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.

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.

The Haemophilus influenzae HMW1 adhesin mediates adherence to respiratory epithelial cells, a critical early step in the pathogenesis of H. influenzae disease. In recent work, we demonstrated that HMW1 undergoes glycosylation. In addition, we observed that glycosylation of HMW1 is essential for HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence to host epithelium. In this study, we examined HMW1 proteolytic fragments by mass spectrometry, achieved 89% amino acid sequence coverage, and identified 31 novel modification sites. All of the modified sites were asparagine residues, in all but one case in the conventional consensus sequence of N-linked glycans, viz. NX(S/T). Liquid chromatography-tandem mass spectrometry analysis using a hybrid linear quadrupole ion trap Fourier transform ion cyclotron mass spectrometer, accurate mass measurements, and deuterium exchange studies established that the modifying glycan structures were mono-or dihexoses rather than the N-acetylated chitobiosyl core that is characteristic of N-glycosylation. This unusual carbohydrate modification suggests that HMW1 glycosylation requires a glycosyltransferase with a novel activity.Until recently, glycosylation of proteins was believed to be limited to eukaryotes. However, glycoproteins are now being recognized increasingly in prokaryotes as well, including a variety of pathogenic bacteria. Examples of bacterial glycoproteins include the flagellins of Pseudomonas aeruginosa, Campylobacter jejuni, and Campylobacter coli; the type IV pili of P. aeruginosa, Neisseria gonorrhoeae, and Neisseria meningitidis; and non-pilus adhesins in Chlamydia trachomatis, enterotoxigenic Escherichia coli, and diffusely adhering E. coli (1-9).Nonencapsulated (nontypeable) strains of Haemophilus influenzae are a common cause of human respiratory tract disease and initiate infection by colonizing the upper respiratory tract. Approximately 75-80% of isolates express two related high molecular weight proteins called HMW1 and HMW2 that promote efficient adherence to respiratory epithelial cells and facilitate the process of colonization (10, 11). The HMW1 and HMW2 adhesins are encoded by highly homologous chromosomal loci that appear to represent a gene duplication event and contain three genes, designated hmw1A, hmw1B, and hmw1C and hmw2A, hmw2B, and hmw2C, respectively (12, 13). HMW1 and HMW2 are synthesized as preproproteins and are secreted by the two-partner secretion system (14 -16). Amino acids 1-68 direct the preproproteins to the Sec apparatus, where they are cleaved by signal peptidase I (15). Subsequently, amino acids 69 -441 target the proproteins to the outer membrane and interact with the HMW1B or HMW2B outer membrane translocator protein, undergoing removal by an unknown process (15, 16 -18). Following translocation across the outer membrane, the mature HMW1 and HMW2 proteins remain noncovalently associated with the bacterial surface, with small amounts released into the culture supernatant (15,16).In recent work, we est...