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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts
58
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.