Abstract
Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5’ untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection.
Highlights
Intracellular pathogens such as Plasmodium spp., Mycobacterium tuberculosis, Salmonella enterica, Trypanosoma cruzi, and Leishmania spp. are responsible for an overwhelming amount of morbidity and mortality worldwide
We selected for mutants in the model intracellular pathogen Listeria monocytogenes that did not up-regulate virulence factors during infection
The majority of the genes identified are part of the bacterial redox stress response, suggesting that redox changes represent one of the biological cues sensed by L. monocytogenes to regulate its virulence program
Summary
Intracellular pathogens such as Plasmodium spp., Mycobacterium tuberculosis, Salmonella enterica, Trypanosoma cruzi, and Leishmania spp. are responsible for an overwhelming amount of morbidity and mortality worldwide. Successful dissemination of many of these pathogens requires complex life cycles that involve survival and replication in environmental or vector niches. To propagate within their hosts, these pathogens establish a variety of unique intracellular niches that are essential for their pathogenesis [1]. There is considerable understanding of how intracellular pathogens manipulate host cell biology to promote their pathogenesis, less is known about the precise mechanisms by which these pathogens sense their host cell. Such an understanding may lead to targets for therapeutic intervention. In this study we used Listeria monocytogenes as a model system for understanding virulence gene regulation of a facultative intracellular bacterium that transitions from extracellular to intracellular growth
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