Neisseria gonorrhoeae (GC) is able to attach to and invade non-ciliated epithelial cells of the Fallopian tube organ culture model (FTOC). The mechanism of this interaction remains to be defined. GC Por or PI, the focus of this proposal, is an outer membrane porin protein that comes in two forms, PIA or PIB. It does not vary within a single GC strain. It can transfer spontaneously from GC to lipid bilayers and human cells. It is able to bind calmodulin, but the impact of this on pathogenesis is unknown. Two surface-exposed regions of PIA have amino acid sequences compatible with a basic amphiphilic a-helical conformation, a motif that calmodulin recognizes in binding to a large variety of proteins. This motif also allows peptides and some transmembrane receptors to activate G proteins. PIA has been cloned and expressed in E. coli. In the FTOC model, these E. coli fail to attach, which has prevented assessment of PIA's invasion-triggering properties. GC opacity-associated proteins (Opa) and lipooligosaccharides (LOS), have been implicated as attachment and invasion factors in some cell culture models. Opa and LOS undergo rapid phase and antigenic variation. One type of LOS defined by the 3F11 monoclonal antibody binds to the asialoglycoprotein receptor in liver cell lines. It is not known if the 3F11 epitope mediates attachment to or invasion of fallopian tube epithelium. Opas and the 3F11 LOS have been cloned and expressed in E. coli. Opa production facilitates attachment of E. coli in the human FTOC model, but observed bacterial transport through the cells has been much less than that seen with GC. Thus, we hypothesize that following intimate Opa- and/or LOS-mediated attachment, PI insertion into host cells triggers significant GC invasion via calmodulin- and/or G protein-mediated pathways. By providing for bacterial attachment and eliminating phase and antigenic variation via subcloning of Opa and/or LOS into PIA-bearing E. coli, "GC surrogates" will be constructed to study invasion-triggering by PIA. Variants which express Opa, 3F11 LOS, and PIA in various combinations will be tested in the FTOC model for invasiveness as measured by computerized image analysis in conjunction with digital confocal microscopy. Invasive subclones of E. coli and GC will be tested in the FTOC model with G protein and calmodulin activators or inhibitors to determine if these pathways are connected to invasion through GC PIA. Site-directed mutagenesis of putative basic amphiphilic alpha-helical PIA regions with a-helix breaking proline residues should ablate calmodulin-binding properties and invasiveness. Morphometric comparisons of these mutants to E. coli expressing wild-type PIA in the FTOC model will confirm the pathogenic role of these regions.