Bordetella pertussis expresses a functional type III secretion system that subverts protective innate and adaptive immune responses - PubMed (original) (raw)

Bordetella pertussis expresses a functional type III secretion system that subverts protective innate and adaptive immune responses

Neil K Fennelly et al. Infect Immun. 2008 Mar.

Abstract

Certain bacteria use a type III secretion system (TTSS) to deliver effector proteins that interfere with cell function into host cells. While transcription of genes encoding TTSS components has been demonstrated, studies to date have failed to identify TTSS effector proteins in Bordetella pertussis. Here we present the first evidence of a functionally active TTSS in B. pertussis. Three known TTSS effectors, Bsp22, BopN, and BopD, were identified as TTSS substrates in B. pertussis 12743. We found expression of Bsp22 in a significant proportion of clinical isolates but not in common laboratory-adapted strains of B. pertussis. We generated a TTSS mutant of B. pertussis 12743 and showed that it induced significantly lower respiratory tract colonization in mice than the wild-type bacteria. Respiratory infection of mice with the mutant bacteria induced significantly greater innate proinflammatory cytokine production in the lungs soon after challenge, and this correlated with significantly higher antigen-specific interleukin-17, gamma interferon, and immunoglobulin G responses later in infection. Our findings suggest that the TTSS subverts innate and adaptive immune responses during infection of the lungs and may be a functionally important virulence factor for B. pertussis infection of humans.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Clinical isolates but not common laboratory-adapted stains of B. pertussis express Bsp22. Protein samples from supernatants of stationary-phase cultures of B. pertussis (Bp) 9340, 12743, 12742, Tohama I (Toh-I), and Wellcome 28 (W28) (A) or B. bronchiseptica (Bb) RB50, B. pertussis W28 and Toh-I, and eight clinical isolates of B. pertussis recovered from respiratory tracts of patients with whooping cough (B), prepared by precipitation with 10% trichloroacetic acid or purified His-Bsp22 or PT (2 μg), were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and probed with Abs specific for Bsp22 or PT (S1 to S5 subunits) by Western blotting.

FIG. 2.

FIG. 2.

Mutation of bscN in Bordetella spp. abolishes Bsp22 production but does not affect secretion of the virulence factor FHA. Proteins from culture supernatants of WT and Δ_bscN B. pertussis_ (Bp) 12743 and B. bronchiseptica (Bb) RB50 or B. pertussis Tohama I (Toh-I) were examined for the presence of Bsp22 and FHA by Western blotting.

FIG. 3.

FIG. 3.

Correlation between in vitro growth curves for B. pertussis (Bp) 12743 and Δ_bscN B. pertussis_ 12743. B. pertussis 12743 and Δ_bscN B. pertussis_ 12743 were cultured for 2 days in SS medium, and samples were removed at the indicated time points. (A) OD600 was determined. (B) The number of viable bacteria was determined by performing CFU counts after plating on BG agar. (C) CFU counts were plotted against OD600 for each strain separately, and linear regression analysis was performed. The correlation (r value) and level of significance (P value) are shown.

FIG. 4.

FIG. 4.

The TTSS of B. pertussis 12743 promotes bacterial adherence to macrophages but does not mediate cytotoxicity. (A) Epithelial cells, J774 macrophages, or bone marrow-derived DC were cultured with WT or Δ_bscN B. pertussis_ (Bp) 12743 or B. bronchiseptica (Bb) RB50 at MOIs of 20, 100, and 500 for 4 h. Cytotoxicity was measured by a lactate dehydrogenase release assay. (B and C) J774 macrophages were cultured with WT or Δ_bscN B. bronchiseptica_ RB50 (B) or B. pertussis 12743 (C) at an MOI of 100:1. Adherence was assessed by assessing CFU counts following 2 h of incubation followed by 1 h of treatment with kanamycin or medium only. *, P < 0.05; **, P < 0.01 (Δ_bscN_ versus WT). Results are representative of three experiments.

FIG. 5.

FIG. 5.

Enhanced inflammatory cytokine and chemokine induction in the lungs of mice infected with Δ_bscN B. pertussis_ (BP) 12743. BALB/c mice were challenged with an aerosol of WT or Δ_bscN B. pertussis_ 12743. Cytokine and chemokine concentrations were determined by ELISA on lung homogenates from mice 3 h and 3, 7, and 14 days (d) after aerosol challenge and in uninfected control mice. Results are means ± standard deviations for four mice per group at each time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Δ_bscN_ versus WT).

FIG. 6.

FIG. 6.

Enhanced antigen-specific IL-17 and IFN-γ production in mice infected with Δ_bscN B. pertussis_ (BP) 12743. BALB/c mice were challenged with an aerosol of WT or Δ_bscN B. pertussis_ 12743. Spleen cells recovered 14 (A and C) and 21 (B and D) days postchallenge were stimulated with sonicated B. pertussis 12743 (Bp), FHA, PT, or medium (Med) only as a control. Supernatants were removed after 3 days, and IL-17 (A and B) and IFN-γ (C and D) concentrations were determined by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Δ_bscN_ versus WT).

FIG. 7.

FIG. 7.

Enhanced B. pertussis_-specific IgG and IgG2a responses in mice infected with Δ_bscN B. pertussis (BP) 12743. BALB/c mice were challenged with an aerosol of WT or Δ_bscN B. pertussis_ 12743. Serum was recovered from infected mice 28 days postchallenge or from uninfected control mice and assayed for anti-B. pertussis IgG, IgG1, and IgG2a by ELISA. Results are means ± standard deviations for four mice per group. *, P < 0.05; **, P < 0.01 (Δ_bscN_ versus WT).

FIG. 8.

FIG. 8.

Δ_bscN B. pertussis_ 12743 has reduced ability to colonize lungs of mice. BALB/c mice were challenged with an aerosol of WT or Δ_bscN B. pertussis_ (Bp) 12743, where the challenge inoculum resulted in intermediate (A), low (B), or high (C) initial colonization with the WT B. pertussis. Groups of four mice were sacrificed 3 h and 3, 7, 14, 21, and 28 days later, and CFU counts were performed on lung homogenates. The dashed line represents the limit of detection. **, P < 0.01; ***, P < 0.001 (Δ_bscN_ versus WT). Results are representative of three experiments for panel A and one experiment each for panels B and C.

Similar articles

Cited by

References

    1. Akerley, B. J., P. A. Cotter, and J. F. Miller. 1995. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell 80611-620. - PubMed
    1. Brinig, M. M., C. A. Cummings, G. N. Sanden, P. Stefanelli, A. Lawrence, and D. A. Relman. 2006. Significant gene order and expression differences in Bordetella pertussis despite limited gene content variation. J. Bacteriol. 1882375-2382. - PMC - PubMed
    1. Byrne, P., P. McGuirk, S. Todryk, and K. H. Mills. 2004. Depletion of NK cells results in disseminating lethal infection with Bordetella pertussis associated with a reduction of antigen-specific Th1 and enhancement of Th2, but not Tr1 cells. Eur. J. Immunol. 342579-2588. - PubMed
    1. Cornelis, G. R. 2002. The Yersinia Ysc-Yop ‘type III’ weaponry. Nat. Rev. Mol. Cell Biol. 3742-752. - PubMed
    1. Cornelis, G. R., A. Boland, A. P. Boyd, C. Geuijen, M. Iriarte, C. Neyt, M. P. Sory, and I. Stainier. 1998. The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 621315-1352. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources