Identification of a Two-Partner Secretion Locus of Enterotoxigenic Escherichia coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecularweight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in travelers and in children in developing countries (72). ETEC is also an emerging cause of diarrheal illness in industrialized countries, including the United States (3, 6, 75). The organisms belonging to this taxon have genetically diverse E. coli pathotypes characterized by the production of heat-labile enterotoxin and/or heat-stable enterotoxin. In the classic paradigm of ETEC infection, these organisms adhere to the small intestinal mucosa via fimbrial colonization factor (CF) molecules, where they elaborate heatlabile enterotoxin and/or heat-stable enterotoxin. While early studies indicated that immunization with CFs provides protection against ETEC infection, development of a broadly protective vaccine has been hampered by the antigenic heterogeneity of these molecules. However, the recent identification of additional ETEC surface molecules (32,56,68) suggests that the present pathogenesis paradigm is incomplete and consequently that there may be additional antigens that can be exploited in vaccine development.Surface expression of virulence molecules by ETEC and other gram-negative pathogens requires export through t...

Beyond the Sequon: Sites of N-Glycosylation

“…In turn, the HMW1A adhesin is important for NTHi colonisation and pathogenesis (St Geme et al, 1993, St Geme, 1994. Similar to several other described bacterial protein glycosyltransferases, HMW1C glycosylates its HMW1A substrate protein in the cytoplasm, before secretion across the inner membrane (Fleckenstein et al, 2006, Charbonneau et al, 2012, Choi et al, 2010, Schwarz et al, 2011a. Most of these other reported bacterial glycosyltransferases are Oglycosyltransferases, transferring nucleotide-activated monosaccharides to the hydroxyl groups of Ser or Thr.…”

Pathogenomics of the Virulence Plasmids ofEscherichia coli

“…1). This plasmid contains eatA (141); the two-partner secretion locus etpABC, involved in adhesion to intestinal epithelia (54); and the genes encoding CFA/I. The presence of CFA/I on pH10407_95, which is a RepFIIA plasmid, suggests that the ETEC CS operons have been acquired on multiple occasions on multiple plasmid backbones.…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…In the same ETEC strain, H10407, an adhesin important for binding to intestinal epithelial cells, EtpA, was also found to be glycosylated by an adjacent enzyme (EtpC). Both genes, together with the etpB gene encoding a transporter, are part of the same locus (425).…”