Research

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 'floxes' out 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.

PrfA Activation

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 Sensing

Spx-family proteins are conserved across Gram positive bacteria and regulate genes in response to redox stress. L. monocytogenes encodes two spx paralogues, spxA1 and spxA2. We found that while ∆spxA1 mutants cannot replicate in aerobic broth in the laboratory, they can replicate in the cytosol of host cells. Ongoing experiments aim to identify the genes that are regulated by SpxA1 during infection and the host signals that are sensed.

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