The interactions between specific bacteria and the surfaces of human epithelia result in both beneficial colonization, as in the gastrointestinal tract, and harmful invasions leading to disease. Whereas many data are available on the ligands of bacteria that mediate their population of animal cells surfaces, few data are available on the animal cell receptors themselves. Although bacteria influence host cell morphology and tissue development through receptor mediated interactions, little is known about how host receptors participate in these changes.
The specific aims of this study are to: 1) separate and identify receptors on host animal epithelia for symbiotic bacteria; 2) confirm relevance in vivo for the role of the receptors in the association; 3) identify proteins related to these receptors to gain insight into the function of the receptors in altering cell activity; and 4) use bacterial genetics to alter interactions of the symbiont with host cell receptors to gain insight regarding the nature of host binding. The current proposal uses a one host/one symbiont model system, the squid Euprymna scolopes and its bacterial symbiont Vibrio fischeri. To identify host-receptors for bacterial surface molecules, I propose to separate proteins from the symbiotic organ epithelium using 2-D gel electrophoresis, blot the proteins onto membranes, and identify which bands bind to bacterial symbionts in standard bacterial overlay methods. Proteins identified as putative binding receptors will be further characterized by antibody production to the proteins, purification for assays designed to determine the function of the receptor, and sequencing for identification. Available mutants of the bacterial symbiont will be screened for inability to bind to receptors. The type of mutations leading to deficient binding will yield information regarding the host receptor recognition sites.
