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Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

ABSTRACT: 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 (EtpA67-447) and 1 to 606 (EtpA1-606). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel β-helix with two extra-helical hairpins and an N-terminal β-strand cap. Analyses by circular dichroism spectroscopy confirmed the β-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 β-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 β-helix into the C-terminal domains of TpsA proteins.

SUBMITTER: Ntui CM

PROVIDER: S-EPMC10284417 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

Ntui Clifford Manyo CM Fleckenstein James M JM Schubert Wolf-Dieter WD

PloS one 20230621 6

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 ...[more]

PMID: 37343026

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Project description:Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Dataset Information

Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

Publications

Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

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