PROJECT SUMMARY/ABSTRACT Neisseria gonorrhoeae is a Gram-negative bacterium that causes the sexually transmitted infection (STI) gonorrhea. With an estimated 78 million cases of gonorrhea annually worldwide, increasing frequency of resistance to all recommended antibiotics, and the lack of a protective vaccine, N. gonorrhoeae is a prominent and growing threat to human health. In women, N. gonorrhoeae establishes infection at the cervix, where it initiates an inflammatory response characterized by the local recruitment of neutrophils. Viable N. gonorrhoeae are recovered from human gonorrheal exudates, indicating that neutrophils cannot effectively clear infection. The resulting cycle of sustained infection and neutrophilic inflammation enables N. gonorrhoeae transmission as well as ascending infection and tissue damage, which underlie sequelae such as pelvic inflammatory disease, ectopic pregnancy, and infertility. Moreover, infection with N. gonorrhoeae is highly epidemiologically associated with coinfection with Chlamydia trachomatis, enhancing likelihood of these negative consequences. Identifying the mechanisms underlying cervical infection and inflammation by these prominent STI pathogens is critical for finding new therapeutic approaches to enhance women’s overall health and reproductive fitness. Our knowledge of the bacterial and host conditions that facilitate productive N. gonorrhoeae infection in women is hampered by the absence of a robust model for human genital infection that incorporates resident and recruited host cells, maintains the architecture of the lower female genital tract, and incorporates the genital microbiota. To overcome this knowledge gap, we will use the 3D human primary cervicovaginal biomimetic system developed by our group to answer fundamental questions about the biology of female genital infection, alone and in the context of cervicovaginal microbiota that we hypothesize are associated with susceptibility (bacterial vaginosis-associated anaerobes, community state type-IV) and resistance (Lactobacillus crispatus-dominant, community state type- I). Aim 1 will define the influence of the microbiota on the progression of cervicovaginal N. gonorrhoeae infection and effects on epithelial host defenses. Aim 2 will characterize the effect of the microbiota on the cervicovaginal recruitment of immune cells in response to N. gonorrhoeae. Aim 3 will define how coinfection with N. gonorrhoeae and C. trachomatis in the context of the microbiota impacts pathogen burden and cervicovaginal immune response. These studies will include vaginal and endocervical cells from women along with their autologous microbiota, as well as unmatched microbiota, to evaluate how the host-microbiota interaction influences the progression and outcome of infections. The findings arising from these studies about the infection process and ensuing inflammatory response and tissue integrity can reveal new interventions to limit bacterial load and mucosa...