Physiological consequences of inactivation of lgmB and lpxL1, two genes involved in lipid A synthesis in Bordetella bronchiseptica.

To develop a Bordetella bronchiseptica vaccine with reduced endotoxicity, we previously inactivated lpxL1, the gene encoding the enzyme that incorporates a secondary 2-hydroxy-laurate in lipid A. The mutant showed a myriad of phenotypes. Structural analysis showed the expected loss of the acyl chain but also of glucosamine (GlcN) substituents, which decorate the phosphates in lipid A. To determine which structural change causes the various phenotypes, we inactivated here lgmB, which encodes the GlcN transferase, and lpxL1 in an isogenic background and compared the phenotypes. Like the lpxL1 mutation, the lgmB mutation resulted in reduced potency to activate human TLR4 and to infect macrophages and in increased susceptibility to polymyxin B. These phenotypes are therefore related to the loss of GlcN decorations. The lpxL1 mutation had a stronger effect on hTLR4 activation and additionally resulted in reduced murine TLR4 activation, surface hydrophobicity, and biofilm formation, and in a fortified outer membrane as evidenced by increased resistance to several antimicrobials. These phenotypes, therefore, appear to be related to the loss of the acyl chain. Moreover, we determined the virulence of the mutants in the Galleria mellonella infection model and observed reduced virulence of the lpxL1 mutant but not of the lgmB mutant.