The Bordetella pertussis type III secretion system tip complex protein Bsp22 is not a protective antigen and fails to elicit serum antibody responses during infection of humans and mice - PubMed (original) (raw)

The Bordetella pertussis type III secretion system tip complex protein Bsp22 is not a protective antigen and fails to elicit serum antibody responses during infection of humans and mice

Rodrigo Villarino Romero et al. Infect Immun. 2013 Aug.

Erratum in

Abstract

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice.

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Figures

Fig 1

Fig 1

No Bsp22-specific antibodies are detected in sera of B. pertussis-infected patients. (A) Bsp22 was expressed with an N-terminal double-6×His purification tag in E. coli BL21(λDE3) cells and purified close to homogeneity by a combination of affinity chromatography on Ni-NTA agarose with cation-exchange chromatography on SP-Sepharose and reversed-phase chromatography on a C4 resin. The purified Bsp22 sample was separated by SDS-PAGE (12.5%) and stained with Coomassie blue. St, molecular mass standards. (B and C) Sera obtained from patients with confirmed whooping cough disease and from healthy volunteers (controls) were diluted 1:1,000 and examined for anti-PT IgG using a validated ELISA kit (B) or by conventional ELISA with purified Bsp22 or FHA used as coating antigens (C). PT IgG values are given as international units (IU) per ml (B), and Bsp22 and FHA IgG values are given as the absorbance at 492 nm (C). Box-whisker plots represent boxes for medians with 25th and 75th percentiles and whiskers for the 10th and 90th percentiles. Closed circles represent outliers. The statistical difference between the pertussis-infected group and the control group was calculated by using the Student t test and was shown to be significant for anti-PT antibody levels (P < 0.001) and anti-FHA antibody levels (P < 0.001) but not for anti-Bsp22 antibody levels (P = 0.118) compared to control sera.

Fig 2

Fig 2

B. pertussis infection of mice does not elicit a serum antibody response against Bsp22. (A) BALB/c mice were intranasally infected with a sublethal dose of 2.5 × 105 CFU of B. pertussis 18323, and the presence of serum antibodies against Bsp22 and FHA was monitored by ELISA at 6 weeks after infection. Average values of four sera ± the standard deviations were calculated from two independent experiments, where only the anti-FHA antibodies were significantly increased (P < 0.01). (Inset) B. pertussis 18323 was recovered from lungs 27 days after infection and subcultured in vitro. Concentrated culture supernatants were tested for production of Bsp22 by Western blotting with hyperimmune mouse anti-Bsp22 serum (lane S). Cultures of the laboratory-adapted strain B. pertussis 18323, having the Bsp22 (T3SS) production switched off (lane C), were used as negative control. Purified recombinant Bsp22 protein was used as a positive control (lane rBsp22) and migrated at a higher molecular mass than did natural Bsp22 because of the N-terminal double-6×His tag extension (∼ 5 kDa). (B) Production and secretion of Bsp22 in B. pertussis 18323 cells recovered from infected mice. Whole-cell lysates (P) or concentrated supernatants (S) from cultures of B. pertussis recovered 2 h (D0) or 8 days (D8) after challenge were separated on a 12.5% SDS-polyacrylamide gel and stained with Coomassie brilliant blue (upper panel) or transferred to nitrocellulose and probed with anti-Bsp22 antisera (lower panel, only the relevant part of the membrane is shown). The supernatant fraction of a B. bronchiseptica RB50 (Bb) culture and purified recombinant Bsp22 (rBsp22) were loaded as positive controls. Position of Bsp22 protein is indicated by an asterisk. Prestained molecular mass standards (St) are shown in the left lane of the stained gel and Western blot.

Fig 3

Fig 3

High preexisting levels of Bsp22-specific serum antibodies do not protect mice against B. pertussis colonization and virulence. (A) Negative staining transmission electron microscopy of filaments that formed upon refolding of recombinant Bsp22 protein from denaturing urea solutions after Ni-NTA agarose and SP Sepharose chromatography through dialysis against buffer. Samples were examined at a magnification of ×64,000. Scale bar, 100 nm. (B to D) BALB/c or (E) CD-1 mice were immunized twice at a 2-week interval with 30 μg of Bsp22 (△) or CyaA-AC− (■) adsorbed on aluminum hydroxide or with buffer containing aluminum hydroxide alone as a control (●). (B) The postvaccination sera were taken from two BALB/c mice from each group immediately before challenge and individually examined for anti-Bsp22 and anti-CyaA-AC− antibody levels using ELISA, yielding identical results. The dashed line indicates the cutoff value calculated as the mean plus two standard deviations of the test results of 1:100 diluted negative sera from mice that received adjuvant alone (cutoff value for Bsp22 = 0.1; cutoff value for CyaA-AC− = 0.1). (C) At 2 weeks after the second immunization, BALB/c mice in all three groups were intranasally infected with 1.5 × 105 CFU of B. pertussis 18323 and sacrificed on days 0, 5, 8, 13, and 16, respectively. The lungs were removed and homogenized, serial dilutions of lung homogenates were plated on BG agar, and the CFU were counted 3 days later. The plots show average values ± the geometric standard deviations for three mice per time point. The results are representative of two experiments with the exception of the group immunized with CyaA-AC−, an experiment that was performed once. (D) The challenge strain reisolated from lungs was analyzed for the secretion of Bsp22 by Western blotting. For lane C and the rBsp22 lane, see the explanations in legend for Fig. 2. (E) At 2 weeks after the last immunization, CD-1 mice in all three groups were intranasally infected with 2 × 108 CFU/mouse of B. pertussis 18323, and the survival of mice was monitored for 7 days.

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