htrB of Haemophilus influenzae: determination of biochemical activity and effects on virulence and lipooligosaccharide toxicity

Abstract

The htrB mutant of Haemophilus influenzae (strain B29) has been shown to lack secondary (non-hydroxylated) acyl groups in its lipid A. We have determined through in vitro biochemical assays that the HtrB protein acts as a specific acyltransferase in the late stages of lipid A biosynthesis and that the preferred acyl group donor is myristoyl-acyl carrier protein. Under the conditions employed, the Escherichia coli precursor, Kdo₂-lipid IV_A, functions as a myristate acceptor. Introduction of the Haemophilus htrB gene into an E. coli mutant lacking htrB complements the biochemical and physiological defects associated with the E. coli htrB mutation. Tumor necrosis factor α (TNFα) assays using murine and human macrophage cells indicated that nontypeable H. influenzae (NtHi) strain 2019 and H. influenzae type b strain A2 elicit levels of expression of TNFα that are 30-40 times greater than levels induced by the isogenic htrB mutants (B29 and A2B29). Studies using cell-free LOS indicated that the LOS from wild type strain 2019 elicits levels of TNFα expression that are 6-8-fold higher than those of B29. In situ hybridization studies of a primary human bronchial epithelial cell line demonstrated a greater increase of TNFα message produced in the presence of 2019 LOS than in the presence of B29 LOS. TNFα levels of the cell supernatant of cells stimulated with 2019 LOS were found to be 7-8-fold higher than levels in B29 stimulated supernatants. Using the Limulus amoebocyte lysate for assessment of endotoxic activity, we found that wild type LOS was 8-fold higher in endotoxic activity compared with the mutant LOS. In virulence assays using intraperitoneal inoculation of infant rats, the htrB isogenic strain caused bacteremia at 50% the frequency of the wild type strain. In intranasal inoculation studies, the htrB mutant strain was unable to cause bacteremia whereas the wild type b parent produced bacteremia in 40-60% of the animals. These findings suggest that the htrB gene of H. influenzae is important for virulence and that host TNFα expression is attenuated in response to htrB mutant strains.