Immunoproteomic analysis to identify Shiga toxin-producing Escherichia coli outer membrane proteins expressed during human infection

Abstract

Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization-tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development.

FIG 1

OMP profiles and OMP IgG seroreactivity with pooled sera from HUS patients and controls. (A) SDS-PAGE of OMPs. A 12% polyacrylamide gel and silver staining were used. (B and C) Western blot of 12% SDS-polyacrylamide gels using pooled sera from HUS patients (B) and controls (C) (1:3,500 dilution). Anti-human IgG secondary antibodies were diluted 1:5,000. Lane M, protein ladder indicating the molecular masses of the various proteins; lanes 1, STEC O26:H11; lanes 2, STEC O103; lanes 3, STEC O113:H21; lanes 4, STEC O157:H7; lanes 5, E. coli HS. Arrows with numbers correspond to the proteins listed in Table 1.

FIG 2

2D-PAGE OMP profiles of STEC strains and E. coli HS. The pH range was 4 to 7, and 12% polyacrylamide gels and Coomassie blue G-250 stain were used. The images show the immunogenic proteins identified using MALDI-TOF/TOF mass spectrometry. (A) STEC O26:H11; (B) STEC O103; (C) STEC O113:H21; (D) STEC O157:H7; (E) E. coli HS. (F) Differences between OMP profiles of the STEC strains and E. coli HS. A unique OMP profile was identified for the STEC strains (blue) and was superimposed on the OMP profile of the E. coli HS strain (orange). Analysis was performed using BioNumerics 2D software (v6.6). The scale bars on the left indicate molecular masses.

FIG 3

Immunogenic proteins identified by 2D-PAGE and Western blot analysis. The O113:H11 OMP profiles on 12% polyacrylamide gels (A) and Western blots (B) using pooled sera from HUS patients (1:3,500 dilution) are shown. Anti-human IgG secondary antibodies were diluted 1:5,000. The scale bars on the left indicate molecular masses. The arrow at the bottom indicates the pH range of the separation.

FIG 4

OMP profiles of transformed E. coli BL21(DE3) strains and Western blots of the recombinant proteins with HUS and control sera. (A) SDS-PAGE of OMPs on 12% polyacrylamide gels stained with Coomassie blue G-250. Lane 1, E. coli BL21(DE3)/pET15C; lane 2, E. coli BL21(DE3)/pET15C_ompT; lane 3, partially purified rOmpT; lane 4, E. coli BL21(DE3)/pET15C_cah; lane 5, partially purified rCah; lane M, molecular mass ladder, with molecular mass standards indicated on the left. The arrows and numbers correspond to the proteins described in Table 4. (B) Representative Western blot using serum obtained from patient HUS-15 (1:3,500 dilution) and anti-human IgG secondary antibodies (1:5,000 dilution). (C) Representative Western blot using serum obtained from patient HUS-15 (1:1,000 dilution) and anti-human IgA secondary antibodies (1:2,000 dilution). (D) Representative Western blot using serum from control subject C-19 (1:3,500 dilution) and anti-human IgG secondary antibodies (1:5,000 dilution). (E) Representative Western blot using serum from control subject C-19 (1:1,000 dilution) and anti-human IgA secondary antibodies (dilution 1:2,000). Lanes in panels B to E: 1, E. coli BL21(DE3)/pET15C; 2, partially purified rCah (*); 3, partially purified rOmpT (**).