Abstract:
Enterotoxigenic Escherichia coli (ETEC) encompass a group of diverse bacterial pathogens that collectively cause hundreds of millions of diarrheal cases annually, mostly in developing countries. 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 linking flagellin at the tip of ETEC flagella to the host cell surface and allowing ETEC 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 this study, a truncated version of EtpA (35 kDa) termed N-terminal EtpA69-445 or N-EtpA69-445 was cloned and produced as an N-terminal GST-tagged cytoplasmic fusion protein in E. coli BL21 cells. The protein was purified by affinity chromatography on glutathione agarose beads. However, the yield of the pure protein was poor due to its limited solubility.
As an alternative, a 57 kDa truncated version of EtpA (N-EtpA69-607) was produced as a secreted C-terminal His6-tagged fusion protein in E. coli TOP10 cells. The protein was purified to homogeneity by metal affinity chromatography (MAC) using Ni-NTA and ion exchange chromatography (IEC) on a Mono S 10/100 GL column. Biophysical characterization of N-EtpA69-607 using circular dichroism (CD) spectroscopy revealed the typical spectrum of a β-helical protein. The in silico modelled structure of the protein confirmed N-EtpA69-607 to be a β-helical protein. CD spectra recorded at increasing temperatures indicated N-EtpA69-607 to be thermally highly stable retaining its fold up to 95°C. Dynamic light scattering (DLS) experiments showed that N-EtpA69-607 is polydisperse in solution forming higher oligomers. Lead crystallization conditions of N- EtpA69-607 were determined but the crystals were too small for X-ray data collection. This study thus represents a significant step towards the characterization of the three dimensional structure of EtpA and understanding its structure-function relationship.
