Immunization with the yersiniabactin receptor, FyuA, protects against pyelonephritis in a murine model of urinary tract infection - PubMed (original) (raw)

Immunization with the yersiniabactin receptor, FyuA, protects against pyelonephritis in a murine model of urinary tract infection

Ariel R Brumbaugh et al. Infect Immun. 2013 Sep.

Abstract

Urinary tract infections (UTI) are common and represent a substantial economic and public health burden. Roughly 80% of these infections are caused by a heterogeneous group of uropathogenic Escherichia coli (UPEC) strains. Antibiotics are standard therapy for UTI, but a rise in antibiotic resistance has complicated treatment, making the development of a UTI vaccine more urgent. Iron receptors are a promising new class of vaccine targets for UTI, as UPEC require iron to colonize the iron-limited host urinary tract and genes encoding iron acquisition systems are highly expressed during infection. Previously, three of six UPEC siderophore and heme receptors were identified as vaccine candidates by intranasal immunization in a murine model of ascending UTI. To complete the assessment of iron receptors as vaccine candidates, an additional six UPEC iron receptors were evaluated. Of the six vaccine candidates tested in this study (FyuA, FitA, IroN, the gene product of the CFT073 locus c0294, and two truncated derivatives of ChuA), only FyuA provided significant protection (P = 0.0018) against UPEC colonization. Intranasal immunization induced a robust and long-lived humoral immune response. In addition, the levels of FyuA-specific serum IgG correlated with bacterial loads in the kidneys [Spearman's rank correlation coefficient ρ(14) = -0.72, P = 0.0018], providing a surrogate of protection. FyuA is the fourth UPEC iron receptor to be identified from our screens, in addition to IutA, Hma, and IreA, which were previously demonstrated to elicit protection against UPEC challenge. Together, these iron receptor antigens will facilitate the development of a broadly protective, multivalent UTI vaccine to effectively target diverse strains of UPEC.

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Figures

Fig 1

Fig 1

Expression and purification of UPEC outer membrane iron receptors. Genes encoding outer membrane iron receptors were cloned from UPEC strains 536 and CFT073. Iron receptors were recombinantly expressed with a His tag and purified using immobilized nickel affinity chromatography. Purified protein fractions were separated by SDS-PAGE and stained with Invitrogen SimplyBlue safe stain. Predicted sizes of tagged proteins are as follows: FyuA, 77 kDa; IroN, 83 kDa; c0294, 82 kDa; FitA, 82 kDa, ChuA middle fragment [ChuA(M)], 30 kDa; ChuA C-terminal fragment [ChuA(C)], 22 kDa. Circle and diamond symbols indicate the locations of the 75-kDa and 37-kDa standard bands, respectively, for each individual gel.

Fig 2

Fig 2

Immunization with the yersiniabactin receptor FyuA protects against experimental pyelonephritis. Female CBA/J mice were intranasally vaccinated as described in the text with a primary dose of 100 μg purified protein cross-linked to 10 μg CT, followed by two boosts of 25 μg antigen cross-linked to 2.5 μg CT. One week following the final boost, animals were transurethrally inoculated with 1 × 108 CFU of E. coli 536 (A) or CFT073 (B to F), and colonization was measured 48 h.p.i. The numbers of animals per group are as follows. (A) CT, n = 30, and FyuA, n = 29, in three independent immunization experiments (for kidneys, P = 0.0430, 0.0045, and 0.1287). (B) CT, n = 10, and IroN, n = 9, in a single immunization experiment. (C) CT, n = 20, and c0294, n = 20, in two immunization experiments. (D) CT, n = 20, and FitA, n = 20, in two immunization experiments. (E) CT, n = 10, and ChuA(M), n = 10, in a single immunization experiment. (F) CT, n = 20, and ChuA(C), n = 20, in two immunization experiments. Symbols represent CFU/g tissue or CFU/ml urine of individual mice, and gray bars indicate median values. Dotted lines show the limit of detection (100 CFU/g) for this assay. Significance was determined using a two-tailed Mann-Whitney test. Only statistically significant differences are noted.

Fig 3

Fig 3

Intranasal immunization with the yersiniabactin receptor FyuA and all other antigens induces significant antigen-specific serum IgG expression in mice. Serum was collected from mice immunized with antigen-CT or CT prior to immunization (PRE) and after immunization but before UPEC challenge (POST). Samples were plated in antigen-coated plates and probed for antigen-specific IgG via indirect ELISA. Absorbance reflects relative quantity of serum IgG. Each experimental group consisted of 20 individual mice from two separate immunization experiments. Error bars indicate the means ± standard deviations. Significance was determined using a one-tailed Mann-Whitney test.

Fig 4

Fig 4

Mice immunized intranasally with the yersiniabactin receptor FyuA produce FyuA-specific urinary antibodies. Urine collected from mice immunized with FyuA-CT (FyuA) or CT was plated on FyuA-coated plates and probed for FyuA-specific IgA (A) or IgG (B) antibodies via indirect ELISA. Absorbance reflects relative quantity of immunoglobulin. Each group (CT or FyuA) consisted of 10 individual mice from a single immunization experiment. Error bars indicate the means ± standard deviations. Significance was determined using a one-tailed Mann-Whitney test.

Fig 5

Fig 5

A correlation between vaccine-specific serum IgG titers and reduced bacterial counts is observed only in mice immunized with the protective vaccine. Normalized kidney CFU values from immunized and E. coli-challenged mice are plotted against their respective vaccine-specific serum IgG levels as measured by indirect ELISA, where absorbance at 450 nm reflects the relative quantity of vaccine-specific serum IgG. Dotted lines indicate the limit of detection (100 CFU/g kidney tissue) for the immunization assay. Correlative significance was determined using a two-tailed Spearman's rank correlation, and the best-fit line was determined by linear regression; the best-fit line is shown only when there is a statistically significant correlation (P < 0.05). (A) The results of linear regression for FyuA are as follows: R 2 = 0.60, F(1,14) = 20.6, P = 0.005.

Fig 6

Fig 6

Immunization with the yersiniabactin receptor FyuA generates long-lived plasma cells. Five female CBA/J mice were intranasally vaccinated as described in the text with a primary dose (P) of 100 μg purified FyuA cross-linked to 10 μg CT, followed by two booster doses (B) of 25 μg FyuA cross-linked to 2.5 μg CT. Weekly serum samples were taken from mice (prior to same-day immunizations), and FyuA-specific serum IgG levels were quantified via indirect ELISA. The shaded area indicates the immunization period, and arrows indicate when vaccine was administered. The dashed line indicates the time point when mice would ordinarily be challenged with UPEC to evaluate vaccine efficacy in a vaccine trial.

References

    1. Schappert SM, Rechtsteiner EA. 2008. Ambulatory medical care utilization estimates for 2006. Natl. Health Stat. Report Aug 6:1–29 -PubMed
    1. Muhldorfer I. 2001. Emerging bacterial pathogens. Preface. Contrib. Microbiol. 8:XI–XIV -PubMed
    1. Mehnert-Kay SA. 2005. Diagnosis and management of uncomplicated urinary tract infections. Am. Fam. Physician 72:451–456 -PubMed
    1. Foxman B. 2002. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Am. J. Med. 113(Suppl 1A):5S–13S -PubMed
    1. Foxman B, Brown P. 2003. Epidemiology of urinary tract infections: transmission and risk factors, incidence, and costs. Infect. Dis. Clin. North Am. 17:227–241 -PubMed

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