In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth. Vibrio cholerae is a Gram-negative bacillus that causes a severe, dehydrating diarrhea in humans (1). V. cholerae can be differentiated by the lipopolysaccharide in the outer membrane; strains of V. cholerae that produce cholera belong to serogroup O1 or O139. V. cholerae O1 is divided into two biotypes, classical and El Tor; the current global pandemic of V. cholerae O1 infection is caused by El Tor strains.A major virulence factor for pathogenic strains of V. cholerae is cholera toxin, a protein exotoxin that consists of a single A subunit noncovalently associated with five B subunits (2). A second major virulence factor of V. cholerae is the toxin coregulated pilus (TCP; ref. 3). TCP is essential for colonization and virulence in both mouse models of cholera (3) and human volunteer studies (4). TcpA, the 20.5-kDa major structural subunit of TCP, has homology to the type IV pili of several other bacterial pathogens (5). TcpA from El Tor and classical strains of V. cholerae show Ϸ80% protein homology; monoclonal antibodies demonstrate epitope differences between these proteins in the two biotypes (6, 7).In addition to TcpA, the V. cholerae genome encodes two other type IV pili, the mannose-sensitive hemagglutinin (MSHA) and PilA (8). MSHA is a thin, flexible pilus composed of a 17-kDa subunit (9). A strain of V. cholerae deleted in mshA showed no defect in colonization of human volunteers (10). Recently, Fullner et al. (11) described a four-gene cluster, pilABCD, encoding a third type IV pilus in V. cholerae. A deletion of pilA had no effect on colonization in infant mice. The role of PilA in human infection has not been previously examined.

BackgroundIn this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle.ResultsAntisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-γ), assayed under same conditions. This suggested that proteins other than intimin-γ that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential.ConclusionProteins other than LEE and intimin-γ proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines.