Resumo

Introduction: Enteropathogenic E. coli has been identified as one of the main responsible agents of acute diarrhea in developing country populations. Diarrhea is still one of the most significant cause of global child mortality between 0-5 years old. The goal of this study is to characterize and identify the outer membrane proteins (OMPs) of extracts derived from two strains of EPEC. Methods: Strains BA320 (serotype 055:H7) and BA 4013 (serotype O83:H6) were selected for this study. The OMPs were analyzed by two dimensional electrophoresis (2-DE), the first dimension by focalization of 13cm, pH range of 4-7 strips (IPGphor III, GE Healthcare) and the second by SDS-PAGE using 15% SDS-polyacrylamide gels (SE 600 Ruby, GE Healthcare). The identification was done by removing the spots from the gels and digestion with trypsin followed by mass spectrometric analysis on ESI QTOF Ultima – Waters. The resulting data were analyzed with a non-redundant protein database (NCBInr) using Mascot v3.0 engine (Matrix Science, www.matrixscience.com). Results and discussion: Thirty three spots were identified with high scores for BA320 strain, allowing for the characterization of nineteen distinct proteins. For BA4013 strain, forty five spots were identified with twenty four distinct proteins. The majority of proteins were localized in the membrane, indicating the efficiency of the extraction method. Ten proteins were identical between the strains. Seven proteins were OMPs or porins (Omp A, Omp C, Omp W, Omp X, nucleoside channel receptor of phage T6 and colicin K, maltoporin and TolC). Three transporters were identified (Tsx, Vitamin B12 and fatty acid transporters). Four enzymes were detected (ATPD, phosphopyruvate hydratase, Isocitrate lyase and phosphopentomutase). Moreover, chaperonin (Groel GROES ADP7), flagellin, protection protein (DPS), one hypothetical protein and two proteins of unknown function (YgaU and YgiW) were identified. Conclusions: The preliminary data indicate that the majority of proteins identified have important roles in membrane permeability and at least three of them, Omp A, Omp X and flagellin are involved in the adhesion of the pathogen to host cells. Identification of proteins of unknown functions creates possibilities to gather important missing information in future. Supported by: FAPESP