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Evaluation of the role of the Bvg intermediate phase in Bordetella pertussis during experimental respiratory infection - PubMed

Comparative Study

Evaluation of the role of the Bvg intermediate phase in Bordetella pertussis during experimental respiratory infection

Nuria Vergara-Irigaray et al. Infect Immun. 2005 Feb.

Abstract

The BvgAS system of Bordetella pertussis was traditionally considered to mediate a transition between two phenotypic phases (Bvg(+) and Bvg(-)) in response to environmental signals. We characterized a third state, the intermediate (Bvg(i)) phase, which can be induced by introducing a 1-bp substitution into bvgS (the bvgS-I1 mutation) or by growing B. pertussis under conditions intermediate between those leading to the Bvg(+) and Bvg(-) phases. Like B. bronchiseptica, B. pertussis displays in its Bvg(i) phase a characteristic colony morphology and hemolytic activity and expresses a Bvg(i)-phase-specific polypeptide called BipA, whose synthesis is regulated by bvgAS at the transcriptional level. Based on our results, we hypothesize that the Bvg(i) phase of B. pertussis may be involved in facilitating transmission between hosts. Thus, a B. pertussis mutant carrying the bvgS-I1 mutation (GMT1i) persisted at wild-type levels only in the upper murine respiratory tract. Interestingly, a bipA deletion derivative of GMT1i displayed a reduced ability to colonize the nasal cavity of mice compared with GMT1i. However, in experimental mixed infections GMT1i expressing the Bvg(i) phase could establish an initial colonization in the nose and trachea of mice as efficiently as GMT1, but the wild-type strain outcompeted GMT1i at a later time point at all sites of the respiratory tract, suggesting that the Bvg(i) phase does not serve as a phenotypic phase specialized in colonization. Finally, even though B. pertussis expresses in vitro the Bvg(i) phase at the human nasal temperature, anti-BipA antibodies were undetectable in a large collection of sera from pertussis patients.

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Figures

FIG. 1.
FIG. 1.

Quantification of B. pertussis bipA transcriptional activity under various growth conditions. The bipA-lacZ fusion-carrying derivatives of B. pertussis GMT1, GMT1i, and GG2 (GMT1-BAL, GMT1i-BAL, and GG2-BAL, respectively) were grown under Bvg+-phase conditions (0 mM nicotinic acid), and levels of β-galactosidase were quantitated as specified in the Materials and Methods section and expressed as β-galactosidase units. In other experiments, GMT1-BAL was grown in the presence of the indicated amount of nicotinic acid. In preliminary tests, these concentrations were found to induce the highest level of BipA expression. Results are the average of three independent experiments performed in triplicate. Error bars represent the means ± 1 standard deviation. The asterisks denote a statistically significant difference (**, P < 0.01; ***, P < 0.001) between the two specified groups.

FIG. 2.
FIG. 2.

Western blot analysis of B. pertussis Bvgi-phase-specific polypeptides expressed under different modulating conditions. (A) Whole-cell extracts of B. pertussis GMT1, GMT1i, BPC1i, and GG2 grown with the indicated concentration of nicotinic acid were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with antiserum S3. (B) Whole-cell extracts of B. pertussis GMT1 or BPC1i grown at various temperatures (26, 30, or 37°C for GMT1; 37°C for BPC1i) were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with antiserum S3. Arrows and stars show Bvgi-phase-specific polypeptides and Bvg-independent polypeptides, respectively. The positions of molecular size markers are indicated on the left (in kilodaltons).

FIG. 3.
FIG. 3.

Western blot-based assessment of the presence of Bvgi-phase-specific polypeptides other than BipA in B. pertussis. Whole-cell extracts of the indicated strain of B. pertussis grown under Bvg+-phase conditions were separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with antiserum S3. In each panel, the arrangement of the lanes is as follows: left lanes, wild-type strain; middle lanes, bvgS-I1-carrying derivative; right lanes, bipA mutant constructed either by insertional activation of bipA (A) or by introduction of an in-frame deletion in bipA (B). The positions of molecular size markers are indicated on the left (in kilodaltons).

FIG. 4.
FIG. 4.

Western blot-based assessment of the presence of Bvgi-phase-specific polypeptides other than BipA and exclusive of B. pertussis. Whole-cell extracts of the indicated B. pertussis strains were grown under Bvg+-phase conditions, separated by SDS-PAGE, transferred to polyvinylidene difluoride, and probed with serum from a rat immunized with killed whole cells of B. pertussis GMT1i (antiserum S219). The positions of molecular size markers are indicated on the left (in kilodaltons).

FIG. 5.
FIG. 5.

Colonization of the mouse respiratory tract by wild-type and mutant (bvgS-I1 carrying) B. pertussis strains. Groups of mice were inoculated intranasally with 50 μl of PBS containing 106 CFU of one of the experimental strains (GMT1, GMT1i, and Tohama I). At days 0 (three mice at least) and 8 (five mice at least), the animals were sacrificed, and colonization at various sites of the respiratory tract was determined. Error bars represent 1 standard deviation. The dashed line indicates the lower limit of detection. The asterisks denote a statistically significant difference (**, P < 0.01; ***, P < 0.001) between the two specified groups.

FIG. 6.
FIG. 6.

Colonization of the mouse respiratory tract by two coinoculated B. pertussis strains, GMT1 and GMT1i. Groups of mice were intranasally administered 50 μl of PBS containing a mixed inoculum of GMT1 and GMT1i (106 CFU and 106 CFU, respectively). At days 0 (four mice at least) and 8 (five mice at least), the animals were sacrificed, and colonization at various sites in the respiratory tract was determined and represented as a scatter diagram. Solid circles, GMT1;shaded triangles, GMT1i. The dashed line indicates the lower limit of detection. Asterisks denote a statistically significant difference (*, P < 0.05; **, P < 0.01) between the two specified groups. Note that the sensitivity of the statistical tests used to analyze data from the upper and lower respiratory tract varies (see Materials and Methods).

FIG. 7.
FIG. 7.

Colonization of the mouse respiratory tract by bipA deletion mutants derived either from wild-type B. pertussis GMT1 (A) or from GMT1i (B). Groups of mice were inoculated intranasally with 50 μl of PBS containing 106 CFU of one of the experimental strains (A, GMT1 and GMT1-DBA; B, GMT1i and GMT1i-DBA). At days 0 (three mice at least) and 8 (five mice at least), the animals were sacrificed, and colonization at various sites of the respiratory tract was determined. Error bars represent 1 standard deviation. The dashed line indicates the lower limit of detection. The asterisks denote a statistically significant difference (**, P < 0.01) between the two specified groups.

FIG. 8.
FIG. 8.

Assessment of the presence of anti-Bvgi-phase-specific antibodies in sera from patients recovering from whooping cough. Whole-cell extracts of GMT1, GMT1i, and GG2 were separated by SDS-PAGE in adjacent gel lanes, transferred to polyvinylidene difluoride, and probed with the corresponding serum. Results are representative of the two main profiles obtained with the 100 sera tested. Arrows and small stars show Bvg-independent and Bvg+-phase-specific polypeptides, respectively. The positions of molecular size markers are indicated on the right (in kilodaltons).

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References

    1. Akerley, B. J., and J. F. Miller. 1993. Flagellin gene transcription in Bordetella bronchiseptica is regulated by the BvgAS virulence control system. J. Bacteriol. 175:3468-3479. - PMC - PubMed
    1. Antoine, R., S. Alonso, D. Raze, L. Coutte, S. Lesjean, E. Willery, C. Locht, and F. Jacob-Dubuisson. 2000. New virulence-activated and virulence-repressed genes identified by systematic gene inactivation and generation of transcriptional fusions in Bordetella pertussis. J. Bacteriol. 182:5902-5905. - PMC - PubMed
    1. Arico, B., J. F. Miller, C. Roy, S. Stibitz, D. Monack, S. Falkow, R. Gross, and R. Rappuoli. 1989. Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins. Proc. Natl. Acad. Sci. USA 86:6671-6675. - PMC - PubMed
    1. Ausubel, F. 1995. Short protocols in molecular biology, 3rd ed. John Wiley & Sons, Inc., Boston, Mass.
    1. Bootsma, H. J., C. A. Cummings, S. van de Pas, D. A. Relman, and J. F. Miller. 2003. Abstr. 1572. In Abstracts of the 103rd Annual Meeting of the American Society for Microbiology 2003. American Society for Microbiology, Washington, D.C.

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