Neisseria gonorrhoeae (Gc) induces an intense inflammatory response that is characterized by the presence of numerous polymorphonuclear leukocytes (PMNs) with intracellular Gc. PMNs kill Gc in vitro, but not by oxygen-dependent defenses. Purified cathepsin G (CG) and two antimicrobial peptides (APs) found within PMNs, LL37 and the bactericidal/permeability- increasing protein (BPI), kill Gc in vitro. Whether these factors challenge Gc has not been tested in an infection model. In the current funding period we showed that Gc sialyltransferase (Lst) plays a strong a role in evasion of killing by murine PMNs by blocking opsonophagocytic uptake and that sialylation increases survival of Gc within PMNs. Importantly, an lst mutant was significantly attenuated during experimental genital tract infection of female mice. We also demonstrated that the Gc MtrC-MtrD-MtrE active efflux pump system, which plays a role in resistance to macrolide antibiotics, penicillin, and antimicrobial factors of the host innate defense, is critical for murine infection, and mutants that over-express the mtrCDE operon due to mutations in the mtrR repressor locus are more fit than wild type Gc in vivo. Consistent with the MtrC-MtrD-MtrE efflux system facilitating evasion of host defenses, we showed MtrCDE-deficient mutants are more sensitive to the cathelicidin-related antimicrobial protein CRAMP, which is the murine homologue of human LL37. We hypothesize that Gc utilizes two dramatically different mechanisms for evasion of PMN killing, sialylation of the bacterial surface and active efflux of APs. Here we will definitively identify the PMN factor(s) that kill Gc and determine how Lst protects internalized Gc from PMN killing (Aim 1). To this end, the activity of purified CG, LL37, CRAMP, and BPI will be measured against sialylated and nonsialylated Gc. Binding studies will be performed to define the mechanism by which sialylation protects Gc. PMNs from humans and CG- or CRAMP-deficient mice CG will be tested for their killing activity against Gc, and infections by wild type and isogenic lst mutant will be compared in normal mice versus CG or CRAMP knock out mice. BPI-deficient mice will be made for similar studies.
In Aim 2, we will identify the basis for the attenuation of the mtrCDE mutants in the murine model and measure the relative contribution of MtrCDE and Lst in evasion of APs and PMNs in strains that differ in mtrCDE expression or Lst activity. Human PMNs and PMNs from normal and CRAMP-deficient mice will be tested and mouse infection experiments will be performed to determine if CRAMP plays a role in the in vivo phenotypes of MtrCDE-deficient Gc or mtrR locus mutants. The sensitivity of mtrE and mtrR locus mutants to the factors tested in aim 1 will also be determined. The capacity of APs and PMNs to modulate the expression of mtrCDE operon and lst will be examined. We will also construct mutant strains that are designed to test the relative contribution of the MtrC-MtrD- MtrE efflux pump system and Lst in evasion of inhibitory host factors. Finally, phosphorothiorate- modified antisense oligo-deoxynucleotides (PS-ODNs) that target the mtrC transcript will be tested as a potential therapy for increasing Gc susceptibility to APs, antibiotics, and PMN killing.

Public Health Relevance

Gonorrhea is the second leading reportable infection in the U.S. and a major cause of pelvic inflammatory disease and the associated complications of ectopic pregnancy and infertility. Gonorrhea is also a co-factor for HIV transmission. There is no gonorrhea vaccine and the rapid emergence of antibiotic resistant strains is a real concern. This project will lead to a better understanding of how N. gonorrhoeae establishes evades the host innate response, which is likely a critical first step in evading the adaptive immune response. Identifying how this pathogen capitalizes or evades the host immune system may lead to new therapeutic or prophylactic strategies, which could prevent or reduce the incidence of gonorrhea and associated morbidity and mortality.

National Institute of Health (NIH)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Hiltke, Thomas J
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Henry M. Jackson Fdn for the Adv Mil/Med
United States
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