The Hemophilus influenzae Hap autotransporter is a chymotrypsin clan serine protease and undergoes autoproteolysis via an intermolecular mechanism

The Hemophilus influenzae Hap adhesin is an auto-transporter protein that u ndergoes an autoproteolytic cleavage event resulting in extracellular relea se of the adhesin domain (Hap(s)) from the membrane-associated translocator domain (Hap(beta)). Hap autoproteolysis is mediated by Ser(243) and occurs at LN1036-7 and to a lesser extent at more COOH-terminal alternate sites. In the present study, we sought to further define the mechanism of Hap auto proteolysis. Site-directed mutagenesis of residues His(98) and Asp(140) ide ntified a catalytic triad conserved among a subfamily of autotransporters a nd reminiscent of the SA (chymotrypsin) clan of serine proteases. Amino-ter minal amino acid sequencing of histidine-tagged Hap(beta) species and site- directed mutagenesis established that autoproteolysis occurs at LT1046-7, F A1077-8, and FS1067-8, revealing a consensus target sequence for cleavage t hat consists of ((Q/R)(A/S)X(L/F)) at the P4 through P1 positions. Examinat ion of a recombinant strain co-expressing a Hap derivative lacking all clea vage sites (Hap Delta 1036-99) and a Hap derivative lacking proteolytic act ivity (HapS243A) demonstrated that autoproteolysis occurs by an intermolecu lar mechanism. Kinetic analysis of Hap autoproteolysis in bacteria expressi ng Hap under control of an inducible promoter demonstrated that autoproteol ysis increases as the density of Hap precursor in the outer membrane increa ses, confirming intermolecular cleavage and suggesting a novel mechanism fo r regulation of bacterial adherence and microcolony formation.