Haemophilus influenza (Hi) causes significant infections in infants, children and adults. Although the Hi vaccine prevents Hi type b infections, it is ineffective against types a, c-f, and non-typable Hi, which cause sinusitis, otitis media, and pneumonia. The first step in the establishment of Hi infections is attachment of the bacteria to human respiratory epithelial cells, a process that is mediated by pili on the surface of Hi. Our goal is to understand the immunobiology, biogenesis, and pathogenic role in Hi pili. Based on this information, methods can be devised to interfere with attachment of Hi to respiratory tissues, thus preventing infection. In our preliminary work, we have defined a Hi Pilus gene cluster, consisting of five genes- a pilin structural gene and four accessory genes. In this application, Aim 1 is to identify and characterize the gene product in Hi. These genes will be mutated and their role in pilus expression determined. Pili are complex structures, composed of pilin subunits and possibly additional components, including adhesions.
Aim 2 is to identify the adhesion molecule of Hi pili, using three possible approaches. In the genetic approach, we will determine if HiiE, the only pilus accessory gene whose function is not suggested by amino acid sequence, encodes an adhesion. In the biochemical approach we will further characterize the 66kDa doublet proteins that are associated with adherence activity and are present on pilated strains but not on non-pilated variants. In the monoclonal antibody approach, we hypothesize that one of our four monoclonal antibodies that recognize two heterogenous pili may define the adhesion.
Aim 3 is to identify immunologically conserved epitopes on Hi pili. After verifying that the anti-pili monoclonal antibodies interfere with adherence of Hi, we will characterize the specificity of the antibodies and localize their epitopes on Hi pili.
Aim 4 is to identify the basis for the immunologic differences between HIb E1a an M43 pili. We will exam the role of amino acid sequence differences in determining immunologic reactivities of these pili; we will construct chimeric pilin genes that contain amino acid sequences of the alternative pilin type, and test the reactivity of the hybrid pili with strain specific antisera. If these studies fail to reveal an explanation for the immunologic heterogenicity, we will """"""""mix and match"""""""" pilin genes with alternative assembly proteins to examine the role of accessory proteins in defining the immunologic differences. Data from these studies will be important in designing potential strategies to prevent Hi infection, such as pilus component vaccines or false ligands.
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