Aspectos genéticos e da dinâmica da interação de Escherichia coli enteropatogênica atípica e E. albertti com células epiteliais

• Ano de defesa: 2017
• Autor(a) principal: Romão, Fabiano Teodoro [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Escherichia Spp; Diarreia; Patógenos; Sistema de secreção tipo 3; Interação bactéria-célula; Attaching and effacing
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5554474; http://repositorio.unifesp.br/handle/11600/50703

Atypical enteropathogenic Escherichia coli (EPEC) and Escherichia albertii are recognized enteropathogens in several geographic regions. They produce the attaching-effacing (AE) lesions in enterocytes, whose formation depends on the expression of several genes located in the LEE (locus of enterocyte effacement) region, including those involved in the formation of a Type 3 Secretion System (SST3) and the adhesin intimin. The AE lesion formation requires the interaction of intimin and its receptor, Tir, which is translocated by the SST3. There are some gaps in the knowledge on how the LEE-genes are expressed during bacterial interaction with host cells. Objectives: To study two AE-producing pathogens, aEPEC 1711-4 and E. albertii 1551-2, regarding the expression of the five LEE operons during the various stages of their interaction with enterocytes (adhesion, invasion, and intracellular persistence), as well as the occurrence of alterations (quantitative and qualitative) in the bacterial interaction during these different phases. In addition, the genome of these strains was sequenced and analysed to verify the possible involvement of effector proteins and other potential virulence factors that could be associated to the process of invasion and intracellular persistence. Methods: Qualitative and quantitative alterations in adhesion, AE lesion production, invasion and intracellular persistence in polarized and differentiated Caco-2 intestinal cells were evaluated using a kinetic study (1.5 h, 3 h, 6 h and 24 h of interaction). RT-PCR was used to analyze and compare the expression of genes representative of the five LEE operons (ler, escC, escV, eae and espA) in these different periods of interaction. For aEPEC 1711-4, the expression of the fliC gene, which encodes the structural subunit of the flagellum, was also analyzed, since previously it had been demonstrated that this structure contributes to cell adhesion and invasion abilities of this strain. For the fluorescence actin polymerization assays, HeLa cells were used at 2 h, 3 h and 6 h times, and for Livecell imaging, GFP transformed cells were used for 2 h. Sequencing of aEPEC 1711-4 and E. albertii 1551-2 strains and secreted protein analysis were also performed for further analysis. Results: For both strains studied, the amount of bacteria adhered to Caco-2 cells increased up to 3 h of contact (approximately 105 CFU / mL), remaining constant between 3 h and 6 h. In addition, for both strains, the invasion process started close to 3 h of contact with these cells (0.2% of the total associated bacteria for aEPEC 1711-4 and 0.02% for E. albertii 1551-2). Expression of the eae gene (which encodes for intimin) in aEPEC 1711-4 progressively increased up to 6 h of contact, while the expression of the other genes representative of the LEE and fliC remained constant. In turn, for E. albertii 1551-2, there was no change in the expression of the genes studied within 6 h of interaction. During intracellular persistence, the expression of the studied genes remained constant in aEPEC 1711-4, while for E. albertii 1551-2, it was observed an increase of at least 8-fold in the expression of these genes. Genome analysis of strain 1551-2 identified the presence of 31 SST3-dependent effectors, among them a TccP2/EspFu2 variant. Conclusion: SST3 is associated with adhesion, colonization, invasion and intracellular persistence in the aEPEC and E. albertii strains studied, with the expression of each operon being distinct, indicating that other transcriptional and/or post-transcriptional regulators could contribute for the observed differences.