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. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease in 10-20% of women with infections, which encompasses a wide range of inflammatory conditions and often leads to chronic pelvic pain, infertility, and ectopic pregnancy. 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. Currently only ceftriaxone and azithromycin are recommended for first-line therapy, and clinical isolates resistant to both of those antibiotics have been reported in countries including Denmark, Canada, and Japan. Thus, there is a compelling need for new antimicrobials for gonococcal infections. Our studies to date of N. gonorrhoeae lipooligosaccharide (LOS) and the human innate immune system have shown that the lipid A portion of LOS is the primary inducer of cytokine-mediated inflammation and investigations by others have shown that the lipid A also facilitates gonococcal infection. These data led us to the concept that targeting lipid A biosynthesis would be an effective approach to combating N. gonorrhoeae infections. We recently reported that inhibition of LpxC, the enzyme that catalyzes the second step of lipid A biosynthesis, was bactericidal for nine multidrug-resistant and human challenge strains of gonococci and reduced cytokine induction without apparent human cell cytotoxicity. From the LpxC inhibitor data, we postulated that membrane disruption due to the inhibition of LOS biosynthesis was lethal for gonococci. To investigate this, we recently evaluated the bactericidal potential of a 12 amino acid cell-penetrating peptide (CPP) for MDR and human challenge strains of N. gonorrhoeae and found that the CPP penetrated the bacterial membrane and was bactericidal for all nine MDR and human challenge strains of gonococci tested. Importantly, no apparent resistance to the CPP developed in surviving bacteria as susceptibility was the same in bacteria from colonies after exposure to CPP and then retreated. Further, the CPP reduced inflammatory cytokine induction and prevented bacterial cell invasion of cervical epithelial cells in the absence of measurable cell cytotoxicity. These novel data highlight LpxC 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, whi...