Gonococcal Nuclease Mediated Escape from Neutrophil Extracellular Traps

University Of Virginia, Charlottesville VA

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Abstract

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Gc) causes the sexually transmitted infection gonorrhea, the second-most prevalent reportable bacterial infection in the United States. Gonorrhea remains a prominent public health problem due to Gc resistance to multiple antibiotics, and completely drug-resistant isolates were identified in 2011. The clinical hallmark of acute gonorrhea is the recruitment of neutrophils to the site of infection. Although neutrophils possess a diverse antimicrobial arsenal, Gc survives in the presence of neutrophils. My laboratory is investigating the cellular and molecular mechanisms used by Gc to resist neutrophil clearance. One approach used by neutrophils to clear extracellular bacteria is the release of neutrophil extracellular traps (NETs). NETs consist of chromatin with associated cationic antimicrobial proteins. Our research team has found that Gc secretes a nuclease with homology to Staphylococcus aureus thermonuclease, called Nuc. We detected increased NET integrity when primary human neutrophils were infected with nuc mutant Gc, compared with parent or nuc complement Gc. Fewer nuc mutant bacteria were recovered after exposure to neutrophils, and nuc mutant recovery was enhanced by adding exogenous DNase. From these observations, we hypothesize that Gc Nuc is a novel virulence factor that enables Gc to escape NETs. In this proposal, we will investigate how Gc Nuc enhances bacterial recovery from NETs. In Specific Aim 1, we will test the ability of recombinant Nuc, as well as Nuc-expressing vs. nuc mutant Gc and their supernatants, to degrade NET DNA released by primary human neutrophils. We will also examine if NETs are present in exudates from individuals with acute gonorrhea and if they are susceptible to Nuc activity. Specific Aim 2 will examine how expression of Nuc allows Gc to escape NETs. We will determine how Nuc expression affects Gc colocalization with NETs formed ex vivo and in humans. We will use dyes that read out the viability of individual bacteria to determine if NETs are bactericidal for Gc, and how Nuc expression affects Gc extracellular viability. The experiments outlined in this proposal will revea the mechanism by which Nuc contributes to Gc pathogenesis. By revealing how extracellular Gc uses Nuc to resist clearance by the innate immune system, these studies may highlight Nuc as a new target for therapeutic intervention against multidrug-resistant gonorrhea.