The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin

Abstract

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.

Figure 1. Purified proteins for examination of glycosylation of HMW1.

Panel A shows a schematic of the HMW1 pre-pro-protein. The white bar represents the signal peptide, corresponding to amino acids 1–68. The gray bar represents the pro-piece, corresponding to amino acids 69–441. The black bar represents the mature protein, corresponding to amino acids 442–1536. The vertical ticks above the black bar represent sites of N-linked glycosylation. The portion of HMW1 that was used as the acceptor protein for in vitro glycosyltransferase assays is highlighted by the bracket and corresponds to amino acids 802–1406 and contains 18 sites of glycosylation. Panel B shows Coomassie blue-strained gels of purified strep-tagged HMW1_802–1406 and purified HAT-tagged HMW1C.

Figure 2. In vitro transferase assays, examining samples by SDS-PAGE and detecting glycosylation with DIG-glycan reagents.

Panel A shows results with various combinations of purified HMW1_802–1406, purified HMW1C, and UDP-α-D-glucose plus UDP-α-D-galactose. Panel B shows results with purified HMW1C, purified HMW1_802–1406, and either UDP-α-D-glucose, UDP-α-D-galactose, GDP-α-D-mannose, UDP-α-D-N-Acetylglucosamine, or UPD-α-D-N-Acetylgalactosamine.

Figure 3. Collision-induced fragmentation spectra from glycosylated peptide NLSITTNSSSTYR (HMW1 amino acids 946–958).

Panel A shows the CID spectrum of the glycopeptide that is modified with one hexose unit. Panel B shows the CID spectrum of the glycopeptide that is modified with a di-hexose. The asterisks indicate b and y fragmentation ions that underwent a neutral loss of one (*) or two (**) hexosyl residues.

Figure 4. Effect of inactivation of the galU gene.

Panel A shows purified HMW1 recovered from strain Rd-HMW1 and Rd-HMW1/galU. With strain Rd-HMW1, HMW1 was recovered from the surface of whole bacteria. With strain Rd-HMW1/galU, HMW1 was recovered from the culture supernatant and concentrated, reflecting the fact that HMW1 is no longer surface associated in this strain. Purified protein was resolved by SDS-PAGE and was then either stained with Coomassie blue (upper gel) or examined for glycosylation using DIG-glycan reagents (lower gel). Panel B shows flow cytometry results with strains Rd-HMW1, Rd-HMW1/hmw1C, and Rd-HMW1/galU using antiserum GP85 against HMW1 to detect surface-associated HMW1. The number in the upper right corner of the plots of Rd-HMW1/hmw1C and Rd-HMW1/galU represents the percentage of surface-associated HMW1 in strain Rd-HMW1. Panel C shows in vitro adherence results comparing adherence by Rd-HMW1, Rd-HMW1/hmw1C, and Rd-HMW1/galU to Chang epithelial cells. Bars and error bars represent mean and standard deviations of measurements performed in triplicate.