B-289. Structural Determinants of Autoproteolysis by Haemophilus influenzae Hap Autotransporter

R. H. Kenjale, J. W. St. Geme, III;
Duke Univ., Durham, NC.

Non-typeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The process of colonization involves both pilus and non-pilus adhesins. Hap is a non-pilus adhesin that belongs to the autotransporter family and mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells. Hap also contains serine protease activity associated with autoproteolysis, resulting in extracellular release of the Haps passenger domain and leaving the Hapβ beta domain embedded in the outer membrane. Autoproteolysis is mediated by a catalytic triad that includes residues H98, D140 and S243. The primary site of cleavage is the LN 1036-37 bond, and alternative sites include the LT1046-7, FA1077-8, and FS1067-8 bonds. In this study, we set out to characterize the structural determinants of Hap autoproteolysis. We utilized site-directed mutagenesis to study the P1-P4 residues at the Hap cleavage sites. In addition, we used the crystal structures of the E. coli Hbp autotransporter, alpha-chymotrypsin and trypsin to model the Hap protease domain and then exploited this model to mutate residues that correspond to the predicted S1, S2, and S4 subsites. We found that non-conservative mutations at the P1 and P2 positions but not at the P3 and P4 positions disrupted proteolysis, suggesting that the P1 and P2 positions are important determinants of Hap substrate specificity. In addition, we observed that mutations at the predicted S2 and S4 subsites eliminated proteolysis, arguing that the model of the Hap protease domain is an accurate representation of the Hap substrate groove. This information may be helpful in identifying potential host targets for Hap proteolysis and in developing novel inhibitors of Hap activity and NTHi disease.