E. coli enteropatogênica atípica (aEPEC) é um importante patótipo causador de diarréia. As cepas de aEPEC não produzem BFP, sugerindo que outras fimbriais estão envolvidas na adesão de aEPEC ao hospedeiro. ECP é encontrada na maioria das cepas patogênicas e não patogênicas de E. coli. O objetivo do estudo foi avaliar a expressão e produção de ECP em cepas de aEPEC. A presença do operon ecp foi avaliada por PCR e a expressão avaliada por PCR utilizando cDNA obtido a partir do cultivo bacteriano em LB, DMEM e DMEM pré-condicionado. A produção de ECP foi avaliada por imunofluorescência. Foi observado que a expressão, por todas as cepas, só ocorreu quando foram cultivadas em DMEM pré-condicionado. Houve expressão diferencial do operon com o cultivo em LB ou DMEM. A produção de ECP foi observada apenas puma cepa quando cultivada em LB. Já em DMEM, três cepas produziram ECP. Houve um aumento do número de cepas produtoras da fímbria com o cultivo em DMEM pre condicionado. Esses resultados sugerem que a sinalização celular pode interferir na expressão e produção da ECP. / Atypical enteropathogenic E. coli (aEPEC) is an pathotype that causes diarrhea. aEPEC does not produce BFP, suggesting that other pili must be involved in aEPEC adhesion to the host cell. ECP is found in most pathogenic and non-pathogenic E. coli. The objective of this study was to evaluate the expression and production of ECP in aEPEC. The presence of ecp operon was assessed by PCR and the expression evaluated by PCR using cDNA obtained from bacterial growth in LB, DMEM and preconditioned DMEM. ECP production was evaluated by immunofluorescence. It was observed that the operon expression by all strains only occurred when they were grown in preconditioned DMEM. There was differential expression of ecp operon when strains were grown in LB or DMEM. ECP production was observed only by one strain when grown in LB. Three strains producted ECP grown in DMEM, but there was a higher production of the pili when strains were grown in DMEM preconditionated. These results suggest that cellular signaling may interfere with the expression and production of ECP.

E. coli enteropatogênica atípica (aEPEC) é um importante patótipo causador de diarréia. As cepas de aEPEC não produzem BFP, sugerindo que outras fimbriais estão envolvidas na adesão de aEPEC ao hospedeiro. ECP é encontrada na maioria das cepas patogênicas e não patogênicas de E. coli. O objetivo do estudo foi avaliar a expressão e produção de ECP em cepas de aEPEC. A presença do operon ecp foi avaliada por PCR e a expressão avaliada por PCR utilizando cDNA obtido a partir do cultivo bacteriano em LB, DMEM e DMEM pré-condicionado. A produção de ECP foi avaliada por imunofluorescência. Foi observado que a expressão, por todas as cepas, só ocorreu quando foram cultivadas em DMEM pré-condicionado. Houve expressão diferencial do operon com o cultivo em LB ou DMEM. A produção de ECP foi observada apenas puma cepa quando cultivada em LB. Já em DMEM, três cepas produziram ECP. Houve um aumento do número de cepas produtoras da fímbria com o cultivo em DMEM pre condicionado. Esses resultados sugerem que a sinalização celular pode interferir na expressão e produção da ECP. / Atypical enteropathogenic E. coli (aEPEC) is an pathotype that causes diarrhea. aEPEC does not produce BFP, suggesting that other pili must be involved in aEPEC adhesion to the host cell. ECP is found in most pathogenic and non-pathogenic E. coli. The objective of this study was to evaluate the expression and production of ECP in aEPEC. The presence of ecp operon was assessed by PCR and the expression evaluated by PCR using cDNA obtained from bacterial growth in LB, DMEM and preconditioned DMEM. ECP production was evaluated by immunofluorescence. It was observed that the operon expression by all strains only occurred when they were grown in preconditioned DMEM. There was differential expression of ecp operon when strains were grown in LB or DMEM. ECP production was observed only by one strain when grown in LB. Three strains producted ECP grown in DMEM, but there was a higher production of the pili when strains were grown in DMEM preconditionated. These results suggest that cellular signaling may interfere with the expression and production of ECP.

Orientador: Rodrigo Tavanelli Hernandes / Resumo: O principal mecanismo de virulência de Escherichia coli enteropatogênica (EPEC) é a capacidade de causar uma lesão histopatológica na mucosa intestinal denominada attaching and effacing (AE), caracterizada pela aderência íntima das bactérias, destruição das microvilosidades e formação de estruturas semelhantes a pedestais, ricos em F-actina, nos enterócitos infectados. Genes do locus of enterocyte effacement (região LEE) codificam todas as proteínas necessárias para a formação da lesão AE. As EPEC são divididas em típicas (tEPEC) e atípicas (aEPEC), com base na presença do EPEC adherence factor plasmid no primeiro grupo. A partir de um conjunto de sete amostras de aEPEC pertencentes ao sorotipo O2:H16, obtidos de surtos e casos esporádicos de diarreia, mostramos que cinco deles produziram uma adesão híbrida localizada/difusa (AL/AD) em células HeLa. Neste estudo, uma amostra de aEPEC deste sorotipo (282/14), que produziu o padrão AL/AD, foi selecionada para investigar as estruturas bacterianas envolvidas em seu fenótipo adesivo. Para este propósito, a amostra de aEPEC 282/14 foi mutagenizada usando o kit EZ::TN <R6Kyori/KAN-2> Tnp transposome kit, gerando uma biblioteca de inserções Tn5. Esses mutantes de inserção Tn5 foram testados quanto a perda ou redução da capacidade aderente, em ensaios realizados em 6 h de incubação com células HeLa. Dentre 320 clones pesquisados, nove foram considerados deficientes em sua capacidade de interagir com células epiteliais e quatro deles a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main virulence mechanism of enteropathogenic Escherichia coli (EPEC) is the capacity to cause a histopathological lesion on the intestinal mucosa, termed Attaching and Effacing (AE); characterized by intimate bacterial adherence, microvillus destruction and formation of F-actin rich pedestal-like structures, in infected enterocytes. Genes of the locus of enterocyte effacement (LEE region) encode all proteins necessary for AE lesion formation. EPEC are divided in typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in the former group. From a collection of seven aEPEC O2:H16, obtained from outbreak and sporadic cases of diarrhea, we showed that five of them produced a hybrid localized/diffuse adherence (LA/DA) in HeLa cells. In this study, we selected one aEPEC isolate of this serotype (282/14) that produced the LA/DA pattern, to investigate the bacterial structures involved in its adhesive phenotype. For this purpose, aEPEC 282/14 was mutagenized using the EZ::TN < R6Kyori/KAN-2 > Tnp transposome kit, generating a library of Tn5 insertions. These Tn5 insertion mutants were screened for non-adherent or less adherent mutants, in assays performed in 6 h of incubation with HeLa cells. Among the 320 clones screened, nine were considered deficient in their ability to interact with epithelial cells, and four of them presented the Tn5 insertion in genes within the LEE region, such as tir, escV, and grlR. In order to confirm the role of ... (Complete abstract click electronic access below) / Doutor

In der vorliegenden Arbeit wurde in einem in vitro-Modell mit porcinen intestinalen Epithelzellen (IPEC-J2) der Einfluss des probiotischen E. coli Nissle 1917 (EcN) auf die Infektion mit atypischen EPEC (aEPEC) untersucht. EcN reduzierte bei Vorinkubation auf IPEC-J2 die aEPEC-Infektion drastisch. Konfokale Laserscanning- und Elektronenmikroskopie zeigten, dass EcN die Adhäsion und Mikrokoloniebildung inhibierte, jedoch nicht die Ausbildung von Attaching and Effacing-Läsionen adhärenter aEPEC. Der inhibierende Effekt von EcN wurde durch dessen sehr gute Adhäsionsfähigkeit an IPEC-J2 vermittelt. Die F1C-Fimbrien wurden als wichtigster Adhäsionsfaktor von EcN identifiziert. Darüber hinaus waren auch H1-Flagellen durch Ausbildung interbakterieller Verbindungen maßgeblich an der Adhäsion des Stammes beteiligt. In gleichem Maß wie die Vorinkubation von EcN reduzierte die Koinkubation seines Kulturüberstandes die aEPEC-Infektion, was auf die Abgabe eines inhibierenden Faktors in den Kulturüberstand schließen lässt. Dieser Faktor wurde auch von anderen pathogenen sowie nicht pathogenen E. coli-Stämmen in Schüttelkultur gebildet und scheint deshalb nicht spezifisch für EcN zu sein. Jedoch ermöglichte erst die gute Adhäsionsfähigkeit von EcN auf der Epithelzelloberfläche die Abgabe ausreichender Mengen des Inhibitors und eine Beeinflussung der aEPEC-Infektion. Die Ergebnisse weisen darauf hin, dass durch EcN die initiale Anheftung von aEPEC an die Wirtszelle unterbunden wird. Der inhibierende Effekt von EcN auf die aEPEC-Infektion war zeitabhängig. Im Gegensatz zur Vorinkubation erhöhten Ko- und Nachinkubation von EcN die Adhäsion von aEPEC und hatten einen geringeren inhibierenden Effekt auf die Mikrokoloniebildung. Dieser gegensätzliche Effekt auf die Adhäsion von aEPEC wird möglicherweise von einem zweiten Faktor hervorgerufen. Dieser scheint nur dann wirksam zu sein, wenn der inhibierende Faktor in zu geringer Konzentration oder erst nach Adhäsion von aEPEC vorliegt. / In this study, the effects of the probiotic E. coli strain Nissle 1917 (EcN) on host cell infection with atypical enteropathogenic E. coli (aEPEC) were investigated in an in vitro porcine intestinal epithelial cell model (IPEC-J2). In pre-incubation experiments, EcN drastically reduced the infection efficiencies of aEPEC. Using confocal laser scanning microscopy and scanning electron microscopy, it was shown that EcN inhibited the attachment and formation of microcolonies, but not the formation of attaching and effacing lesions by adherent aEPEC. The inhibitory effect was mediated by the adherent properties of EcN to epithelial cells. The F1C fimbriae were identified as the most important adhesion factor of EcN in vitro. Furthermore, the H1 flagellae were also shown to be involved in the adhesion of EcN, serving as bridges between bacterial cells. Co-incubation of culture supernatants of EcN reduced the infection efficiencies of aEPEC to the same extent as in pre-incubation with EcN bacteria, indicating the secretion of an inhibitory factor by EcN. This factor was also secreted by other pathogenic and non-pathogenic E. coli strains in shaking culture and therefore does not appear to be specific for EcN. However, the outstanding ability of EcN to adhere to epithelial cells largely contributes to the secretion of sufficient concentrations of this inhibitory factor und to the influence on the aEPEC infection. The results suggest that EcN interferes with the initial adhesion of aEPEC to host cells. The inhibitory effect of EcN was found to be time-dependent. In contrast to pre-incubation experiments, co- and post-incubation of EcN actually increased the adhesion efficiencies of aEPEC and showed only minor effects on microcolony formation. This second effect of EcN on aEPEC adhesion, possibly due to a second factor, appears only to be effective when the putative inhibitory factor is either present at low concentrations or after aEPEC is already adherent to host cells.