Nontypeable Haemophilus influenzae (NTHi) is a predominant etiologic agent of otitis media (OM) in children as well as a major cause of pneumonia and other respiratory tract diseases in adults. Although much has been learned about several surface- exposed proteins and the endotoxin (lipooligosaccharide) produced by this organism, overall we have a poor understanding of the pathogenesis of Haemophilus-induced infection. Type IV pili (Tfp) play a significant role in the pathogenesis of disease caused by Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae and numerous other Gram-negative pathogens. Tfp-based immunogens induce protective activity against these organisms, although in some systems, including N. gonorrhoeae, protection against infection induced by these immunogens is limited due to sequence diversity of the pilin protein. We recently demonstrated that Tfp are produced by NTHi. More recently, we demonstrated that these Tfp play a role in adherence and biofilm formation in the chinchilla model of otitis media caused by NTHi. We also demonstrated that the product of the pilA gene, PilA, exhibits limited antigenic heterogeneity suggesting that it might be a useful vaccine candidate. We are currently funded to characterize the genes and gene products required for Tfp biogenesis in NTHi and to epitope map the PilA protein. We have made considerable progress toward these goals. However, as we endeavor to identify both the surface-exposed domains and immunodominant epitopes within the type IV pilus protein, it will be important to have detailed structural information on the NTHi type IV pilus. We established a collaboration with Dr. Lisa Craig to determine conditions for crystallization of the Haemophilus type IV pilin with the expectation of ultimately seeking future supplemental funding to determine the structure. These studies were funded by a modest 2-year subcontract as part of this funded initiative. Dr. Craig's group has succeeded in crystallizing recombinant pilin and has collected a 1.7 Angstrom data set. This competing supplemental application requests an additional 17 months of subcontract funding in order that Dr. Craig's group can complete the structural analysis of the PilA protein. Project Narrative Nontypeable Haemophilus influenzae is an important cause of otitis media in children as well as other diseases of the human respiratory tract. Recently, we developed evidence indicating that a structure on the bacterial surface, called a type IV pilus, was important for infection. Further, our data indicate that the major protein component of the type IV pilus should be considered for inclusion in an otitis media vaccine. Understanding the structure of this protein is critical to understanding the role of this protein in infection and immunity. We have made considerable progress toward this goal and request funds to complete determination of the structure of this protein. ? ? ?
