The objective of the proposed research is to define interactions that occur between bacteria and the mucosal cell surface and to relate these to mechanisms involved in disease. We are currently studying a group of bacterial proteases, produced by different serogroups of Haemophilus influenzae and by Streptococcus sanguis, that cleave human IgA1, in order to understand the role of these enzymes in infection. We propose to elucidate how the proteases interact with the IgA substrate by expressing the IgA heavy chain from cloned cDNA and using it, or mutant variants as a substrate. We also want to determine the basis for cleavage specificity of the H.influenzae proteases which fall into two main types based on the specific bond cleaved in the IgA hinge region. Hybrids will be constructed between the two gene types to identify the determinants of specificity. The IgA proteases are secreted outside the cell in a process that is dependent on the carboxyl terminal region of the primary gene product. This region is thought to form a pore in the membrane. We want to understand the role of this segment in secretion and will approach the problem through a detailed genetic analysis just this portion of the gene. We have observed that growth of H.influenzae in human milk blocks secretion of IgA protease, resulting in aggregation of the organism. The effect is due to IgA1 in the milk. We will examine this effect further to see if it more accurately mimics the in vivo conditions than growth in synthetic medium. We will use a series of iga gene hybrids to identify sites in the enzymes that are highly antigenic in vivo with the hope that such knowledge might be useful in vaccine development. We will use human lung epithelial cells , cultured in the presence of human IgA, to study the role of H. influenzae IgA1 protease in bacterial attachment and colonization.
