The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides

Abstract

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.

FIG. 1

Susceptibility of Bordetella strains to the action of protamine as determined in a liquid bactericidal assay. Stars above the bars indicate statistically significant differences in survival of the various strains at different protamine concentrations in comparison to that of the B. bronchiseptica wild-type strain, BB7865 (P < 0.01).

FIG. 2

Susceptibility of B. bronchiseptica and B. pertussis wild-type and mutant strains to various cationic peptides as determined in radial diffusion assays. (A) Comparison of wild-type B. bronchiseptica BB7865, B. pertussis Tohama I, and their bvg mutant derivatives, BB7866 and BP347, respectively. Stars above the bars indicate statistically significant differences in growth inhibition of the various strains in comparison to that of the B. bronchiseptica phase-variant strain BB7866 (P < 0.01). (B) Comparison of wild-type B. bronchiseptica BB7865, its phase variant BB7866, and transposon-induced protamine-sensitive mutants (BB-PS1 to BB-PS3) in radial diffusion assays. Stars above the bars indicate statistically significant differences in growth inhibition of the various strains in comparison to that of the B. bronchiseptica wild-type strain, BB7865 (P < 0.01).

FIG. 3

Structures of the wlb locus in B. pertussis (top) and in B. bronchiseptica (bottom) (1, 2). Grey arrows identify genes for which the DNA sequence is available; e.g., in the case of B. bronchiseptica, only the wlbH gene has been sequenced so far (1, 2). The integration sites of Tn5 in the wlbA and wlbL genes of B. bronchiseptica BB-PS1 and BB-PS3, respectively, are indicated by downward arrows. The black boxes in the wlbA and wlbL structures of B. bronchiseptica indicate those parts of the two genes sequenced during this study.

FIG. 4

Silver-stained polyacrylamide gels with LPS preparations of various Bordetella strains. The positions of band A and band B are indicated. O-specific side chains in the BB7865, BB7866, and BB-PS2 strains are visible as a diffuse cloud above band A. Abbreviations: BB 7865 PS1, PS2, and PS3, mutants BB-PS1, BB-PS2, and BB-PS3, respectively; Bp TI, B. pertussis Tohama I; Bp 347, B. pertussis BP347.