The Siderophore receptor IroN of extraintestinal pathogenic Escherichia coli is a potential vaccine candidate

Abstract

It would be medically and economically desirable to prevent the millions of annual extraintestinal infections and the thousands of associated deaths due to Escherichia coli. Outer membrane proteins are potential vaccine candidates for the prevention of these infections. This study tested the hypotheses that the siderophore receptor IroN is antigenic and that an IroN-specific antibody response confers protection in vivo. Subcutaneous immunization with denatured IroN resulted in a significant IroN immunoglobulin G (IgG)-specific response in serum (P < 0.0001) but not a systemic or mucosal IroN-specific IgA response. In a mouse model of ascending urinary tract infection, subcutaneous immunization with denatured IroN conferred significant protection against renal (P = 0.0135 and 0.0095 in two independent experiments), but not bladder, infection. These data, together with the previously demonstrated role of IroN in virulence, its expression in human biologic fluids, and its prevalence among extraintestinal pathogenic E. coli strains, support further studies on the role of IroN as a vaccine candidate.

FIG. 1.

Overexpression and purification of IroN. (A) Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of IroN expression over 3 h postinduction in strains AD494(DE3)pLysS/pET28a (control; odd-numbered lanes) and AD494(DE3)pLysS/pET28a::iroN (overexpresses IroN; even-numbered lanes). These strains were grown overnight in Luria-Bertani medium plus kanamycin. IPTG was added to a final concentration of 1 mM to induce the expression of IroN. Lanes (time after addition of IPTG): 1 and 2, 30 min; 3 and 4, 60 min; 5 and 6, 90 min; 7 and 8, 120 min; 9 and 10, 150 min; 11 and 12, 180 min. (B) Affinity purification of IroN. IroN expression was induced with IPTG in AD494(DE3)pLysS/pET28a::iroN, cells were lysed, and the lysate was applied to a TALON cobalt-based immobilized metal affinity chromatography column. The affinity-bound IroN was eluted under denaturing conditions (to maintain solubility) with 8 M urea elution buffer at a pH between 5.1 and 5.3. Lanes: 1, protein size markers; 2, nonpurified sample of the induced culture; 3 to 12, eluted IroN with breakdown products. (C) Western blot of IroN SDS-PAGE in panel B. A T7-Tag antibody (Novagen) to recombinant IroN was used for detection.

FIG. 2.

Protective effect of subcutaneous immunization with denatured IroN in a model of ascending, unobstructed UTI. BALB/c mice were injected with either IroN (immunized mice) or buffer (nonimmunized controls). Infection was initiated by intravesicular challenge of BALB/c mice with wild-type strain CP9 (2.0 × 10⁶ CFU in experiment 1 and 1.25 × 10⁷ CFU in experiment 2). Two, four, and six days postchallenge, urine, bladder, and kidneys were harvested and bacterial titers were determined. To test for immunization effectiveness, multiple regressions were run for all three endpoints (bladder, kidney, urine bacterial titers) in both experiments 1 and 2. Panels A (experiment 1) and B (experiment 2) are bladder titers, panels C (experiment 1) and D (experiment 2) are urine titers, and panels E (experiment 1) and F (experiment 2) are renal titers. The renal titers of CP9, but not the bladder or urine titers, were significantly (P = 0.0135 and 0.0095, experiments 1 and 2, respectively) diminished across time in IroN-immunized mice compared to those of controls (E and F).