Laboratory adaptation of Bordetella pertussis is associated with the loss of type three secretion system functionality - PubMed (original) (raw)

Laboratory adaptation of Bordetella pertussis is associated with the loss of type three secretion system functionality

M E Gaillard et al. Infect Immun. 2011 Sep.

Abstract

Although Bordetella pertussis contains and transcribes loci encoding type III secretion system (TTSS) homologues, expression of TTSS-associated proteins has been reported only for non-laboratory-adapted Irish clinical isolates. Here we confirm such a result for clinical isolates obtained from patients treated in Argentinean hospitals. Moreover, we demonstrate that the expression of TTSS-associated proteins is independent both of the year in which the isolate was obtained and of the types of polymorphic alleles for other virulence factors but is dependent on environmental growth conditions. Interestingly, we observed that TTSS-associated protein expression is lost after successive in vitro passages but becomes operative again when bacteria come into contact with the host. This in vivo activation of TTSS expression was observed not only for clinical isolates previously adapted to the laboratory after successive in vitro passages but also for vaccine strains that did not express the system in vitro. The reversibility of TTSS expression, demonstrated by its switching off-on when the bacterium comes into contact with the host, appears to be an adaptive response of this pathogen.

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Figures

Fig. 1.

Fig. 1.

Quantitative PCR analysis of bsc N, bsc C, and _bsp_22 transcript levels in the vaccine strain Tohama I and the clinical isolate _Bp_106. cDNA obtained from total RNA of Tohama I and _Bp_106 was used as a template for PCR. The rec A transcript was used as a reference. Comparisons of Tohama I with _Bp_106 were performed for each gene evaluated; P values were <0.05 (a and b) and <0.01 (c).

Fig. 2.

Fig. 2.

Reactivities of an anti-Bsp22 serum against supernatant proteins from B. bronchiseptica and B. pertussis strains. Proteins from B. bronchiseptica 9.73 grown under Bvg+ conditions (_Bb_9.73 BvgAS+) and from its avirulent constitutive derivative mutant (Bb_9.73 Δ_bvgA) (A) and from a Bsp22-defective mutant of B. pertussis Bp106 (Bp_106 Δ_bsp22) and its parental strain _Bp_106 (B) were resolved by 15% (wt/vol) SDS-PAGE (left) and were probed with the indicated sera (right). Purified His6-Bsp22 and Ptx were used as positive controls.

Fig. 3.

Fig. 3.

Immunoblot analysis of supernatant proteins from Argentinean B. pertussis clinical isolates and laboratory-adapted strains. Proteins from reference and vaccine strains (A) and from clinical isolates (B) were resolved by 15% (wt/vol) SDS-PAGE, transferred to a PVDF membrane, and probed with a polyclonal serum specific for Bsp22. Samples in panel A were also probed with a polyclonal antiserum specific for subunit S1 of pertussis toxin (PtxS1). Positive controls were purified recombinant His6-Bsp22 and PtxS1 proteins, as well as samples obtained from B. bronchiseptica strain 9.73 (_Bb_9.73) and the clinical isolate _Bp_106.

Fig. 4.

Fig. 4.

Immunoblot analysis of laboratory-adapted B. pertussis isolates/strains for TTSS expression. (A) Supernatant proteins of B. pertussis strains _Bp_6901, _Bp_106, and Tohama I with their derivatives obtained after 35 successive in vitro passages (Adapted). (B) TTSS expression through 35 in vitro passages of _Bp_106. The sera used in immunoblotting are indicated on the left.

Fig. 5.

Fig. 5.

Immunoblot analysis of TTSS expression through 5 in vitro passages of _Bp_106. Supernatant proteins of B. pertussis Bp106 with their derivatives obtained after 5 successive in vitro passages were resolved by 15% (wt/vol) SDS-PAGE, transferred to a PVDF membrane, and probed with a polyclonal serum specific for Bsp22. Samples were also probed with a polyclonal antiserum specific for subunit S1 of pertussis toxin (PtxS1). Purified recombinant His6-Bsp22 and PtxS1 proteins were included in this assay as positive controls.

Fig. 6.

Fig. 6.

Analysis of TTSS expression in vivo. Protein samples obtained from B. pertussis supernatant cultures (in vitro) or from whole-cell bacteria recovered from infected mice (in vivo) were analyzed for Bsp22 expression by immunoblotting. Samples obtained from Tohama I, _Bp_106 (_Bp_106 Not Adapted), and isolate _Bp_106 subjected to 35 in vitro passages (_Bp_106 Adapted) were probed with a polyclonal antiserum specific for Bsp22 or subunit S1 of pertussis toxin (PtxS1). Purified His6-Bsp22 and Ptx were used as positive controls.

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