Mechanisms regulating peptidoglycan fragment production

Neisseria gonorrhea infection induces inflammatory responses that are responsible for the symptoms of gonococcal diseases. The
inflammatory response is particularly damaging in female upper reproductive tract tissues, and tubal scarring and loss of ciliated Fallopian tube cells cause infertility and increased risk of ectopic pregnancy or chronic pelvic pain.

The inflammation is induced in response to gonococcal envelope components and metabolites shed from the bacteria as they grow, including endotoxin, porin, heptose-bisphosphate, and peptidoglycan (PG) fragments. Lytic transglycosylases are the enzymes that produce the toxic PG fragments that kill ciliated cells during gonococcal pelvic inflammatory disease.

Our previous studies demonstrated that lytic transglycosylase A (LtgA) produces about half of the toxic PG fragments that are released. This highly active enzyme also produces a vast majority of the PG fragments that are recycled by the bacteria. Two studies identified regulation of ltgA. Bill Shafer’s lab found that ltgA is co-regulated with the multidrug-resistance efflux pump Mtr. These studies demonstrated reduced expression of ltgA in an mtrR mutant. Those results, combined with preliminary results from the Williams lab in this proposal, indicate that MtrR directly acts in transcriptional activation of ltgA and results in increased toxic PG fragment release.

An RNA-Seq analysis by Thomas Rudel’s group characterized the gonococcal transcriptome during in vitro growth. Those studies identified an anti-sense transcript in ltgA, designated ngncR_246. Our preliminary data suggest that the anti-sense message functions to down-regulate ltgA transcript levels. A proteomic analysis we performed on N. gonorrhea mutants that are unable to recycle PG identified a putative helix-turn-helix family transcriptional regulator, NGO1982, as present in increased amounts in these mutants. A knockout of NGO1982 resulted in an 8–9-fold increased mRNA for ltgA. These results suggest that NGO1982 may be a repressor of ltgA transcription.

We hypothesize that regulation of ltgA alters N. gonorrhea cell wall growth and shedding of toxic PG fragments and that changing the levels of LtgA will lead to differences in infection and inflammation. We will test this hypothesis by fulfilling two specific aims:

1. Determine the molecular mechanisms of ltgA regulation by MtrR, NGO1982, and ngncR_246. We will determine transcript and protein levels for LtgA in wild-type and mutant strains. Purified proteins MtrR and NGO1982 will be tested for the ability to bind the ltgA promoter region. Antisense RNA ngncR_246 will be tested for binding to ltgA mRNA, and genetic methods will determine the amounts of full-length ltgA message in mutant and WT strains. We will also determine under what conditions these regulatory mechanisms are deployed, measuring their levels over time during growth, upon infection of specific tissues, or under growth conditions that may be encountered in human infections such as anaerobiosis or iron sequestration.
2. Determine the effects of these mechanisms on growth characteristics, PG structure, PG metabolism, interactions with neutrophil elastase and lysozyme, and infection of Fallopian tube tissue. Mutant and complemented strains for mtrR, NGO1982, and ngncR_246 will be examined for growth defects by multiple methods, and cell morphology will be evaluated by electron microscopy. PG composition will be determined by LC-MS, and glycan strand length will be determined by HPLC. Pulse-chase methods will be used to follow degradation of the cell wall and measure PG fragment release and recycling. Infection of Fallopian tube tissue will be evaluated, enumerating CFUs, examining ciliated cell sloughing, and measuring inflammatory cytokine production.