Gonococcal Infection and Gene Expression in Female Mice

Project Details

Description

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/permeabilityincreasing protein (BPI), kill Gc in vitro. Whether these factors challenge Gc during infection has not been tested in PMNs from mice that are genetically deficient in these factors or in an infection model. In the current funding period we showed that Gc sialyltransferase (Lst) plays a strong role in evasion of PMN killing 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-MtrDMtrE 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 a dramatically different mechanism 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 Gc 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 mice that are deficient in CG or CRAMP will be tested for their killing activity against Gc, and infections by wild type Gc and an isogenic lst mutant will be compared in CG or CRAMP knock out mice versus normal mice of the same strain background. 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. To determine if CRAMP plays a role in the observed attenuation or fitness advantages exhibited by MtrCDE-deficient Gc or mtrR locus mutants, respectively, normal and CRAMP-deficient mice will be used in PMN killing assays and murine infection experiments. 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 oligodeoxynucleotides (PS-ODNs) that target the mtrC transcript will be tested as a potential therapy for increasing Gc susceptibility to APs, antibiotics, and PMN killing.
StatusFinished
Effective start/end date26/09/0931/05/10

Funding

  • NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES: $274,742.00