Neisseria gonorrhoeae (Gc) is a Gram-negative bacterium that causes the sexually transmitted disease gonorrhea. With an estimated 78 million cases of gonorrhea annually worldwide, increasing frequency of resistance to all recommended antibiotics, and the lack of a protective vaccine, N. gonorrhoeae is a prominent and growing threat to human health. A hallmark of Gc infection is the influx of neutrophils, but this inflammatory response is unsuccessful in clearing infection. Phase-variable opacity-associated (Opa) proteins on the Gc surface mediate non-opsonic phagocytosis by neutrophils. Expression of Opa proteins like OpaD of strain FA1090, which activates neutrophils by binding to the phagocytic receptors carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-1 and CEACAM-3, significantly decreases Gc survival in the presence of human neutrophils in vitro. Neutrophils respond to OpaD+ Gc by generating reactive oxygen species, releasing toxic granule contents extracellularly, and internalizing the bacteria into a phagolysosome where they are killed by proteases and antimicrobial peptides. Despite being killed by neutrophils in vitro, Opa-expressing Gc survive and even predominate among Gc in neutrophil-rich gonorrheal exudates. As one explanation for these discordant findings, we have found that opsonization with human serum, which is found in inflammatory secretions, enhances Opa+ Gc survival from primary human neutrophils and suppresses the neutrophil oxidative burst. Through ion-exchange chromatography and mass spectrometry, we identified C4-binding protein (C4BP) as the serum component that binds to Opa+ Gc to limit neutrophil activation and increase Gc survival from neutrophils. Remarkably, these effects are independent of complement deposition or complement lytic activity, suggesting a novel mechanism by which binding of C4BP modulates neutrophil functions. In this proposal, we will define how C4BP mediates these effects on Opa+ Gc, by defining the molecular interactions between C4BP and the Gc surface and the cellular mechanisms that limit the response of human neutrophils to Opa+ Gc. Completion of this work will reveal a previously unappreciated way in which Opa+ Gc survives in neutrophil-rich secretions to enable its continued colonization as well as transmission to new hosts. With this knowledge, we predict that the development of therapies that interfere with C4BP binding to Gc will increase the efficacy of neutrophil antigonococcal activity, in addition to enhancing complement lytic activity, thereby improving outcomes of gonorrhea.
Gonorrhea is the second most commonly reportable bacterial infection in the United States. Cases are increasing, there is no vaccine for gonorrhea, and resistance to all recommended antibiotics has emerged. This proposal will investigate how the bacterium that causes gonorrhea, Neisseria gonorrhoeae, recruits complement C4 binding protein from human serum to limit the ability of immune cells to clear infection, which can lead to new approaches for combating drug-resistant gonorrhea.
