Identiﬁcation of In Vivo-Induced Bacterial Protein Antigens during Human Infection with Salmonella enterica Serovar Typhi

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immu- nogenic 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 trans- port, 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. above) to reduce background reactivity against the host strain used in the whole- colony immunoblot assay. Evaluation of serum IgG responses to in vitro-transcribed and -translated PagC by Western blot analysis using paired acute- and convalescent-phase sera from patients with serovar Typhi bacteremia and from control patients. To conﬁrm the signiﬁcance of the serum immunoglobulin response to PagC in a whole-colony immunoblot assay, we applied an in vitro transcription-translation system using the pagC ORF cloned into the 5 (cid:7) NdeI and 3 (cid:7) NotI restriction enzyme cloning sites of pET30a to produce a T7 promoter-inducible template DNA, which was used in a commercial cell extract system (ActivePro; Ambion, Austin, TX) to yield a 20-kDa protein. Immune responses to PagC in paired acute- and convalescent-phase serum samples from 14 patients with serovar Typhi bacteremia and 7 control patients with cholera were then compared by Western blot analysis. For these studies, we used nonadsorbed serum from each patient at a 1:10,000 dilution. We detected immune responses with peroxidase- conjugated goat anti-human IgG (KPL, Gaithersburg, MD) at a dilution of 1:100,000, using an ECL kit (Amersham, Piscataway, NJ). Evaluation of serum IgG responses to puriﬁed TcfB by ELISA, using paired acute- and convalescent-phase sera from patients with serovar Typhi bacteremia and from control patients. To conﬁrm the immunoreactivity of TcfB in the whole-colony immunoblot assay, we constructed an inducible TcfB fusion protein expressing an N-terminal hexahistidine residue coupled with the C-terminal 148 amino acids encoded by tcfB . The initial 43 amino acids of the full predicted peptide sequence were predicted to be a signal peptide, and we did not include them in the expressed fusion. We generated the encoding sequences on an NdeI-XhoI fragment from serovar Typhi CT18 genomic DNA, using the 5 (cid:7) primer GGAGATATACATATGGTTCAGAAGGATATTACCGTC and the 3 (cid:7) primer ATGCGGATCCTCGAGTTAGCCGGCAGTGACAGCCTGTG, and cloned this fragment into the pET15b (Novagen, San Diego, CA) expression vector. We induced the expression of TcfB at 37°C with 1 mM isopropyl- (cid:6) - D thiogalactopyranoside in E. coli BL21(DE3). We lysed TcfB-containing cells via sonication and puriﬁed the fusion protein by nickel-afﬁnity chromatography under nondenaturing conditions to obtain a ﬁnal 17.5-kDa product at a concen- tration of 100 (cid:2) g/ml. We then diluted this product 1:100 in 50 mM carbonate buffer (pH 9.6) and allowed it to adsorb overnight at room temperature onto ELISA plates (Nunc Maxisorb; NalgeNunc, Rochester, NY), using 100 (cid:2) l of sample per well. We applied acute- and convalescent-phase sera (1:100 dilution) from 14 patients with serovar Typhi bacteremia and 7 control patients with cholera, detected anti-TcfB IgG with horseradish peroxidase-conjugated rabbit anti-human IgG (Jackson Immunoresearch, West Grove, PA) at a 1:1,000 dilution, and measured peroxidase activity with the substrate 2,2 (cid:7) -azinobis(ethyl- benzthiazolinesulfonic acid). The optical density at 405 nm was determined kinetically with plates read for 5 min at 19-second intervals, and the maximum slope for an optical density change was converted to ELISA units by comparison with pooled standard reference sera (obtained from healthy volunteers from Bangladesh), as described previously (10). We evaluated the statistical signiﬁcance of the difference between acute- and convalescent-phase serum anti-TcfB IgG levels by using a nonparametric test (the Wilcoxon signed rank test) for comparing paired samples.