Structural and biophysical characterization of the secreted, & beta;-helical adhesin EtpA of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with high morbidity and mortality especially among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that binds to the tips of flagellae to bridge ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion system (TPSS, type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) transporter that is integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal TPS domain followed by an extensive C-terminal domain with divergent sequence repeats. Two soluble, N-terminal constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA(67-447)) and 1 to 606 (EtpA(1-606)). The crystal structure of EtpA(67-447) solved at 1.76 & ANGS; resolution revealed a right-handed parallel & beta;-helix with two extra-helical hairpins and an N-terminal & beta;-strand cap. Analyses by circular dichroism spectroscopy confirmed the & beta;-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical AlphaFold model of full-length EtpA largely concurs with the crystal structure adding an extended & beta;-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal & beta;-helix into the C-terminal domains of TpsA proteins.

Automated summary

Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen without a vaccine, but a potential vaccine antigen called EtpA has been identified. EtpA is a conserved secreted adhesin that binds to flagella tips to attach ETEC to the host intestine. The folding structure of EtpA provides a template for extending the protein into the C-terminal domains.