Regulatory mechanisms that impact Neisseria gonorrhoeae survival of host innate immunity and antibiotics: The roles of LptA, TbpBA, and MisR Open Access

Kandler, Justin Lee (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/1c18dg40x?locale=en
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Abstract

Neisseria gonorrhoeae is a Gram-negative bacterium and causes the sexually
transmitted infection, gonorrhea. Gonococci can be cultured from purulent exudate in
symptomatic individuals, which also contains numerous polymorphonuclear cells
(neutrophils). This potent proinflammatory response is effective largely due to the killing
action of cationic antimicrobial peptides (CAMPs) and proteins, but not all gonococci are
killed. To further explore how gonococci survive the innate immune response, we
investigated the importance of a two component regulatory system response regulator,
MisR, for gonococcal resistance to CAMPs. Though loss of MisR did not affect the
function of the LptA lipid A phosphoethanolamine transferase required for gonococcal
resistance to CAMPs, we observed that lptA can be transcribed from two distinct
promoters, and is post-transcriptionally regulated by a phase variable poly-T8 tract
present within the lptA open reading frame. Importantly, lptA-deficient gonococci are
much less fit than WT gonococci in competitive infections of mice and men. RNA-Seq
analysis demonstrated that MisR significantly regulates the transcription of nearly 100
genes (including the transferrin-binding protein genes tbpB and tbpA, which are essential
for in vivo survival). Interestingly, we found a previously unknown RNA species that
impacts tbpBA transcript and TbpBA protein levels by an undefined mechanism.
Phagocytosis of gonococci by human macrophage-like monocytic cells greatly
upregulated transcription of tbpBA (and other iron-responsive genes), and did not kill
100% of the internalized bacteria, suggesting that the iron-limiting environment of the
macrophage interior can be sensed by gonococci to increase survival during the ironlimiting
innate immune response. Loss of MisR increased susceptibility to CAMPs and
aminoglycosides by approximately the same factor (4-8 fold). Furthermore, function (but
not expression) of the MtrCDE antimicrobial efflux pump was impaired by loss of MisR
in both WT and mtrCDE-overexpressor genetic backgrounds, which are common among
gonococcal clinical isolates. We propose that loss of MisR increases membrane
permeability due to misfolded protein accumulation. The characterization herein of a
novel multi-antimicrobial resistance mechanism in gonococci is of special interest in light
of the dwindling number of curative antibiotics for gonorrhea, and the approaching
implementation of an aminoglycoside (gentamicin) as a first-line therapy in the United
States.

Table of Contents

Abstract


Acknowledgments


List of Tables and Figures


Chapter 1: Introduction........................................................................................................1
Chapter 2: Lipid A's structure mediates Neisseria gonorrhoeae fitness during
experimental infection of mice and men................................................................................71
Chapter 3: Neisseria gonorrhoeae modulates iron-limiting innate immune defenses in
macrophages....................................................................................................................77
Chapter 4: Phase-variable expression of lptA modulates the resistance of Neisseria
gonorrhoeae
to cationic antimicrobial peptides.......................................................................94
Chapter 5: Identification of regulatory elements that control expression of the tbpBA
operon in Neisseria gonorrhoeae..........................................................................................99
Chapter 6: Mechanisms and significance of bacterial resistance to human cationic
antimicrobial peptides.......................................................................................................113
Chapter 7: Unpublished Results..........................................................................................184
Chapter 8: Discussion.......................................................................................................209
Chapter 9: Appendix.........................................................................................................236

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