Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. This Gram-negative, human-specific pathogen is able to colonize mucosal sites in the genitourinary tract, rectum, oropharynx and conjunctiva and can spread systemically, causing sepsis, endocarditis and meningitis. The CDC estimates around 820,000 cases annually, and has recently named N. gonorrhoeae as an urgent antibiotic resistance threat. In females, cervical infections can ascend into the Fallopian tubes, where inflammatory tissue damage and death of ciliated cells can lead to pelvic inflammatory disease (PID), causing chronic pelvic pain, tubal-factor infertility and increased risk of ectopic pregnanc. In a human Fallopian tube organ culture model of infection, two bacterial products, lipooligosaccharide (LOS) or peptidoglycan (PG) can each reproduce the ciliated cell damage seen during PID. While it is unknown exactly how PG is detected by the Fallopian tube epithelium during infection, in cultured epithelial cells the application of PG monomers or of membranous blebs known as outer membrane vesicles (OMVs) can both activate the intracellular PG sensor NOD1, leading to an inflammatory response via activation of NF-?B. It has recently been shown that NOD1- agonist PG fragments are packaged into OMVs in other Gram-negative bacteria and may therefore represent a mechanism for the delivery of PG to cytoplasmic sensors such as NOD1. N. gonorrhoeae is a prolific producer of both soluble PG fragments and OMVs during normal growth. Though shown to activate NOD1, it is unknown what PG fragments are packaged in N. gonorrhoeae OMVs, or whether the released PG or OMV-PG is more important for signaling through NOD1 and inducing inflammatory damage. The goal of this research plan is to test the hypothesis that N. gonorrhoeae packages PG into OMVs, which act to modulate the NOD1-dependent immune response to N. gonorrhoeae during host infection. To accomplish this, experiments have been designed to radiolabel PG and identify the PG in OMVs using size-exclusion chromatography and HPLC. Strains with specific mutations impacting PG fragment release will also have their OMV-PG content analyzed. To determine the relative contribution of soluble PG and OMV-PG, cell lines with NF-?B reporters will be employed to measure the NOD1-agonist activity of OMVs compared to soluble PG, with and without input from other microbe-associated molecular patterns (MAMPs). Lastly, the human Fallopian tube organ culture model of infection will be used to determine the ability of OMVs to induce an immune response as measured by inflammatory cytokine production, and to determine the contribution of OMVs to hNOD1-dependent transcriptional changes. This work will provide important insight into how N. gonorrhoeae PG is delivered to and sensed by the human host, and how this PG delivery influences the inflammatory response in the Fallopian tube.