HTRB OF HAEMOPHILUS-INFLUENZAE - DETERMINATION OF BIOCHEMICAL-ACTIVITY AND EFFECTS ON VIRULENCE AND LIPOOLIGOSACCHARIDE TOXICITY

The htrB mutant of Haemophilus influenzae (strain B29) has been shown to lack secondary (nonhydroxylated) acyl groups in its lipid A. We hav e determined through in vitro biochemical assays that the HtrB protein acts as a specific acyltransferase in the late stages of lipid A bios ynthesis and that the preferred acyl group donor is myristoyl-acyl car rier protein. Under the conditions employed, the Escherichia coli prec ursor, Kdo(2)-lipid IVA, functions as a myristate acceptor. Introducti on of the Haemophilus htrB gene into an E. coli mutant lacking htrB co mplements the biochemical and physiological defects associated with th e E. coli htrB mutation. Tumor necrosis factor alpha (TNF alpha) assay s using murine and human macrophage cells indicated that nontypeable H . influenzae (NtHi) strain 2019 and H. influenzae type b strain A2 eli cit levels of expression of TNF alpha that are 30-40 times greater tha n 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 TNF alpha expression that are 6-8-fold higher than those of B29. In situ hybridization studies of a primary human bronchi al epithelial cell line demonstrated a greater increase of TNF alpha m essage produced in the presence of 2019 LOS than in the presence of B2 9 LOS. TNF alpha levels of the cell supernatant of cells stimulated wi th 2019 LOS were found to be 7-8-fold higher than levels in B29 stimul ated supernatants. Using the Limulus amoebocyte lysate for assessment of endotoxic activity, we found that wild type LOS was 8-fold higher i n endotoxic activity compared with the mutant LOS. In virulence assays using intraperitoneal inoculation of infant rats, the htrB isogenic s train caused bacteremia at 50% the frequency of the wild type strain. In intranasal inoculation studies, the htrB mutant strain was unable t o 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 TNF alpha expr ession is attenuated in response to htrB mutant strains.