Outer-membrane proteins from 48 isolates of nontypable Haemophilus influenzae from adults and children were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the biotypes were determined. Each strain had two principal outer-membrane proteins in the 32,000-42,000 molecular-weight range. Eight subtypes were identified based on the differences in molecular weights of the two principal outer-membrane proteins. The lower molecular-weight protein of those two major proteins was heat modifiable. There was no definite relationship between subtype and biotype. Future studies on the pathogenesis and epidemiology of infections due to nontypable H. influenzae must take into account the differences in outer-membrane proteins among various strains. A subtyping system based on major outer-membrane proteins may provide a basis for such studies.

A 16,600-D outer membrane protein is present in all strains of Haemophilus influenzae and antibodies to this protein are present in human serum. This study was designed to assess the role of this outer membrane protein (P6) in nontypeable H. influenzae as a target for human serum bactericidal antibody. P6 was isolated and coupled to an affinity column. Depleting normal human serum of antibodies to P6 by affinity chromatography resulted in reduced bactericidal activity of that serum for nontypeable H. influenzae. Immunopurified antibodies to P6 from human serum were bactericidal. Finally, preincubation of bacteria with a monoclonal antibody that recognizes a surface epitope on P6, inhibited human serum bactericidal killing. Taken together, these experiments establish that P6 is a target for human bactericidal antibodies. This observation provides evidence that P6 plays a potentially important role in human immunity to infection by nontypeable H. influenzae.

The lipooligosaccharides (LOS) of nontypable Haemophilus influenzae are an antigenically heterogeneous group of macromolecules. Immunodiffusion and enzyme-linked immunosorbent assay inhibition studies with phenol-water-extracted LOS and absorbed antisera specific for the oligosaccharide portion of the LOS identified six LOS strain-specific antigens. To facilitate screening large numbers of strains to search for LOS antigenic heterogeneity, a system utilizing proteinase K whole cell digests in Western blots was developed. Seventy-two nontypable H. influenzae LOS extracts were analyzed in this Western blot assay. Thirty-seven of these extracts could be segregated into 10 antigenically distinct LOS groups based on immunologic recognition by one or more of the rabbit antisera. Thirty-five of the strains did not contain these LOS antigens. These results demonstrate that antigenic differences exist among the LOS of nontypable H. influenzae strains, and this heterogeneity has the potential to be used to establish an LOS-based serogrouping system.

A mouse monoclonal antibody that recognizes an epitope on a 16,600-dalton outer membrane protein was developed to nontypable Haemophilus influenzae. This epitope was present on all 115 isolates of H. influenzae tested, including typable and nontypable strains. Screening of 89 strains of other bacteria demonstrated that this epitope is a highly specific marker for H. influenzae because the epitope was absent in virtually all other bacterial species tested. Western blot assays were performed with two normal human serum samples and convalescent-phase serum from an adult with bacteremia due to nontypable H. influenzae. Antibody to the 16,600-dalton outer membrane protein was present in all three human serum samples.

The identification of enterobacterial mutants that contain alterations in the lipopolysaccharide (LPS) oligosaccharide core structure facilitated the development of the model of the physicochemical and immunochemical structures of enteric LPS. Results of recent immunochemical studies have suggested that the structural model of the lipooligosaccharides (LOSs) of Neisseria gonorrhoeae may differ from the enteric LPS model. The difficulties in the analysis of the wild-type gonococcal LOS have precluded understanding of the precise nature of the LOS structure. This study was undertaken to isolate a series of mutants of N. gonorrhoeae 1291 that had sequential saccharide deletions in the LOS. Results of preliminary studies suggested that the pyocin, designated pyocin C, allowed selection of gonococci with such mutant LOS structures. Results also indicated that the receptor for pyocin C binding was an LOS component. Pyocin C selection led to the isolation of five strains with LOS patterns on sodium dodecyl sulfate-polyacrylamide gels which differed from the LOS of parent strain 1291. In this system, the Mr of the parent LOS was 4,715, while the LOSs from the mutant strains demonstrated progressive saccharide deletions, with Mrs of 4,230, 4,089, 3,627, 3,262, and 3,197. Protein patterns of these mutants on sodium dodecyl sulfate-polyacrylamide gels were qualitatively similar to those of the parent strains. Results of studies with five monoclonal antibodies specific for neisserial LOS indicated that shared as well as unique epitopes were present on the mutant LOSs. Results of ketodeoxyoctonate analysis of the mutant LOSs indicated that the majority of the ketodeoxyoctonate residues may be substituted on C-4 or C-5. Chemical and immunological analysis of such LOS mutants should expedite the development of the model for the structure of gonococcal LOS.