Serological response of Shiga toxin-producing Escherichia coli type III secreted proteins in sera from vaccinated rabbits, naturally infected cattle, and humans

Abstract

Escherichia coli O157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producing Escherichia coli (STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.

Fig. 1.

Western blots using anti-T3SP E. coli O157:H7 and non-O157 sera against recombinant STEC O57 LEE proteins. In total, 40 LEE genes were selected for overexpression and purification. All genes were cloned and sequenced using the Qiagen pQE-30 6×His-tagged vector cloning system (except EscS and Rorf1, which were cloned into pGEX-5X-1 for GST fusion purification). Thirty-seven proteins were purified and tested against sera. (A) Proteins visualized by SDS-PAGE using Coomassie brilliant blue. (B) Western blots using rabbit anti-T3SP E. coli O157. (C) Western blots using rabbit anti-T3SP E. coli O26. (D) Western blots using rabbit anti-T3SP E. coli O103. (E) Western blots using rabbit anti-T3SP E. coli O111. (F) Western blots using rabbit preimmune sera. (G) Western blots using bovine anti-T3SP E. coli O157. (H) Western blots using anti-6×His monoclonal antibody. #, GST-fused proteins; +, membrane proteins not purified; *, Tir protein supplied by Bioniche Life Sciences. The following shading was used in the arrows: small dots, orf of unknown function; large dark circles, chaperone; diagonal lines, intimin; vertical lines, secreted protein; mesh, regulator; white dots on a black background, translocator protein; horizontal lines, structural protein.

Fig. 2.

Antibody response of sera from STEC O157:H7 experimentally infected cattle and human sera from HUS patients against STEC O157 secreted proteins. Sixty-six purified proteins were tested, and only reactive proteins (ELISA OD higher than 0.050) were graphed. Negative proteins not shown on the graph consist of Ler, Orf2, CesA/B, Orf4, Orf5, EscT, Rorf13, GrlR, CesD, EscC, SepD, EscJ, Orf8, SepZ, Orf12, EscN, Orf16, EspH, CesF, Map, CesT, EscD, SepL, CesD2, EscF, Orf29, EspF, NleB, NleB2-1, NleC, NleE, NleG, NleH1-2, NleI, NleG2-2, NleG3, NleG5-1, NleG6-1, NleG8-2, NleG9, EspK, EspL2, EspR1, TccP, EspV, EspW, EspX2, EspX7, EspY1, EspY2, and EspY3. Single-well dilutions of sera were used for each protein. Preimmune cattle sera were used to calculate background values against each protein. The graphed ELISA OD represents the means plus standard deviations from samples (six samples of human HUS patients and two samples from experimentally infected cattle) which were calculated by subtracting the preimmune value from the infected cattle value. Duplicate values were averaged, and three standard deviations were calculated before subtraction. Solid grey bars, sera from experimentally infected cattle; dotted bars, sera from human from HUS patients.