Mapping of binding domains of nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins

Nontypeable Haemophilus influenzae is an important cause of localized respi ratory tract disease, which begins with colonization of the upper respirato ry mucosa. In previous work we reported that the nontypeable H. influenzae HMW1 and HMW2 proteins are high-molecular-weight nonpilus adhesins responsi ble for attachment to human epithelial cells, an essential step in the proc ess of colonization. Interestingly, although HMW1 and HMW2 share significan t sequence similarity, they display distinct cellular binding specificities . In order to map the HMW1 and HMW2 binding domains, we generated a series of complementary HMW1-HMW2 chimeric proteins and examined the ability of th ese proteins to promote in vitro adherence by Escherichia coli DH5 alpha. U sing this approach, we localized the HMW1 and HMW2 binding domains to an si milar to 360-amino-acid region near the N terminus of the mature HMW1 and H MW2 proteins. Experiments with maltose-binding protein fusion proteins cont aining segments of either HMW1 off HMW2 confirmed these results and suggest ed that the fully functional binding domains may be conformational structur es that require relatively long stretches of sequence. Of note, the HMW1 an d HMW2 binding domains correspond to areas of maximal sequence dissimilarit y, suggesting that selective advantage associated with broader adhesive pot ential has been a major driving force during H. influenzae evolution. These findings should facilitate efforts to develop a subcomponent vaccine effec tive against nontypeable H. influenzae disease.