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| Author(s): |
Fernanda Batista de Andrade
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2019-03-15 |
| Examining board members: |
Waldir Pereira Elias Junior;
Rita de Cassia Cafe Ferreira;
Carla Taddei de Castro Neves;
Rosa Maria Silva
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| Advisor: | Waldir Pereira Elias Junior; Roxane Maria Fontes Piazza |
| Abstract | |
Enteroaggregative Escherichia coli (EAEC) is defined as the pathotype of diarrheagenic E. coli that presents the aggregative adherence pattern on epithelial cells. After colonization of the intestinal mucosa, EAEC secretes several enterotoxins and cytotoxins, among them the proteins called serine proteases autotransporters of Enterobacteriaceae (SPATEs), classified in class-1 (cytotoxic) and class-2 (immunomodulatory). SepA (Shigella extracellular protein A) is a SPATE of 110 kDa class-2, which was originally described in Shigella flexneri 5a and later in EAEC. The presence of the sepA gene is a marker associated with diarrhea caused by EAEC, indicating that this toxin plays an important role in its pathogenesis. However, studies characterizing SepA as a virulence factor are restricted to S. flexneri. In this species, SepA participates in the destruction and invasion of epithelial tissue but has no cytotoxic effect on HEp-2 cells. However, it destabilizes the integrity of the tight junctions in polarized T84 cells, through the activation of the cofilin. Thus, the present study aimed to evaluate the prevalence of the sepA in a collection of EAEC isolated in Brazil, to purify SepA and to analyze its interaction with the Caco-2, HEp-2, HeLa, HT-29, MDCK, Vero and Y-1, as well as their ability to induce interleukins in HT-29 cells. The sepA gene was detected in 13.5% of the 193 EAEC strains and was significantly associated with the atypical EAEC subgroup. SepA was purified from S. flexneri M90T in its native form, showing biological activity of serine protease. SepA caused morphological changes in HeLa and HT-29 cells, induced by its binding to the membrane of these cells, without its internalization. The same effects were caused by the interaction of the SepA-producing EAEC strain (BA732:: pic) and the secretion of this SPATE was observed in the focus of bacterial adhesion on epithelial cells. The morphological changes in HeLa and HT-29 did not lead to cell death. SepA was also able to induce IL-8 production in HT-29 cells. Our data indicate that, as in Shigella, SepA is an important virulence factor of EAEC, since it induces cytopathic and proinflammatory effect in cells of human intestinal origin, changes known to be promoted by EAEC in experimental models. (AU) | |
| FAPESP's process: | 14/18286-0 - Role of the SepA serine protease in the cytotoxic and proinflammatory activities induced by enteroaggregative Escherichia coli |
| Grantee: | Fernanda Batista de Andrade |
| Support Opportunities: | Scholarships in Brazil - Doctorate |