PROJECT SUMMARY/ABSTRACT Neisseria gonorrhoeae (Gc) is an obligate human bacterial pathogen and the cause of gonorrhea. Gc is a major public health concern because numbers of cases remain high, it has gained resistance to multiple antibiotics, no vaccine exists, and infection is associated with debilitating, lifelong negative outcomes such as infertility and ectopic pregnancy. Mucosal colonization by Gc drives the recruitment of polymorphonuclear leukocytes (PMNs or neutrophils) to the site of infection and the production of a characteristic purulent exudate. Despite PMNs’ robust phagocytic and antimicrobial activities, infectious Gc can be cultured from PMN-rich exudates, implying Gc possesses mechanisms to evade killing by PMNs. The failure of PMNs to clear Gc not only allows the infection to persist, but also sustains the PMN inflammatory response, causing the tissue damage that underlies infertility and other sequelae. To identify the mechanisms used by Gc to resist killing by PMNs, we developed an infection model using adherent, chemokine-treated primary human PMNs to approximate the physiological state of PMNs recruited to mucosal surfaces. We used this model to uncover two major ways in which Gc survives after exposure to PMNs. First, Gc expresses gene products that confer resistance to PMN antimicrobial compounds, such as proteases and antimicrobial peptides. Second, Gc varies expression of surface structures to modulate the route of phagocytosis by PMNs and consequent exposure to these compounds. Specifically, Gc that have phase-varied off expression of opacity-associated (Opa) proteins is internalized into immature phagosomes in which it survives and suppresses the PMN oxidative burst, while Gc expressing an Opa protein that interacts with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) 1 and 3 is phagocytosed into a mature, degradative phagolysosome and induces a potent oxidative burst. In this renewal proposal, we will continue to dissect the mechanisms that underlie Gc survival after PMN challenge. Aim 1 will define how Opa-negative Gc is phagocytosed into PMNs without eliciting the oxidative burst or phagolyososome formation and will evaluate the survival profile of Gc expressing an Opa protein that does not bind CEACAM3. We will evaluate the long-term consequences of these receptor- mediated interactions on the viability of both Gc and PMNs. Aim 2 will build upon results from a Tn-seq experiment conducted in the previous funding period to examine how bacterial efflux pumps and lipooligosaccharide variants contribute to Gc survival from PMNs. All experiments will be conducted with the adherent, interleukin-8 treated PMN infection model alongside analysis of freshly collected human gonorrheal exudates. Together, the results from this research will reveal the diverse approaches used by Gc to survive from PMNs, which ensures the continued persistence of gonorrhea in the human population even when facing a neutrophil...