Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

25-POS Board 25 Structural Characterization of EtpA, an Adhesin from Enterotoxigenic Escherichia Coli (ETEC) Lungelo Mandyoli , Wolf-Dieter Schubert, University of Pretoria, Pretoria, Gauteng, South Africa. Enterotoxigenic Escherichia coli (ETEC) are the most common bacterial pathogens causing diarrhoea in developing countries and in travelers to endemic countries. They cause hundreds of thousands of deaths, mostly in children. As part of its infection strategy, ETEC invades and colonizes small intestinal epithelial cells where it secretes heat-labile and/or heat-stable enterotoxins, inducing diarrhoea. The ability of ETEC to invade human epithelial cells is a hallmark of its pathogenicity and is controlled by a set of plasmid and chromosome encoded virulence factors. They include EtpA, a 170 kDa plasmid encoded autotransporter. During infection, EtpA functions as an adhesin that links flagellin at the tip of ETEC flagella to the host cell surface. ETEC hence interacts with host cells through its flagella appendages to deposit its toxins. Antibodies targeting either EtpA or the conserved regions of flagellin impair delivery of the heat-labile toxin in vitro , and prevent intestinal colonization of mice following gastrointestinal challenge with ETEC. EtpA is thus critical to the pathogenicity of ETEC. In our study we are aiming to elucidate the structure of EtpA to explain how it is able to perform its bridging function. We have cloned and are producing a truncated version of EtpA (57 kDa) termed N-terminal EtpA or N-EtpA as a C-terminal His6-tagged fusion protein in E. coli TOP10 cells. The protein is purified to homogeneity by metal affinity chromatography (MAC) using Ni- NTA and size exclusion chromatography (SEC) on a Superdex 75 10/30 column. Biophysical characterization of N-EtpA using circular dichroism spectroscopy (CD) revealed the typical spectrum of a β-helical protein. Recording CD spectra at increasing temperatures indicate N- EtpA to be highly thermal stable retaining its conformation up to 95 ℃ . Crystallization experiments of N-EtpA are currently under way.

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