How are virulence genes activated?
Virulence genes, such as actA, are exclusively activated in the host cytosol during infection. However, the mechanisms by which the bacterium senses its location in the host in order to activate this virulence program are not well-understood. We engineered a strain of L. monocytogenes that kills itself upon activation of virulence genes (the 'suicide strain'). The chromosome contains loxP sites flanking the origin of replication and cre recombinase is under control of the actA promoter, which is activated only in the host cytosol (A). The suicide strain grows well in broth when actA is OFF but upon entering the host cytosol it quickly excises the origin of replication and dies (B). Using growth of this strain as a read-out we can identify both host and bacterial factors required to activate this virulence program.
What host factors are required for infection?
Specific expression of actA in the host cell cytosol suggests the presence of host cell factors which regulate this virulence gene. In addition, host factors required for other stages of the L. monocytogenes lifecycle, such as recognition and uptake by phagocytes, remain uncharacterized. To identify host genes involved in L. monocytogenes virulence, we constructed and screened a genome-wide CRISPR/Cas9 knockout library in immortalized bone marrow-derived macrophages (iBMMs). We engineered a fluorescent reporter strain of L. monocytogenes expressing GFP under a constitutive promoter and RFP driven by the actA promoter. Infection of host cells with the reporter strain allows for simultaneous detection of cytosolic invasion, intracellular replication, and actA expression using flow cytometry. The iBMM library was infected with the fluorescent reporter to identify genes required for phagocytosis of L. monocytogenes and expression of actA in the host cytosol. The lab is currently investigating the results of this screen to understand what host cell signaling pathways regulate L. monocytogenes infection of macrophages.
PrfA is the master virulence gene regulator in L. monocytogenes and as such, directly activates all nine virulence genes. The mechanism by which PrfA is activated specifically during pathogenesis is still being elucidated. Our recent work demonstrated that PrfA activation is a two-step process:
1. PrfA thiols must be reduced for DNA-binding
2. Allosteric binding to glutathione for transcriptional activation.
Several questions remain:
What are the roles of the cysteine residues in PrfA activation?
What is the mechanism of glutathione-mediated PrfA activation?
How is gshF up-regulated during infection?
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Redox stress refers to an imbalance in reductive and oxidative species, which is often caused by reactive oxygen species (ROS). ROS are generated endogenously (by the bacteria) during aerobic respiration and exogenously (by the host respiratory burst) during infection. To combat these assaults, bacteria produce many enzymes that detoxify ROS as well as regulators to sense ROS and induce the appropriate responses as redox stress arises.
Spx-family proteins are conserved across Gram-positive bacteria and regulate genes in response to redox stress. We found that L. monocytogenes spxA1 is unique in that it is essential for aerobic growth in rich media. However, mutants lacking spxA1 are able to access the host cytosol and replicate intracellularly in macrophages, suggesting that the redox environment of the host cytosol may be less stressful than the growth environment in aerobic broth.
To investigate the mechanisms behind the ∆spxA1 phenotypes, we took a global approach and analyzed the SpxA1-dependent transcriptome. We found that SpxA1 is required to activate genes for production of heme and catalase to detoxify endogenous ROS generated in the presence of oxygen. Taken together, we propose a model in which L. monocytogenes ∆spxA1 produces less cytochrome bd than wt, generating ROS from the incomplete electron transport chain. While these ROS would be readily detoxified by wt bacteria, ∆spxA1 is deficient in catalase production and is thus more sensitive to peroxide-mediated toxicity. Finally, the ∆spxA1 mutant is severely limited for heme production, which further contributes to decreased aerobic survival. In summary, this work showed that the severe ∆spxA1 aerobic growth defect is the result of increased ROS in the absence of catalase and heme.