Department of Molecular and Cell Biology and Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720

How intracellular pathogens sense their intracellular environment to activate virulence gene expression

Intracellular pathogens collectively cause an enormous amount of world-wide morbidity and mortality.  In my lab, we study Listeria monocytogenes as a very amenable intracellular pathogen as a model for both bacterial pathogenesis and host response.  L. monocytogenes is a Gram-positive, food-borne bacterium that lives a biphasic lifestyle, cycling between a saprophytic existence in the environment and as an intracellular pathogen of mammals. In today‚Äôs lecture, I will ask how L. monocytogenes recognizes and responds to the intracellular environment.  I will begin by describing a novel genetic strategy to identify bacterial mutants that fail to up-regulate a virulence factor (ActA) that is not expressed in the environmental phase of growth but becomes the most highly expressed bacterial protein during intracellular growth.  Most of the mutants identified in this screen were in genes that control redox regulation including a gene that encodes glutathione synthase (gshF).  It turns out that both bacterially and to a lesser degree, host-derived glutathione, is the co-factor that activates the central transcriptional virulence activator, PrfA.  In culture, we could recapitulate this response by the addition of glutathione and surprisingly, by adding one of many different reducing agents to the growth media.  The addition of reducing agents caused the up-regulation of gshF, but we still did not know what the precise biological cue was to activate gshF expression.  GshF mutants were 200-fold less virulent in mice while a mutation in PrfA that is locked in its fully active configuration (referred to as PrfA*) completely rescued virulence of a gshF mutant. This led us to look for additional mutants that formed small plaques in tissue culture, but were rescued by the introduction of a PrfA* mutation.  Among the genes identified in this screen was gloA, which encodes glyoxalase A, a component of a glutathione-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. The gloA mutants grew normally in broth but were sensitive to MG and severely attenuated upon IV infection in mice, but were fully rescued in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, yet gloA mutants had decreased levels of GSH, presumably because GSH reacted irreversibly with MG. These data suggest that MG is a host cue that leads to GshF production and activation of PrfA. 

Location: Zoom Virtual Meeting, details to be emailed to Branch Members

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