Use of in vivo -induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae

Abstract

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.

The Vibrio cholerae chitin utilization program

“…Fullner et al (17) showed that an in-frame deletion of pilA did not affect colonization of the infant mouse intestine. However, Hang et al (25), using in vivo-induced antigen technology (IVIAT), showed that a PilA immunogen is expressed during human infection with V. cholerae, indicating that it might have a pathogenic function in man. This result suggests that V. cholerae might encounter GlcNAc ␤1-4GlcNAc-containing glycoconjugates in the intestine, thus inducing pilA expression, but the pathogenic significance of this response is unknown.…”

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

“…It is a type IV bundle-forming pilus, whose major subunit (TcpA) was identified in a screen for secreted virulence factors which are coregulated with CT (214,215). It is expressed in the human intestine and belongs to the major antigens in human infections (18,97). The genetic element encoding TCP (also termed VPI for "V. cholerae pathogenicity island") has been described as the genome of a filamentous phage (VPI⌽ or TCP⌽ [129]), but the phage nature has been disputed recently (63,77).…”

Biodiversity of Vibrios

“…Although chromosome 1 contains most of the genes that are required for growth (162,251), chromosome 2 contains more genes for bacterial adaptation to environmental changes, indicating that the chromosomes play different roles (251). The global expression pattern of the genes on the two chromosomes of V. cholerae has been analyzed by using a whole-genome microarray (37,231,264,447) and other methods (156). By using the rabbit ileal-loop model, Xu et al (447) compared the global transcriptional pattern of in vivo-grown cells with that of cells grown to mid-exponential phase in rich medium under aerobic conditions.…”