The mucosal inflammatory response to Neisseria gonorrhoeae (Gc) is characterized by recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils by limiting the ability of neutrophils to make antimicrobial products and by expressing factors that defend against these products. The multiple transferable resistance (Mtr) system is a tripartite efflux pump, comprised of the inner membrane MtrD, the periplasmic attachment protein MtrC, and the outer membrane channel MtrE. Gc MtrCDE exports a diverse array of substrates, including certain detergents, dyes, antibiotics, and host-derived antimicrobial peptides. Here we report that MtrCDE contributes to the survival of Gc after exposure to adherent, chemokine-treated primary human neutrophils, specifically in the extracellular milieu. MtrCDE enhanced survival of Gc in neutrophil extracellular traps and in the supernatant from neutrophils that had undergone degranulation (granule exocytosis), a process that releases antimicrobial proteins into the extracellular milieu. The extent of degranulation was unaltered in neutrophils exposed to parental or mtr mutant Gc. MtrCDE expression contributed to Gc defense against some neutrophil-derived antimicrobial peptides but not others. These findings demonstrate that the Mtr system contributes to Gc survival after neutrophil challenge, a key feature of the host immune response to acute gonorrhea.

Summary Infection with Neisseria gonorrhoeae (Gc) is marked by an influx of neutrophils to the site of infection. Despite a robust immune response, viable Gc can be recovered from neutrophil-rich gonorrheal secretions. Gc enzymatically modifies the lipid A portion of lipooligosaccharide by the addition of phosphoethanolamine (PEA) to the phosphate group at the 4’ position. Loss of LptA, the enzyme catalyzing this reaction, increases bacterial sensitivity to killing by human complement and cationic antimicrobial peptides. Here, we investigated the importance of LptA for interactions between Gc and human neutrophils. We found that lptA mutant Gc was significantly more sensitive to killing by human neutrophils. Three mechanisms underlie the increased sensitivity of lptA mutant Gc to neutrophils. 1) lptA mutant Gc is more likely to reside in mature phagolysosomes than LptA-expressing bacteria. 2) lptA mutant Gc is more sensitive to killing by components found in neutrophil granules, including CAP37/azurocidin, human neutrophil peptide 1, and the serine protease cathepsin G. 3) lptA mutant Gc is more susceptible to killing by antimicrobial components that are exocytosed from neutrophils, including those decorating neutrophil extracellular traps. By increasing the resistance of Gc to the bactericidal activity of neutrophils, LptA-catalyzed modification of lipooligosaccharide enhances survival of Gc from the human inflammatory response during acute gonorrhea.

Neisseria gonorrhoeae (Gc) is the sole causative agent of the disease gonorrhea. More than 100 million people are infected annually, and reported infection numbers are consistently on the rise. There are clinical isolates of Gc that have developed resistance to the latest third generation cephalosporins, which is the last recommended line of monotherapy for disease treatment, and there is still no available gonococcal vaccine.Following transmission, Gc comes into contact with host mucosal surfaces. Host epithelial cells recognize the pathogen, and respond by releasing both antimicrobials to combat Gc and proinflammatory cytokines to alert the immune system. The cytokine gradient favors recruitment of large numbers of neutrophils to the site of infection. Neutrophils mount a robust immune response characterized by phagocytosis of Gc, production of reactive oxygen species by NADPH oxidase, formation of NETs and release of antimicrobials that are stored in cytoplasmic granules. Despite the potent immune response, Gc resists complete clearance, which suggests that Gc has defensive mechanisms against killing by neutrophils and host-derived antimicrobials at mucosal surfaces. Herein, I investigated the contribution of two important virulence factors, an LOS-modifying enzyme LptA and a clinically relevant antimicrobial efflux pump MtrCDE, for protection of Gc during interactions with human neutrophils. In addition, I explored the antigonococcal activity of a host-derived antimicrobial, β-defensin 22, and characterized its potential for therapeutic application.Gc interactions with neutrophils are not well understood. Though several important observations by our lab and other groups have advanced our understanding, there are still many unknowns.