE regulation is multifactorial, being the ler gene its main regulator. Until now there are no studies on the role of Hfq in EPEC, thus, the present study analyzes the function of Hfq on the regulation of virulence factors in typical EPEC (O127:H6) and atypical (O55:H7) strains. Hfq gene mutagenesis was obtained utilizing the allelic recombination l Red system. The mutant strains demonstrated a decrease in the capability of mutant strains to adhere and form A/E lesion. Transcriptional analysis showed a decrease on the espA gene and eae gene transcription. It was possible to notice a decrease in motility of the mutant strains. In silico analysis revealed the possibility of a natural folding of ler mRNA, concealing the ribosome binding site. With this study we could demonstrate the need of Hfq for the transcription of genes responsible for the A/E lesion.

Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarreia infantil tanto em países desenvolvidos como em países em desenvolvimento. Esta bactéria possui dois fatores de virulência comprovadamente envolvidos na patogênese, intimina e bundle-forming pilus (BFP). Este patotipo está dividido em EPEC típica e EPEC atípica, ambos apresentam uma ilha de patogenicidade cromossomal denominada locus of enterocyte effacement (região LEE) onde está localizado o gene eae (E. coli attachment effacement), que codifica a intimina, uma proteína de membrana externa que medeia a adesão íntima da bactéria ao enterócito. Diferente da EPEC típica, as cepas de EPEC atípica não possuem o plasmídeo EAF (EPEC adherence factor) no qual encontra-se o operon bfp (bundle forming pilus) constituído por 14 genes incluindo bfpA o qual codifica a bundlina (BfpA), principal subunidade da fímbria Bfp, que possibilita a agregação bacteriana. Na infecção por EPEC ocorre grave disfunção da barreira epitelial, e uma das conseqüências é a inflamação. Na literatura, é bem descrito a interação entre as proteínas efetoras de EPEC com as células epiteliais e os processos iniciais da interação bactéria à célula hospedeira. Entretanto, poucos são os estudos que analisam a produção de citocinas em infecções por EPEC ou suas moléculas efetoras com relação a ativação de macrófagos, fundamentais para o controle do processo inflamatório e geração da resposta imune durante esta infecção. A análise das citocinas produzidas constitui uma parte importante da resposta imune e representa a tentativa do hospedeiro em lidar com um determinado microrganismo. Em função disto analisou-se o papel da intimina e do BfpA na capacidade de ativar a resposta inata mediada por macrófagos in vitro, onde avaliou-se a produção de citocinas pró-inflamatórias (TNF-α, IL-1, IL-6 e IL-12), citocina antiinflamatória (IL-10) e quimiocina (MCP-1). Os resultados demonstraram que as proteínas recombinantes intimina e BfpA são potentes ativadores de macrófagos, de forma dose dependente, produzindo TNF-α, IL-12 e IL-6, IL-10 e MCP-1, mas não IL-1β. Neste estudo não foi observado efeito sinérgico na produção de citocinas pró-inflamatórias ao associar intimina e BfpA, entretanto em dose mais elevada potenciou a produção de IL-10, um mediador antiinflamatório. O efeito imune obtido foi atribuído majoritariamente a estas proteínas uma vez que o tratamento destas com polimixina B não alterou a produção de TNF-α. Conclui-se que intimina e BfpA são potentes ativadores de macrófagos durante a resposta inata podendo colaborar para o controle do processo inflamatório durante a infecção por EPEC. / Enteropathogenic Escherichia coli (EPEC) is a common cause of childhood diarrhea in developed countries as well as developing countries. This bacterium has two proven virulence factors involved in pathogenesis, intimin and bundle-forming pilus (BFP). This pathotype EPEC is divided into typical and atypical EPEC, both having a chromosomal pathogenicity island called locus of enterocyte effacernent (LEE region) which contains the gene eae (E. coli attachment effacement). eae encodes intimin, an outer membrane protein that mediates the intimate adherence of bacteria to the enterocyte. Unlike typical EPEC, atypical EPEC strains do not possess the plasmid EAF (EPEC adherence factor) which is in the operon bfp (bundle forming pilus) consisting of 14 genes including bfpA, which encodes bundlin (BfpA), the main subunit of BFP allowing bacterial aggregation. EPEC infection occurs in severe dysfunction of the epithelial barrier, and one consequence is inflammation. In the literature, the interaction between effector proteins of EPEC and epithelial cells and the initial processes of bacterial interaction with the host cell are well described. However, there are few studies that have examined cytokine production in EPEC infections or their effector molecules with respect to macrophage activation, essential for controlling inflammation and immune response during this infection. The production of cytokines is an important part of the immune response and represents the host\'s attempt to deal with a particular microorganism. Therefore, we examined in vitro the role of intimin and BfpA in the ability to activate the innate response mediated by macrophages, where we analyzed the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, and the antiinflammatory cytokine IL-10 and chemokine MCP-1. The results show that recombinant intimin and BfpA are potent activators of macrophages in a dose-dependent manner, where the stimulated cells produce TNF-α, IL-12 and IL-6, IL-10 and MCP-1, but not IL-1β. In this study, no synergistic effect was observed in the production of proinflammatory cytokines by combining BfpA and intimin, although production of IL-10, an antiinflammatory mediator, was potentiated at a higher dose. The effect obtained was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. We conclude that intimin and BfpA are potent activators of macrophages during the innate response and may contribute to the control of inflammation during infection with EPEC.

Escherichia coli Enteropatogênicas são um importante patógeno causador de diarréia. As EPEC podem ser classificadas em típica e atípica, baseado na presença do plasmídeo EAF. As amostras de EPEC apresentam em seu genoma uma ilha de patogenicidade denominada região LEE, na qual estão contidos os genes relacionados a formação da lesão (A/E). A regulação gênica da região LEE é multifatorial, sendo o principal regulador o gene ler. Até o momento não existem trabalhos sobre a participação de Hfq em EPEC, assim sendo, o presente estudo analisa o papel de Hfq na regulação dos fatores de virulência de EPEC típica (O127:H6) e atípica (O55:H7). A mutagênese do gene hfq foi obtida através do sistema l Red de recombinação alélica. As amostras mutantes apresentaram uma diminuição na capacidade aderir e formar a lesão A/E. Analise transcricional dos mutantes revelou uma significativa diminuição na transcrição do gene espA e do gene eae. Foi possível evidenciar uma diminuição da motilidade das amostras mutantes. A analise in silico revelou a possibilidade do dobramento natural do mRNA ler, ocultando o sitio de ligação do ribossomo. Aqui demonstramos a necessidade Hfq para a transcrição dos genes responsáveis pela lesão A/E. responsible for the A/E lesion. / Enteropathogenic Escherichia coli are an important pathogen responsible for causing diarrhea. EPEC can be classified as typical and atypical, based on the presence of the EAF plasmid. EPEC strains have in their genome a pathogenicity island known as LEE region, where it harbours genes related to the formation of a lesion A/E. LEE regulation is multifactorial, being the ler gene its main regulator. Until now there are no studies on the role of Hfq in EPEC, thus, the present study analyzes the function of Hfq on the regulation of virulence factors in typical EPEC (O127:H6) and atypical (O55:H7) strains. Hfq gene mutagenesis was obtained utilizing the allelic recombination l Red system. The mutant strains demonstrated a decrease in the capability of mutant strains to adhere and form A/E lesion. Transcriptional analysis showed a decrease on the espA gene and eae gene transcription. It was possible to notice a decrease in motility of the mutant strains. In silico analysis revealed the possibility of a natural folding of ler mRNA, concealing the ribosome binding site. With this study we could demonstrate the need of Hfq for the transcription of genes responsible for the A/E lesion.

Quarenta e nove amostras EPEC típica (tEPEC) e atípica (aEPEC) pertencentes a diferentes sorotipos, isoladas de humanos e animais (cães, gatos, bovinos, ovinos, coelhos e sagüis) foram investigadas quanto ao perfil de virulência pela PCR e similaridade clonal por Multilocus Sequence Typing (MLST) e Pulsed-Field Gel Electrophoresis (PFGE). O objetivo deste estudo foi verificar se animais atuam como reservatório e fonte infecção de aEPEC para humanos. Os marcadores de virulência analisados revelaram que cepas aEPEC isoladas de animais possuem potencial para causar diarréia em humanos. As técnicas MLST e PFGE revelaram que amostras isoladas de animais e humanos compartilham relações clonais próximas ou idênticas. Estes resultados indicam que os animais estudados atuam como reservatório de aEPEC e representam fonte de infecção para humanos. Pelo fato de humanos, também atuarem como reservatório de aEPEC, ciclos de infecção cruzada animal-humano não podem ser descartados, pois a dinâmica de transmissão entre reservatórios de aEPEC não é muito bem compreendida. / Forty-nine typical and atypical EPEC strains belonging to different serotypes, isolated from humans, pets (cats and dogs), farm (bovines, sheep and rabbits) and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. Close clonal relationship between human and animal isolates was found with MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out, since the transmission dynamics between the reservoirs are not yet clearly understood.

Summary The group of E. coli strains is a highly heterogeneous group of strains, including pathogenic and non-pathogenic strains. The classification of these strains is made upon specific virulence factors of these bacteria. Some of these pathogenic strains are not capable to cross over the intestinal border and are responsible for intestinal disorders associated with diarrhoea. Other strains can cross the intestinal border and produce septicaemia and other complications depending on the infected organs. These pathogenic strains of E. coli interact with the host in a typical manner and produce specific lesions. These specific interactions are observed after the colonisation of the gut by pathogenic bacteria and are the results of the presence of specific virulence factors. The colonisation of the gut is mediated by specific adhesins, which are specific for each group of pathogenic E. coli. It seems that these adhesins are not only responsible to initiate the interaction between the pathogen and the host but are also responsible for the host specificity shown by some pathogenic strains. Its for that that a better understanding of these adhesins would permit a better understanding of the host specificity and a better evaluation of the zoonotic potential of these strains. The aim of this work was to identify bacterial structures involved in the intestinal adhesion step and in the intestinal colonisation of the enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) and verotoxigenic E. coli (VTEC) bovine isolates, focusing mainly (but not exclusively) on strains of serogroup O26. In order to achieve this objective, two different approaches have been followed: i) the immunologic approach and ii) the genetic approach. The immunologic approach is using the benefits of the 2F3 monoclonal antibody (MAb), which was obtained against an outer membrane protein extract from a human O26 EHEC strain. The work on this approach focused on the study of this MAb as epidemiological tool and on the identification of the epitope recognised by this antibody and of its genetic basis. The work on the genetic approach involves the screening by PCR and by colony hybridization of a collection of EHEC, VTEC and EPEC of human and bovine isolates for the presence of homologues for gene clusters coding for fimbriae of type I, II, III and IV: (i) putative adhesins of human EPEC and EHEC strains like LifA, Iha, LpfA, BfpA; (ii) fimbriaires adhesins (CS31A et F17) and afimbriaire adhesins of Afa family (Afa III, Afa VII, Afa VIII and F1845) described for other groups of bovine pathogenic E. coli. The accomplished work allowed, first of all, to confirm that the 2F3 MAb is specific for the O26 EPEC and O26 EHEC strains without being capable to distinguish between human and animal isolates, that means without being capable to distinguish them depending on theirs host. Moreover, it has been shown that: i) the epitope recognised by the 2F3 MAb is component of the O antigen from the O26 EPEC and O26 EHEC strains; ii) the genetic basis of this epitope is located inside the O antigen gene cluster of O26 EPEC and O26 EHEC strains; and iii) the 2F3 + and O26 + characters are two characters that can be dissociated by random insertional mutagenesis in a bovine EHEC strain. Nevertheless, the results of this work not allowed us to explain the specificity of the 2F3 MAb for the O26 EPEC and O26 EHEC strains. Actually, by sequencing the O-antigen gene cluster (the rfb locus) of an O26 non-EPEC and non-EHEC strain and comparing the obtained sequence with the already published sequence of the O-antigen gene cluster of an O26 EHEC strain no major differences (which could explain the specificity of the 2F3 MAb for the O26 EPEC or EHEC strains) were observed. The obtained results in the genetic approach have been shown that: i) all O145 and O157 EPEC and EHEC strains were positive for the LpfA probe (lpfA gene is coding for the type IV pili) and all other human and bovine strains belonging to other serogroups (O5, O26, O103, O111 and O118) have no homologue sequences for this probe; ii) a big majority of EPEC and EHEC strains belonging to O5, O26, O103, O111 or O118 serogroups were positives for the LifA probe (the lifA gene is involved in the synthesis of adhesins not very well characterised) but none of those belonging to the O145 et O157 serogroups; iii) different proportions of these strains belonging to various serogroups were positives for the Iha probe (the iha gene is involved in the synthesis of an adhesin not very well characterised); and iv) ten out of twelve bovine O26 EPEC strains were positive for the ClpE probe and all other strains (human EPEC and EHEC strains and bovines EHEC strains) were negative for the ClpE probe. The clpE gene is involved in the synthesis of the chaperon protein of the CS31A fimbria, which was described for the enterotoxigenic and septicaemic bovine and porcine E. coli isolates. Since the O145 and O157 EHEC strains show the same pathotype, it looks like that the presence of the lpfA gene and the absence of the lifA gene is linked more to a specific pathotype than to a specific serotype. Concerning the results obtained results with the ClpE probe for the O26 EPEC strains, it is possible that these strains have not an entire clp gene cluster since all the strains positive for the ClpE probe were negative for the ClpG (clpG gene is involved in the biosynthesis of the major unit of the CS31A fimbriae). Nevertheless, it is possible that the clpE-like gene of these strains belongs to an entire gene cluster for which the major unit is different from the ClpG. Finally, the screening of a collection of O8 and O20 VTEC bovine isolates for the presence of sequences homologues to the genes involved in the biosynthesis of adhesins of the Afa family (Afa III, Afa VII, Afa VIII and F1845), adhésines produced mainly by human uropathogenic isolates and by some septicaemic strains isolated from animals, allowed the identification of two O8 VTEC bovine strains harbouring the afa-VIII D/afa-VIII E genes and expressing the Afa VIII E adhesin. During this work, a lot of progresses, that could be useful for a better understanding of the pathogenesis of the EPEC, EHEC and VTEC strains, have been done. Despite that, the general objective of this project description of new factors involved in the initial adhesion stage and/or in the intestinal colonisation by the EPEC, EHEC and VTEC bovine strains in order to allow an easy and reliable diagnostic of these strains have been not accomplished. In perspective of this work, complementary works should focus: i) to identify the epitope recognised by the 2F3 MAb; and ii) to investigate which is the role of the adhesins, for which homologues sequences have been described in the human or bovine EPEC and EHEC isolates of different serogroups, in the adhesion to eukaryotic cells like bovine and human enterocytes. Résumé Lespèce Escherichia coli (E. coli) est hétérogène, contenant des souches pathogènes et des souches inoffensives. La classification des différentes souches pathogènes est basée sur leurs propriétés spécifiques de virulence. Les souches pathogènes dE. coli sont en effet associées à des pathologies variées. Certaines souches ne franchisent pas la barrière intestinale produisant des lésions dentérite, associées à de la diarrhée et dautres souches pathogènes dE. coli peuvent franchir la barrière intestinale, produisant de la septicémie, avec des complications variables selon les organes infectés. Ces souches interagissent avec lhôte dune manière particulière produisant des lésions typiques au niveau cellulaire et tissulaire. Ces interactions spécifiques sont dues aux propriétés de virulence spécifiques après la colonisation de lintestin. Létape de colonisation de lintestin se fait grâce à des adhésines particulières, spécifiques pour chaque groupe de souches pathogènes dE. coli. Il semble que ces adhésines nont pas seulement le rôle dinitialiser linteraction entre lhôte et le pathogène mais quelles soient responsables aussi de la spécificité dhôte présentée par certaines souches pathogènes. Ainsi la connaissance de ces adhésines permet la reconnaissance de la véritable spécificité dhôte de ces souches et lévaluation de leur potentiel zoonotique. Lobjectif général du travail était didentifier des structures bactériennes impliquées dans ladhérence aux entérocytes et, donc, dans la colonisation intestinale par des souches entérohémorragiques (EHEC), entéropathogènes (EPEC) et vérotoxinogènes (VTEC) bovines, et qui représenteraient une base de la spécificité dhôte de ces souches, en ciblant plus particulièrement le sérogroupe somatique O26. Pour accomplir lobjectif de ce travail, deux approches ont été suivies : immunologique et génétique. Lapproche immunologique a utilisé lanticorps monoclonal 2F3 qui a été dérivé contre des extraits protéiques de membrane dune souche EPEC humaine du sérogroupe O26. Le travail a consisté à identifier l'antigène reconnu par l'anticorps monoclonal 2F3, à déterminer sa base génétique et à lutiliser en tant qu'outil d'épidémiologie moléculaire. Le travail dans lapproche génétique a consisté à rechercher, par des épreuves de PCR et d'hybridation sur colonies sur une collection de souches EHEC, EPEC et VTEC bovines et humaines, la présence de gènes dopérons qui codent pour différents fimbriae de type I, II, III et IV : (i) des adhésines potentielles des souches EHEC et EPEC humaines, telle que LifA, Iha, LpfA, BfpA; (ii) des adhésines fimbriaires (CS31A et F17) et afimbriaires la famille Afa (Afa III, Afa VII, Afa VIII et F1845) présentes chez dautres catégories de souches dE. coli pathogènes pour le bovin. Les travaux effectués ont tout dabord permis de confirmer que lanticorps monoclonal 2F3 est spécifique pour les souches EPEC et EHEC du sérogroupe O26 et ne reconnaît pas les souches non-EHEC non-EPEC, sans cependant quil puisse faire la différence entre les souches EHEC et EPEC dorigine humaine et animale, cest-à-dire en fonction de leur hôte. Dans la partie des recherches consacrée à létude de cet anticorps, il a été aussi démontré que : i) lépitope reconnu par lanticorps monoclonal 2F3 est une composante du lipopolysaccharide (LPS) des souches EPEC et EHEC du sérogroupe O26 ; ii) la base génétique de cet épitope est situé dans lopéron codant pour lantigène O26 ; et iii) le caractère 2F3+ peut néanmoins être dissocié du caractère O26+ par mutagenèse par transposition aléatoire dans une souche EHEC bovine. Néanmoins, les résultats de ces travaux nont pas permis dexpliquer la raison pour laquelle lanticorps monoclonal 2F3 reconnaît les souches EPEC et EHEC O26, mais non les souches non-EPEC et non-EHEC O26. En effet, le séquençage de lopéron codant pour lantigène O26 (locus rfb) dune souche non-EHEC non-EPEC et sa comparaison avec la séquence publiée de lopéron rfb dune souche O26 EHEC na pas permis de reconnaître une différence pouvant expliquer la reconnaissance par lanticorps 2F3. Les résultats obtenus dans le cadre des travaux selon lapproche génétique ont montré que : i) toutes les souches EHEC bovines et humaines appartenant aux sérogroupes O145 et O157 étaient positives pour la sonde LpfA (les gènes lpfA codent pour des pili de type IV), mais aucune souche appartenant aux autres sérogroupes (O5, O26, O103, O111 et O118); ii) une grande majorité des souches EHEC et EPEC bovines et humaines aux sérogroupes O5, O26, O103, O111 et O118 étaient positives pour la sonde LifA (les gènes lifA codent pour des adhésines encore peu caractérisées), mais pas celles appartenant aux sérogroupes O145 et O157; iii) des proportions variables de souches appartenant à ces différents sérogroupes étaient positives pour la sonde Iha (les gènes iha codent aussi pour des adhésines peu caractérisées); et iv) dix souches EPEC bovines O26 sur les douze étudiées étaient positives pour la sonde ClpE (dérivée du gène codant pour la protéine chaperone du fimbria CS31A produit par des souches entérotoxinogènes et septicémiques bovines et porcines dE. coli), alors que les souches EHEC bovines et humaines du même sérogroupe, ainsi que toutes les souches des autres sérogroupes, étaient négatives pour cette sonde. Comme les souches EHEC O157 et O145 présentent le même pathotype, il semblerait que la présence des gènes lpfA et labsence des gènes lifA soient associées plus à un pathotype particulier quà un, ou quelques sérogroupes. En ce qui concerne les résultats avec la sonde ClpE sur les souches EPEC bovines O26, il est possible que ces souches ne possèdent pas lopéron clp complet puisque toutes les souches étaient négatives pour la sonde clpG (codant pour la sous-unité majeure du fimbria CS31A). Il est cependant aussi possible que le gène clpE-like de ces souches appartienne à un opéron complet dont la sous-unité majeure soit différente de ClpG. Enfin, la recherche des gènes intervenant dans la biosynthèse dadhésines de la famille Afa (Afa III, Afa VII, Afa VIII and F1845), surtout produites par des souches uropathogènes humaines et sur certaines souches septicémiques animales, dans la collection des souches VTEC dorigine bovine de sérogroupe O8 et O20 a permis la mise en évidence de deux souches VTEC de sérogroupe O8 qui possèdent les gènes afa-8D/afa-8E et qui expriment ladhésine Afa 8E. Au cours de nos travaux une série de résultats, qui pourront amener à une meilleure compréhension des mécanismes de pathogénicité et des souches EPEC, EHEC et VTEC ont été réalisés. Cependant, lobjectif général du projet - la description de facteurs spécifiques de lhôte impliqués dans le processus dadhérence initiale des souches EPEC, EHEC et VTEC bovines permettant la colonisation intestinale, afin de pouvoir les diagnostiquer et de les typer de manière spécifique - na pas été atteint. A court terme, les compléments de travaux à envisager sont la poursuite des essais didentification de lépitope reconnu par lanticorps monoclonal 2F3 et le rôle dans ladhérence aux cellules eucaryotes, comme les entérocytes bovines et humains, des adhésines dont les gènes ont été détectés dans les souches EHEC et EPEC bovines et humaines appartenant à différentes sérogroupes.

Quarenta e nove amostras EPEC típica (tEPEC) e atípica (aEPEC) pertencentes a diferentes sorotipos, isoladas de humanos e animais (cães, gatos, bovinos, ovinos, coelhos e sagüis) foram investigadas quanto ao perfil de virulência pela PCR e similaridade clonal por Multilocus Sequence Typing (MLST) e Pulsed-Field Gel Electrophoresis (PFGE). O objetivo deste estudo foi verificar se animais atuam como reservatório e fonte infecção de aEPEC para humanos. Os marcadores de virulência analisados revelaram que cepas aEPEC isoladas de animais possuem potencial para causar diarréia em humanos. As técnicas MLST e PFGE revelaram que amostras isoladas de animais e humanos compartilham relações clonais próximas ou idênticas. Estes resultados indicam que os animais estudados atuam como reservatório de aEPEC e representam fonte de infecção para humanos. Pelo fato de humanos, também atuarem como reservatório de aEPEC, ciclos de infecção cruzada animal-humano não podem ser descartados, pois a dinâmica de transmissão entre reservatórios de aEPEC não é muito bem compreendida. / Forty-nine typical and atypical EPEC strains belonging to different serotypes, isolated from humans, pets (cats and dogs), farm (bovines, sheep and rabbits) and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. Close clonal relationship between human and animal isolates was found with MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out, since the transmission dynamics between the reservoirs are not yet clearly understood.

No description available.

Le génome des bactéries vivantes n’est pas une entité statique mais au contraire c’est quelque chose très dynamique évoluant avec le temps. Les bactéries évoluent en acquérant par transfert horizontal des gènes du matériel génétique. C’est le cas des EPEC qui ont acquis l’îlot LEE via ce mécanisme. La protéine H-NS joue un rôle important dans la reconnaissance de cet ADN étranger, dans la liaison à cet ADN et dans la répression de son expression quand ce n’est pas en profit du « fitness » de la bactérie. Comme résultat H-NS régule la majorité des gènes associés à la virulence chez les entérobactéries Salmonella, Yersinia et les EPEC. Les EPEC possèdent une protéine paralogue à H-NS et codée dans le premier opéron de leur îlot LEE, il s’agit de la protéine Ler. Une fois exprimée Ler induit l’expression des 4 opérons restant de la région parmi lesquels LEE5. Ler partage une grande homologie avec H-NS surtout au niveau de leurs domaines de reconnaissance de l’ADN. Malgré cette homologie H-NS réprime LEE5 tandis que Ler l’active. De plus si H-NS est un régulateur global agissant sur plus de 500 gènes chez E. coli Ler est une protéine spécifique qui ne va agir que sur un petit nombre de promoteurs tous impliqués dans la virulence L’étude qualitative et quantitative de l’interaction de H-NS et de Ler avec la région promotrice de LEE5 montre qu’elles partagent globalement les mêmes sites de fixation sur des régions étendues en amont et en aval du +1 de la transcription. Ces sites de fixation sont bien définis d’une dizaine de paires de bases. L’affinité de ces sites pour H-NS est variable. Trois sites de haute affinité pour H-NS ont été identifiés. La séquence de ces sites est similaire à celle du site consensus élaboré en étudiant le promoteur proU(Bouffartigues et al - 2007). Des différences dans l’interaction de ces deux protéines avec le promoteur LEE5 résident surtout autour du +1 et des boîtes -10 et -35. Il s’agit de la première étude comparant la fixation de H-NS et de Ler sur des régions étendues de ce promoteur dans le but d’expliquer la régulation différentielle de ces deux protéines paralogues. L’étude de l’expression de LEE5 in vivo nous a permis de proposer que le mécanisme essentiel d’action de Ler est dirigé contre la répression induite par H-NS et que le taux maximum d’expression du promoteur LEE5 wtobservé dans la souche mutante pour hnsen présence de Ler (en comparaison avec la souche double mutante où Ler est absente) n’est pas dû à une activation directe par Ler mais plutôt à une répression par StpA, sensible à la mutation des sites de haute affinité de H-NS. / The genes of the LEE5 operon of enteropathogenic E.coliencode for proteinsthat are essential for their virulence. Their expression istightlyregulated, with H-NS silencing the transcriptional expression of LEE5 while Ler, product of the first operon of thispathogenicityislandcancounteract the silencing of H-NS. We show that H-NS and Ler use the samebinding sites on the LEE5 promoterin vitro. However, around the transcription start site differences in DNA constraints are detectabledepending on the presence of H-NS or Ler. Modification of the central AT bases, characteristic of H-NS consensus binding sites, affect the binding of bothproteinsin vitro and the expression in vivo of the LEE5 promoter. Additionallywe show that an additionalrepressor, the H-NS homologue StpA, isimplicated in the LEE5 regulationleading to a new model of how Ler canrelieve the H-NS imposedrepression on the LEE5 promoter

Orientador: Roberto de Oliveira Roça / Resumo: O objetivo deste estudo foi determinar a excreção de patógenos e inocuidade da carcaça de bovinos alimentados com diferentes níveis de silagem inoculada de grãos úmidos de destilaria desengordurados (WDG). Um total de 100 bovinos machos não castrados, 50% Angus e 50% Nelore, foram divididos aleatoriamente entre quatro dietas (N = 25) compostas por diferentes níveis de silagem de WDG (0, 15, 30 e 45% da matéria seca dietética). Amostras de fezes foram colhidas por meio de suabe da junção reto anal, 15 dias antes do abate, para determinar as ocorrências e quantificação de Escherichia coli produtora de toxina Shiga (STEC), E. coli enteropatogênica (EPEC) e Salmonella spp. por meio da técnica qPCR. Também foram colhidas, 52 dias antes do abate, amostras de fezes de cada animal do piso do curral, logo após defecação, as quais foram submetidas a análises físico-químicas. Logo após o abate, a ocorrência e a contagem de indicadores higiênicos e sanitários, E. coli não patogênica, coliformes totais e bactérias aeróbias mesófilas, assim como, a ocorrência de STEC, EPEC e Salmonella spp., foram determinados a partir de amostras colhidas por meio de esponja da superfície das carcaças das regiões do coxão, flanco, peito e pescoço. Os resultados quantitativos foram submetidos a análises de variância e os dados binários foram submetidos a análises logísticas com razão de chances. Todas as análises estatísticas foram realizadas no software estatístico SAS 9.4 considerando um nível de signifi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to determine the pathogen excretion and carcass safety of cattle fed different levels of inoculated silage from degreased wet distillery grains (WDG). A total of 100 male, 50% Angus and 50% Nelore male bulls were randomly divided into four diets (N = 25) composed of different levels of WDG silage (0, 15, 30 and 45% of dietary dry matter) . Stool specimens were collected by rectal anal junction swab 15 days prior to slaughter to determine the occurrences and quantification of Shiga toxin-producing Escherichia coli (STEC), E. coli enteropathogenic (EPEC) and Salmonella spp. by the qPCR technique. Samples of faeces from each animal on the corral floor were also collected, 52 days before slaughter, immediately after defecation, which were submitted to physical-chemical analysis. Immediately after slaughtering, the occurrence and counting of hygienic and sanitary indicators, non-pathogenic E. coli, total coliforms, and mesophilic aerobic bacteria, as well as the occurrence of STEC, EPEC and Salmonella spp., were determined from samples collected by medium of the surface of the carcasses of the regions of the tail, flank, chest and neck. The quantitative results were submitted to analysis of variance and the binary data were submitted to logistic analyzes with odds ratio. All statistical analyzes were performed in SAS 9.4 statistical software considering a significance level of 5%. The results showed that there was no difference between treatments fo... (Complete abstract click electronic access below) / Doutor