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| Author(s): |
Ester Castro Araujo
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2025-02-07 |
| Examining board members: |
Rodrigo da Silva Galhardo;
Cristiane Rodrigues Guzzo Carvalho;
Rodrigo Tavanelli Hernandes;
Fernanda Fernandes dos Santos
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| Advisor: | Waldir Pereira Elias Junior; Cecilia Mari Abe |
| Abstract | |
Enteroaggregative Escherichia coli (EAEC) is an intestinal pathogen responsible for cases of acute and persistent diarrhea in children and adults worldwide, corresponding to the most prevalent diarrheagenic E. coli pathotype in our country, which is characterized by the production of the aggregative adhesion pattern (AA) in cultured epithelial cells. EAEC infection and colonization are associated with a high adhesion capacity to the intestinal mucosa, mediated by aggregative adherence fimbriae (AAF). Genes related to its biogenesis are organized in an operon. Five AAF variants are known (AAF/I, AAF/II, AAF/III, AAF/IV and AAF/V) and each EAEC strain presents only one of them. However, a recent study on the frequency of AAF variants identified strains harboring the genes encoding the pilins of two distinct AAF variants: agg3A and agg5A, which encode the main subunits of AAF/III and AAF/V, respectively. The plasmids of these strains were sequenced, identifying a hybrid genetic arrangement with the presence of the complete AAF/III operon and an incomplete part of the operon that encodes AAF/V. However, the structure of the fimbria produced in the simultaneous presence of AAF/III and AAF/V biogenesis genes has not been described. Therefore, the present study aimed to characterize the ultrastructure of the fimbria encoded by the hybrid agg3/agg5 operon using an EAEC strain of our collection (strain BA381) previously characterized by the presence of the agg3A, agg5A, aggR and aatA genes. By PCR analyzes and whole genome sequencing, it was shown that BA381 harbors different genes associated with EAEC virulence and belongs to phylogroup A and sequence type 446. Complementary PCR assays and sequencing of the region where the agg3/agg5 genes are located using the Sanger method indicated the presence of a genetic arrangement with the presence of the complete AAF/V operon and part of the operon that encodes AAF/III, different from that previously observed for the reference strain C700-09. RT-PCR assays were also performed, detecting transcripts only from agg5A when BA381 was grown in different culture media. Comparative analysis between strains BA381 and C700-09 were performed employing anti-AAF/III and anti-AAF/V antisera in immunoblotting and immunogold labelling (transmission electron microscopy) assays. These assays showed the exclusive presence of AAF/V in strain BA381, as well as AAF/III in the reference strain C700-09, supporting data obtained by RT-PCR. The results obtained here showed that the type of AAF fimbriae synthesized and assembled by strains that simultaneously harbor agg3A and agg5A is determined by the operon present in a complete organization (chaperone, usher, minor pilin and major pilin). Also, the agg3A and agg5A genes are not arranged in a conserved manner among the different EAEC strains studied. (AU) | |
| FAPESP's process: | 22/09435-9 - Ultrastructural characterization of the AAF fimbriae encoded by the hybrid agg/agg5 operon of enteroaggregative Escherichia coli and its role in interaction with host cells |
| Grantee: | Ester Castro Araujo |
| Support Opportunities: | Scholarships in Brazil - Master |