Gonorrhea remains a significant worldwide public health problem with over 87 million cases/year. Compounding the problem of disease incidence is the emergence of strains of Neisseria gonorrhoeae in the community that are resistant to currently used antibiotics in dual therapy (azithromycin and ceftriaxone). We have shown that the MtrCDE efflux pump is a critical system used by gonococci to resist both beta-lactams and macrolides. Moreover, this multidrug efflux pump can recognize and export host defense compounds (e.g., bile salts, cationic antimicrobial peptides and progesterone), and loss of the pump by genetic inactivation or repression of gene expression renders gonococci less fit in a female mouse model of lower genital tract infection. Based on these observations we propose that MtrCDE is a virulence factor used by gonococci to survive the onslaught of host defense compounds and antibiotics so as to proliferate during infection. We have shown that both cis- and trans- acting transcriptional control systems can modulate the degree of mtrCDE expression. In this respect, we have identified novel single nucleotide mutations in promoter sequences that elevate mtrCDE expression. More recently, we found that single amino acid changes in the MtrD transporter can result in enhanced resistance of gonococci to antimicrobials. In the two Specific Aims of this competitive renewal application we will perform experiments that address the emergence of mutations that influence antimicrobial resistance expressed by gonococci. We will use a female mouse of infection to ascertain whether such mutations impact antibiotic efficacy during infection. Mutations that cause amino acid changes in the MtrD transporter will serve as tools to understand its structure-function through the use of cryo-electron microscopy and in vitro drug binding assays. The successful completion of the work will provide important basic knowledge on the role of the MtrCDE efflux pump in providing gonococci with resistance to antibiotics and mediators of innate host defense. With such knowledge, future translational work focused on therapeutics that target MtrCDE (and other similar pumps possessed by Gram-negative pathogens) can be rationally pursued.