SummaryNon-typeable Haemophilus influenzae is a common respiratory pathogen and an important cause of morbidity in humans. The non-typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that mediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Secretion of these adhesins requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C. In the present study, we investigated the specific function of HMW1C. Examination of mutant constructs demonstrated that HMW1C influences both the size and the secretion of HMW1. Co-immunoprecipitation and yeast two-hybrid assays revealed that HMW1C interacts with HMW1 and forms a complex in the cytoplasm. Additional experiments and homology analysis established that HMW1C is required for glycosylation of HMW1 and may have glycotransferase activity. The glycan structure contains galactose, glucose and mannose and appears to be generated in part by phosphoglucomutase, an enzyme important for lipooligosaccharide biosynthesis. In the absence of glycosylation, HMW1 is partially degraded and is efficiently released from the surface of the organism, resulting in reduced adherence. Based on these results, we conclude that glycosylation is a prerequisite for HMW1 stability. In addition, glycosylation appears to be essential for optimal HMW1 tethering to the bacterial surface, which in turn is required for HMW1-mediated adherence, thus revealing a novel mechanism by which glycosylation influences cellcell interactions.

SummaryThe pathogenesis of non-typable Haemophilus influenzae disease begins with colonization of the nasopharynx and is facilitated by bacterial adherence to respiratory mucosa. The H. influenzae Hap autotransporter is a non-pilus adhesin that promotes adherence to epithelial cells and selected extracellular matrix proteins and mediates bacterial aggregation and microcolony formation. In addition, Hap has serine protease activity. Hap contains a 110 kDa internal passenger domain called Hap S and a 45 kDa Cterminal translocator domain called Hap b . In the present study, we sought to define the structural basis for Hap adhesive activities. Based on experiments using a panel of monoclonal antibodies against Hap S , a deletion derivative lacking most of Hap S and a purified fragment of Hap S , we established that adherence to epithelial cells is mediated by sequences within the C-terminal 311 residues of Hap S . In additional experiments, we discovered that bacterial aggregation is also mediated by sequences within the C-terminal 311 residues of Hap S and occurs via Hap S -Hap S interaction between molecules on neighbouring organisms. Finally, we found that adherence to fibronectin, laminin and collagen IV is mediated in part by sequences within the C-terminal 311 residues of Hap S and in full by sequences within the C-terminal 511 residues of Hap S . Taken together, these results demonstrate that all Hap adhesive activities reside in the C-terminal portion of Hap S . Coupled with earlier observations, the current results establish that Hap S adhesive activities and Hap S protease activity are contained in separate modules of the protein.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Approximately 75 to 80% of NTHi clinical isolates produce proteins that belong to the HMW family of adhesins, which are believed to facilitate colonization. The prototype HMW adhesins are designated HMW1 and HMW2 and were identified in NTHi strain 12. HMW1 and HMW2 are 71% identical and 80% similar overall, yet display differing cellular binding specificities. In the present study we set out to define more clearly the relationships between HMW1 and HMW2 and other members of the HMW family of adhesins. PCR analysis of 49 epidemiologically distinct isolates revealed that all strains possessing hmw genes as determined by Southern analysis contain two hmw loci in conserved, unlinked physical locations on the chromosome. Functional analysis of the HMW adhesins produced by three unrelated strains demonstrated that each isolate possesses one protein with HMW1-like adherence properties and another with HMW2-like adherence properties. These findings suggest that the hmw1 and hmw2 loci may have arisen via a gene duplication event in an ancestral strain. In addition, they support the hypothesis that the distinct binding specificities of HMW1 and HMW2 emerged early and have persisted over time, suggesting an ongoing selective advantage.Nontypeable Haemophilus influenzae (NTHi) strains are commensal organisms in the nasopharynx and are also a frequent cause of localized respiratory tract disease, including otitis media, conjunctivitis, sinusitis, pneumonia, and exacerbations of chronic bronchitis (13,29,38). The pathogenesis of NTHi disease begins with colonization of the nasopharynx, followed by contiguous spread within the respiratory tract. Successful colonization requires that the organism overcome the mucociliary escalator, a task accomplished in part by adherence to respiratory epithelium (29,38). NTHi adherence is mediated by both pilus and nonpilus adhesins. In experiments with cultured human epithelial cells, the major nonpilus adhesins were found to be HMW1/HMW2 and Hia (20,22,33,35). Based on examination of several collections of epidemiologically distinct NTHi strains, approximately 75 to 80% of isolates produce HMW1/HMW2-like proteins, while most of the remaining isolates produce Hia (7, 20, 36). Of note, isolates produce either HMW1/HMW2-like proteins or Hia, but not both (7,20,36).The HMW adhesins were first identified as major targets of the human serum antibody response during acute otitis media (4). The prototype proteins are designated HMW1 and HMW2 and are produced by NTHi strain 12, the strain from which they were originally cloned and sequenced (5). HMW1 and HMW2 are encoded by separate chromosomal loci, with each locus consisting of three genes, designated hmwA, hmwB, and hmwC. The hmwA genes encode the surface-exposed adhesins (HMW1 and HMW2), and the hmwB and hmwC genes encode accessory proteins required for processing and secretion of the adhesins ...

Adhesion to the respiratory epithelium plays an important role in