Adherence and invasion are thought to be key events in the pathogenesis of non‐typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS‐coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB::ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB::ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co‐localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti‐ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.

The lipooligosaccharide (LOS) of Haemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgB affected only the sialylated glycoforms containing N-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Chromosomal complementation of the siaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lacking N-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant in siaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in the siaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction in N-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases.

The htrB mutant of Haemophilus influenzae (strain B29) has been shown to lack secondary (nonhydroxylated) 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, Kdo 2 -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.

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A ␤-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV A , which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a welldefined murine typhoid model of animal pathogenesis. To this end, an r ؊ m ؉ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter-and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.Lipooligosaccharide (LOS), a major component of the outer membrane of nontypeable Haemophilus influenzae, is a complex molecule that requires the functions of many genes for proper assembly. One gene, htrB, has recently been shown to play a role in the acylation of the lipid A portion of the LOS of H. influenzae (19). The lipid A of H. influenzae is typically hexaacyl, containing ester-and amide-linked 4-hydroxymyristic acids. The two ␤-hydroxymyristic acids on the second glucosamine are replaced at their hydroxy group with myristic acid. In contrast, the lipid A of the H. influenzae htrB mutant is approximately 90% tetraacyl, with only four hydroxymyristic acid ester-and amide-linked fatty acids. This mutant lipid A is similar in structure to lipid IV A . The remaining 10% of the htrB lipid A is pentaacyl, with a single myristic acid substitution.Recent studies of nontypeable H. influenzae have indicated that changes in the LOS structure affect bacterial virulence. The similarity of the LOS from the nontypeable H. influenzae htrB mutant to lipid IV A suggests that it binds to the CD14 receptor but does not initiate signalling which results in a macrophage cytokine response (18). Experimental evidence has shown that LOS from the nontypeable H. influenzae htrB mutant has reduced toxicity in an infant-rat model and elicits less tumor necrosis factor alpha from human macrophages than does wild-type LOS (unpublished data). In addition, Somerville et al. (3...

Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-mannoheptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.