The overall research program of the applicant has focused on the role of LOS and the innate immune system and has shown that the degree of phosphorylation of the lipid A component is correlated with the potential of the LOS to induce innate immune cytokine-mediated inflammation and, in general, with the severity of Neisserial disease. Based on the results of these studies, recent work has undertaken the development of potential therapeutics targeting gonococcal LOS as infections due to N. gonorrhoeae are a major cause of morbidity with an estimated 850,000 cases in the U.S. and 87 million cases worldwide annually. Within the VA Health Care System, cases of gonorrhea increased between 2013 and 2017 with the total number in that time period at 10,587. Within the U.S. military, service members are a defined high-risk group for gonorrhea as noted by the U.S. Preventive Services Task Force as the high-risk environment of active military service is thought to increase risky behavior and, thus, the rate of sexually transmitted infections including gonorrhea. A total of 27,658 cases of gonorrhea were identified among active duty personnel during FY 2007 to 2015 (222.7 per 100,000 military compared with 179.3 per 100,000 non-military in 2018). There is no vaccine to N. gonorrhoeae and a great need for new antibiotics due to the alarming rise in multidrug-resistance (MDR), which is making emergence of untreatable gonococcal infections a real prospect. Thus, there is a compelling need for new antimicrobials for gonococcal infections. To this end, the projects to be pursued during the proposed funding period of this SRCS award are as follows: 1) LpxC inhibitors and cell-penetrating peptides; we recently reported that treatment of gonococci with an inhibitor of LpxC, the enzyme that catalyzes the second step of lipid A biosynthesis, was bactericidal for MDR and human challenge strains of gonococci and reduced cytokine induction without apparent human cell cytotoxicity. Most recently, we evaluated the bactericidal potential of a 12 amino acid cell-penetrating peptide (CPP) and found that it penetrated the bacterial membrane and was bactericidal for all multi-drug resistant and human challenge strains of gonococci tested and reduced inflammatory cytokine induction and prevented bacterial cell invasion of cervical epithelial cells. These novel data highlight LpxC inhibitors and CPP as promising antimicrobials for N. gonorrhoeae and strongly support the hypothesis of this application that inhibiting the biosynthesis of lipid A components with LpxC inhibitors and disrupting outer membrane integrity with CPP will impact bacterial viability and host response to N. gonorrhoeae infection in vitro and in vivo, which will have a therapeutic impact on infection outcomes. Translational studies of these two potential therapeutics for gonorrhea will be the primary focus of the investigations proposed for the award period. 2) EptA inhibitors; we will continue our collaborat...