Directed evaluation of enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.

Methodology: Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice.

Principal findings: Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC.

Conclusions: Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.

Figure 1. Chromosomal context of conserved autotransporter genes in ETEC and nonpathogenic E. coli genomes.

a. location of antigen 43 (agn43) genes in the chromosomes of ETEC strains (H10407, E24377A, and B7A) and nonpathogenic E. coli strains (MG1655, and HS). Genes are shaded by similarity. Individual autotransporter genes are depicted in blue. Putative full-length autotransporter genes include their assigned ncbi protein reference numbers. Mobility elements are depicted in green. Open arrows represent hypothetical genes. b. location of pAT genes (in blue). Figures are based on RAST annotations (http://rast.nmpdr.org/).

Figure 2. Surface expression of autotransporter proteins by ETEC.

a. specificity of antibodies directed against ETEC autotransporter passenger domains. Data shown are kinetic ELISA data obtained at a 1∶1024 dilution of all primary antisera. b. flow cytometry study comparing the presence of pAT and Ag43 AT passenger domains on the surface of ETEC strain H10407 compared to the HS E. coli commensal strain. Pre-immune (NI for non-immune) sera, as well as unstained bacteria (no primary antibody) are shown as controls c. Examination of pAT surface expression by ETEC strains B7A and E24377A relative to the H10407 (+ control) and the commensal HS (negative control); unstained control used no primary antibody. d. Examination of Ag43 surface expression by ETEC strains B7A and E24377A relative to the H10407 and HS controls. Flow cytometry histograms depict intensity of fluorescence signal on the abscissa (x-axis) while the relative frequency of bacteria counted is depicted on the ordinate (y-axis).

Figure 3. Immunogenicity of autotransporter protein passenger domains.

a. passenger domains of autotransporters pAT and Ag43 are recognized by during the course of experimental murine infection with ETEC H10407. Shown are metal affinity chromatography-purified antigens (MAC) used in analysis, followed by corresponding immunoblots using pooled sera obtained from mice before and after intestinal challenge with ETEC H10407. b. human ETEC convalescent sera (from patients infected with ETEC obtained at ICDDR,B), but not age-matched sera from uninfected controls (from LeBonheur Children's Hospital, Memphis) recognize the ETEC H10407 autotransporter passenger domain of pAT (RAST designation 459). c. human ETEC convalescent sera, but not age-matched control sera recognize passenger domain of the ETEC H10407 autotransporter Ag43 (RAST designation 2318).

Figure 4. Vaccination with autotransporter passenger domains protects against intestinal colonization with ETEC in a murine model.

a. kinetic ELISA data showing serologic responses of animals vaccinated with passenger domain of autotransporter designated pAT_p (closed circles) relative to adjuvant-only controls (ivx908, open circles). b. kinetic ELISA data for serologic responses of animals immunized with Ag43 passenger (Ag43_p, closed triangles) relative to ivx908-only controls. c. kinetic ELISA of fecal antibodies (total IgG, IgM, IgA) obtained from pATp and Ag43p immunized mice (closed symbols) relative to adjuvant-only controls (open symbols); antigen (in shaded region) on the x-axis refers to the antigen used to coat ELISA wells. d. kinetic ELISA of fecal IgA antibody following vaccination with either the pAT or Ag43 passenger domains (closed symbols) relative to adjuvant-only controls (ivx908, open symbols) e. KmR-bacteria recovered from intestinal lysates following challenge with (1.2×10⁴ cfu/mouse) of jf876 (ΔlacZYA::KmR mutant of ETEC strain H10407). Dashed horizontal lines reflect geometric means. All statistical comparisons were performed using two-tailed Mann Whitney analysis.