Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi

Abstract

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with Salmonella enterica serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in S. enterica serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute- versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses.

FIG. 1.

Evaluation of the specificity of immunoreactivity of three fimbria-related antigens and PagC. We compared the immunoreactivity to each antigen identified by IVIAT in a whole-colony blot assay using pooled sera from patients with serovar Typhi bacteremia to the reactivity of pooled sera from adult North American volunteers. Four of the IVIAT-identified antigens (PagC, TcfB, STY0860, and STY3683) had no detectable immunoreactivity with the North American volunteer sera compared to control colonies with no insert. Each antigen was screened in triplicate with both patient and control sera, and a single representative immunoblot is shown.

FIG. 2.

Responses to PagC in paired serum samples obtained from patients infected with S. enterica serovar Typhi or V. cholerae (controls). PagC was produced by a cell-free in vitro transcription-translation system, and individual patient immune responses were visualized by Western blotting. (A) Eleven of 14 patients infected with serovar Typhi had a visible increase in anti-PagC IgG antibodies during convalescence (conv.). +, visually significant increase in reactivity between paired acute- and convalescent-phase samples by Western blotting; −, no response. (B) None of the patients infected with V. cholerae had a visible increase in PagC reactivity with convalescent-phase serum. None of the North American volunteers had visible reactivity against PagC in this assay (data not shown).

FIG. 3.

Responses to TcfB in paired serum samples obtained from patients infected with S. enterica serovar Typhi or V. cholerae (controls). Sera were collected at the time of presentation to a health care facility (acute phase) and again 2 to 3 weeks later (convalescent phase [conv.]). Columns represent geometric means, and error bars represent the standard errors of the means.