Suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by Bordetella pertussis and identification of an immunodominant lipoprotein

Abstract

Pertussis toxin (PT), a virulence factor secreted by Bordetella pertussis, contributes to respiratory tract infection and disease caused by this pathogen. By comparing a wild-type (WT) B. pertussis strain to a mutant strain with an in-frame deletion of the ptx genes encoding PT (DeltaPT), we recently found that the lack of PT confers a significant defect in respiratory tract colonization in mice after intranasal inoculation. In this study, we analyzed serum antibody responses in mice infected with the WT or DeltaPT strain and found that infection with the DeltaPT strain elicited greater responses to several B. pertussis antigens than did infection with the WT, despite the lower colonization level achieved by the DeltaPT strain. The same enhanced antibody response was observed after infection with a strain expressing an enzymatically inactive PT; but this response was not observed after infection with B. pertussis mutant strains lacking filamentous hemagglutinin or adenylate cyclase toxin, nor when purified PT was administered with the DeltaPT inoculum, indicating a specific role for PT activity in this immunosuppressive effect. In particular, there were consistent strong serum antibody responses to one or more low-molecular-weight antigens after infection with the DeltaPT strain. These antigens were Bvg independent, membrane localized, and also expressed by the closely related pathogens Bordetella parapertussis and Bordetella bronchiseptica. Two-dimensional gel electrophoresis and mass spectrometry were used to identify one of the immunodominant low-molecular-weight antigens as a protein with significant sequence homology to peptidoglycan-associated lipoprotein in several other gram-negative bacterial species. However, a serum antibody response to this protein alone did not protect mice against respiratory tract infection by B. pertussis.

FIG. 1.

Serum antibody response of individual BALB/c or C57BL6 mice to respiratory tract infection with the Tohama I WT or ΔPT strain. (A and B) Western blots of whole-cell lysates of WT run on SDS-12% PAGE gels probed with individual sera (1:50 dilution) from BALB/c or C57BL6 mice obtained 28 days after infection with either the WT or ΔPT strain. (C) Western blots of whole-cell lysates of the WT run on SDS-15% PAGE gels probed with individual sera (1:50 dilution) from BALB/c or C57BL6 mice obtained 28 days after infection with the ΔPT strain. The molecular mass (kDa) of protein markers is indicated at right.

FIG. 2.

Serum antibody response of mice to respiratory tract infection with Tohama I WT or different mutant strains. Western blots of whole-cell lysates of the WT strain run on SDS-12% PAGE gels probed with individual sera (1:50 dilution) from BALB/c mice obtained 28 days after infection with the indicated strain.

FIG. 3.

Effect of PT on the serum antibody response of mice to respiratory tract infection with B. pertussis. (A) Western blots of whole-cell lysates of the Tohama I WT strain run on SDS-12% PAGE gels probed with individual sera (1:50 dilution) from BALB/c mice obtained 21 days after infection with either the 18323 WT, 18323 ΔPT, or Tohama I strain expressing inactive mutant PT9K/129G (TohI PT*). (B) Respiratory tract colonization levels of BALB/c mice 7 days after infection with 5 × 10⁵ CFU of the Tohama I WT or ΔPT strain or the ΔPT strain mixed with 10 ng of PT (ΔPT+PT). (C) Western blots of whole-cell lysates of Tohama I WT run on SDS-12% PAGE gels probed with individual sera (1:50 dilution) from BALB/c mice obtained 21 days after infection with the Tohama I WT, ΔPT, or ΔPT+PT strain.

FIG. 4.

(A) Western blots of whole-cell lysates of the indicated strains (WT and mutants of Tohama I, B. bronchiseptica [Bb], and B. parapertussis [Bpp]) run on SDS-12% PAGE gels probed with serum (1:30 dilution) from a BALB/c mouse obtained 28 days after infection with the Tohama I ΔPT strain. (B) Western blots of soluble (S) or membrane (M) fractions of Tohama I WT probed with serum (1:30 dilution) from a BALB/c mouse obtained 28 days after infection with the Tohama I ΔPT strain (serum) or the indicated monoclonal antibodies.

FIG. 5.

(A) Silver-stained two-dimensional SDS-15% PAGE gel of the ΔBVG strain whole-cell lysate. Arrow indicates protein spot that was excised and identified. (B) Western blot of the lower half of a gel probed with serum (1:30 dilution) from a BALB/c mouse obtained 28 days after infection with the Tohama I ΔPT strain. The arrow indicates a protein spot that reacted with the serum. (C) Western blot of whole-cell lysates of E. coli DH10B pGEX-Pal after the IPTG induction of fusion protein expression for the indicated times run on SDS-12% PAGE gels and probed with serum (1:100 dilution) from a BALB/c mouse obtained 28 days after infection with the Tohama I ΔPT strain. (D) Western blot of whole-cell lysates of Tohama I WT or two different isolates of the Pal+15 strain (M) run on SDS-15% PAGE gels probed with serum (1:50 dilution) from a BALB/c mouse obtained 28 days after infection with the Tohama I ΔPT strain.

FIG. 6.

(A) Western blots of whole-cell lysates of Tohama I WT run on an SDS-12% PAGE gel probed with sera (1:50 dilution) from BALB/c mice immunized with the indicated protein by the indicated route or obtained 28 days after infection with the ΔPT strain. (B) Respiratory tract colonization levels of BALB/c mice previously immunized with the indicated protein or infected with the Tohama I ΔPT strain (or PBS as a control) 4 days after infection with 2 × 10⁶ CFU of Tohama I WT. i.n., intranasal; s.c., subcutaneous.

FIG. 7.

Protection of mice against B. pertussis respiratory tract infection by previous infection with either the Tohama I WT or ΔPT strain. BALB/c mice were infected with 10⁴ CFU of the WT or 2 × 10⁵ CFU of the ΔPT strain (or administered PBS as a control), and day 7 colonization levels were assessed (inset). On day 34 postinoculation, all mice were challenged with 7 × 10⁵ CFU of Tohama I WT, and respiratory tract colonization levels were assessed on the indicated days postchallenge.