Proteoimics, molecular and functional analysis of potential adhesins from human sepsis associated Escherichia coli (SEPEC)

Author(s)

Rogério Arcuri Conceição

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2014-08-15

Examining board members

Tomomasa Yano; Cristina Elisa Alvarez Martinez; Wanderley Dias da Silveira; Terezinha Knöbl; Roxane Maria Fontes Piazza

• Advisor: Tomomasa Yano

Abstract

Our preliminary studies about bacterial adhesion and invasion showed 100% of 49 strains of human sepsis-associated Escherichia coli (SEPEC) were able to adhere and invade Vero (African green monkey kidney) and HUVEC (human umbilical vein) cells. In genotypic analysis, high prevalence of fimH (98.0%), gene encoding for the type 1 fimbrial adhesin was observed. However, all the strains analyzed were able to adhere and invade these cellular lineages both in the presence and absence of the type 1 fimbriae adherence-inhibitor ?-D-mannopiranoside. Consequently, the outer membrane proteins of SEPEC were analyzed in order to find potential adhesion and invasion factors expressed by these strains. Through proteomic techniques, three proteins with affinity to cellular glycoproteins were identified: OmpA (Membrane Protein A); FimA (major subunit of type 1 fimbriae - F1) and YdeR (minor subunit of type 9 fimbriae - F9). These data were consistent with the genotypic analysis of SEPEC strains because a high percentage of the genes encoding these three proteins was observed, being that ompA+ (100%) fimA+ (98%) and ydeR+ (100%). Assays of adhesion and invasion of SEPEC (OmpA+ / F1+) in Vero and HUVEC cells showed that ?-D-manopiranosideo and GlcNAc, when acting together, act as potent inhibitors of adhesion and invasion, suggesting that these molecules could be occupying sites of carbohydrate-binding displayed by adhesins from both F1 and/or OmpA, respectively. To confirm this hypothesis, null mutants ?fimA (OmpA+/F1-) and ?ompA (OmpA-/F1+) were constructed, and the phenotypic results of bacterial adhesion and invasion were similar to those obtained in the presence of inhibitors ?-D-mannopiranoside and GlcNAc. The mutant ?ydeR (OmpA+ / F1+), null mutant for YdeR protein of F9 fimbriae, showed a significant reduction in bacterial adhesion and invasion in Vero and HUVEC (p ? 0.05) just in the presence of inhibitors of cell adhesion mediated by F1 and OmpA. These data suggest that F9 also can contribute to the adhesion and invasion of SEPEC in Vero and HUVEC cells. Hold together, our data demonstrated that adhesion and invasion of SEPEC are OmpA, F1 and F9 dependents, which can be complementary and express synergistic activity leading to enhancing the ability of adhesion and invasion in SEPEC strains (AU)

• FAPESP's process: 10/00587-3 - Proteomic analysis of adherence and biofilm formation of human sepsis-associated Escherichia coli(SEPEC)
• Grantee: Rogério Arcuri Conceição
• Support Opportunities: Scholarships in Brazil - Doctorate