<p> <i>Listeria monocytogenes</i> is a Gram-positive intracellular pathogen that is readily amenable to genetic manipulation and for which there are excellent <i>in vitro</i> and <i>in vivo </i> virulence models. These attributes have allowed a thorough examination of the molecular underpinnings of <i>L. monocytogenes</i> pathogenesis, however, there are still a number of major unresolved questions that remain to be answered. For example, it has been known for many years that <i> L. monocytogenes</i> rapidly changes its transcriptional profile upon access to the host cytosol, however the host cues and bacterial components that are involved in driving this change have remained continually unanswered. One large piece of evidence came when the long-sought co-factor for the primary virulence regulator, PrfA, was discovered to be the antioxidant tripeptide, glutathione. Glutathione was demonstrated to play a crucial role in the activation of PrfA <i>in vivo</i>— a finding that has since led to two important discoveries that are described herein. First, the activation of PrfA <i>in vitro</i> requires both exogenous glutathione and a metabolic licensing step that can be recapitulated by a chemically defined synthetic media. Second, glutathione also functions as a post-translational regulator of the pore-forming virulence factor, Listeriolysin O (LLO), by reversibly binding via an S-glutathionylation reaction and preventing membrane association of the LLO monomers. These discoveries elucidate numerous regulatory roles for glutathione during infection and describe how <i>L. monocytogenes </i> is able to sense and respond to critical host compartments to mount a successful infection. </p><p> Upon entry to the host cell cytosol, the facultative intracellular pathogen <i> Listeria monocytogenes</i> coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator, PrfA. Here we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium (iLSM) containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge compared to bacteria grown in conventional media. During cultivation <i>in vitro </i>, PrfA activation was completely dependent on intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in iLSM supplemented with oligopeptides, but suppression was relieved by stimulation of the stringent response. These data suggest that cytosolic <i>L. monocytogenes</i> interpret a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression <i>in vivo</i>. </p><p> Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutating the cysteine residue to alanine has minor effects on overall protein function. Thus, the function of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC Listeriolysin O (LLO), secreted by the facultative intracellular pathogen <i>Listeria monocytogenes</i>, was post-translationally modified by a S-glutathionylation at this conserved cysteine residue, and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein <i>in vitro</i>, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation and retained full hemolytic activity. A mutant strain of <i>L. monocytogenes </i> expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from wild-type <i>in vitro</i>, yet was attenuated 4-6 fold in a competitive murine infection model <i>in vivo</i>. This study suggests that S-glutathionylation may represent a mechanism by which CDC family proteins are post-translationally modified and regulated, and help explain an evolutionary pressure behind the highly conserved undecapeptide cysteine.</p><p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:10620349 |
| Date | 09 February 2018 |
| Creators | Portman, Jonathan Lewis |
| Publisher | University of California, Berkeley |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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