PROJECT SUMMARY The increasing rate of infection and spread of antibiotic resistance in Neisseria gonorrhoeae poses an urgent threat to public health. Knowledge of the genes and pathways that support evasion of the host immune response and enable infection is needed to develop new strategies for surveillance, prevention, and treatment. Despite our understanding of some of the gonococcal genes and alleles that confer resistance to serum-mediated killing, significant knowledge gaps remain regarding the factors that influence the variation in serum resistance. We lack a systematic evaluation of the pathways to resistance in clinical isolates: how complete is our understanding of the genetic determinants of serum resistance? What is the prevalence and phylogenetic distribution of these resistance genes and alleles? In this proposal, we address these gaps through a comprehensive strategy that links experimental and computational identification of genetic variants that contribute to serum resistance and defines their distribution in a dataset of >16,000 clinical isolate genome sequences. The overall goal of this project is to systematically define the genetic contributors to serum resistance in N. gonorrhoeae. We will achieve this goal through three specific aims, building on strategies that have been suc- cessful in our work defining the genetic basis of antibiotic resistance and decades studying the mechanisms of serum resistance. In Aim 1, we will leverage our collection of hundreds of clinical isolates of N. gonorrhoeae from across the species phylogeny to define the phenotypic range of serum resistance and determine the genetic contributors to serum resistance using a combination of phylogenetics, statistic methods, and transformation. Our preliminary data supports this aim, with a genome-wide association study using serum resistance in 120 clinical isolates identifying known and novel candidate genetic variants that contribute to serum resistance. In Aim 2, we will passage serum susceptible isolates from multiple genetic lineages in pooled human serum to determine factors that contribute to serum resistance and describe the role of genomic background in shaping the evolutionary trajectories to resistance. In Aim 3, we will characterize the prevalence and phylogenetic distri- bution of genetic modulators of serum resistance—combining those in the literature with those identified in Aims 1 and 2—in a set of >16,000 clinical isolates for which we have genome sequences. This project brings together the complementary and non-overlapping expertise of leading investigators in the biology and genetics of N. gonorrhoeae, linking the study of serum resistance (Drs. Lewis and Ram) with ge- nomics (Dr. Grad).