Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains

Non-typable Haemophilus influenzae is a common cause of human disease and i nitiates infection by colonizing the upper respiratory tract. The non-typab le H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithel ial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N-terminus and undergo cleavage of a 441-amino-acid N-termi nal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacteri al surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino a cids, which are characterized by a highly charged region from amino acids 1 -48, followed by a more typical signal peptide with a predicted leader pept idase cleavage site after the amino acid at position 68. Additional experim ents established that the SecA ATPase and the SecE translocase are essentia l for normal export and demonstrated that maturation involves cleavage firs t between residues 68 and 69, via leader peptidase, and next between residu es 441 and 442. Site-directed mutagenesis revealed that HMW1 processing, se cretion and extracellular release are dependent on amino acids in the regio n between residues 150 and 166 and suggested that this region interacts wit h the HMW1B outer membrane translocator. Deletion of the C-terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1-me diated adherence, arguing that the C-terminus may serve to tether the adhes in to the bacterial surface. These observations suggest that the HMW protei ns are secreted by a variant form of the general secretory pathway and prov ide insight into the mechanisms of secretion of a growing family of Gram-ne gative bacterial exoproteins.