PROJECT SUMMARY: A major obstacle to the development of new vaccines and treatments for Neisseria gonorrhoeae (Ng) has been the efficiency with which this pathogen evades complement killing. The complement cascade, an important arm of innate immunity, targets invasive pathogens for killing. Infectious disease researchers have identified many ligands on Ng for fluid phase complement inhibitory proteins of the human host. However, there is a fundamental gap in our understanding of the contribution of membrane-associated complement inhibitory proteins (mCIs) to Ng complement evasion strategies. This gap needs to be filled in order to design effective treatments against gonorrhea, which is now resistant to most antibiotic therapies. The long term goal is to develop new therapies that hinder Ng's ability to use complement inhibitors of the human host to evade complement killing. The objective of this application is to define the contribution of mCIs and other epithelial proteins to Ng complement evasion. The central hypothesis is that during infection of epithelial cells Ng recruits mCIs into bacterial aggregates called microcolonies as a method to evade complement killing. This hypothesis is based on preliminary data generated by the applicant and his students. The rationale for this hypothesis is based on a new assay that can address the lack of knowledge about mCI-dependent Ng complement evasion. Using a co-culture serum bactericidal assay (ccSBA), in which Ng serum survival is monitored in the presence of human epithelial cells, the applicant has shown that infected epithelial cells positively contribute to Ng complement evasion. Guided by solid preliminary data, the hypothesis will be tested by probing three specific aims: 1) Determine if altering epithelial mCIs levels alters Ng's survival in serum; 2) Define the contributions of individual mCIs to Ng survival in the ccSBA; and 3) Elucidate pathways by which Ng recruits serum-protective epithelial proteins. For Specific Aim 1, expression levels for three mCIs (CD46, CD55 and CD59) will be stably altered in epithelial cells using shRNAs or protein overexpression and tested in the ccSBA for their influence on Ng survival. One cell line with altered mCI levels has already been made and supports higher levels of Ng survival. For Specific Aim 2, an epithelial cell line lacking all three mCIs will be made with genome editing technology. Each mCI will be individually expressed in the ?mCI line to determine if all three mCIs, or a subset, can protect Ng from serum killing. For Specific Aim 3, the applicant will use electron microscopy to characterize epithelial membranous structures induced by Ng infection and determine if microvilli contribute to protection of Ng from serum killing. Expression of specific epithelial genes that influence microvilli biogenesis will be altered in cells and evaluated for Ng survival phenotypes. This proposal's research is innovative because it uses a new assay that can test the contribution of epithelial-derived proteins to Ng complement evasion. This research is significant because it will expand our understanding of Ng complement evasion strategies dependent upon the infected cell and may lead to development of new antibody-based therapies or vaccines.