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.

Fig. 1.

ELISA results after sequential steps in adsorption of pooled, patient sera; OD values are corrected for background and for dilution during adsorption steps. (A) Shown are ELISA plates coated with whole V. cholerae extracts. (B) Shown are ELISA plates coated with GM₁ ganglioside and CtxB. FP, French press.

Fig. 2.

Results using pooled, convalescent sera after complete adsorption. (A) Individual, purified proteins were spotted on a nitrocellulose membrane and the membrane developed by using pooled convalescent sera. (B) Individual E. coli BL21 strains containing plasmids expressing specific proteins (or control plasmid, pET30a) were transferred to a nitrocellulose membrane and developed by using pooled patient sera after adsorption.

Fig. 3.

Comparison of reactivity of unadsorbed sera from individual patients collected on days 0, 9, and 23 after V. cholerae infection, and control patients from day 23, against purified El Tor PilA or TcpA.