Multiple Roles for Bordetella Lipopolysaccharide Molecules during Respiratory Tract Infection

Bordetella pertussis , Bordetella parapertussis , and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide ( B. pertussis ), a trisaccharide plus an O-antigen-like repeat ( B. bronchiseptica ), or an altered trisaccharide plus an O-antigen-like repeat ( B. parapertussis ). Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligo- saccharide. We have used wlb deletion ( D wlb ) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in coloni- zation of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed signiﬁcantly. Although the D wlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5 2 / 2 mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufﬁcient to explain the in vivo defects. B. pertussis and B. parapertussis D wlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, the B. bronchiseptica D wlb strain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deﬁcient mice, suggesting that wlb -dependent LPS modiﬁcations in B. bronchiseptica modulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria. Sera from B. bronchiseptica -infected rabbits (immune serum) were shown to contain antibodies recognizing all three Bordetella subspecies by ELISA and Western analysis (refer-ence 14 and data not shown). wt B. pertussis and all three subspecies of D wlb mutants, which were killed by naive serum, were killed by immune serum, as expected (data not shown). Immune serum also killed wt B. bronchiseptica ( . 98% killing) and B. parapertussis ( . 95% killing), which were not killed by naive serum, suggesting that antibody-mediated complement activation is responsible for the killing. Similar results were obtained in multiple experiments with naive sera from mice, rats, and rabbits or immune sera from mice, rats, rabbits, and humans. Inhibition of complement with EDTA or by heat treatment prevented killing in these assays (data not shown).