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<p>Abstract</p><p>Listeria monocytogenes is a gram-positive soil saprophytic bacterium
that is capable of causing fatal infection in humans. The main virulence regulator
PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the
expression of essential proteins required for host cell invasion and cell-to-cell
spread. The mechanism of PrfA activation and the identity of its small molecule coactivator
have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares
mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene
expression by binding cAMP, increasing the DNA binding affinity of the protein and
causing a significant DNA bend that facilitates RNA polymerase binding and downstream
gene activation. Our data suggests PrfA activates virulence protein expression through
a mechanism distinct from the canonical Crp activation mechanism that involves a combination
of cysteine residue reduction and glutathione (GSH) binding. </p><p>Listeria lacking
glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when
the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly,
Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine
(Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double
mutant strains are avirulent. DNA-binding affinity, measured through fluorescence
polarization assays, indicate reduction of the cysteine side chains is sufficient
to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar
affinity. Oxidized PrfA binds the promoters poorly. </p><p>Unexpectedly, Quad also
binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role
in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH
at physiologically relevant and comparable affinities, however GSH did not affect
DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type
PrfA differ structurally upon binding GSH, which supports the in vivo difference in
infection between the regulator and its mutant. </p><p>Structures of PrfA in complex
with cognate DNA, determined through X-ray crystallography, further support the disparity
between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound
to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation
when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding
data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.
</p><p>From these results, we hypothesize a two-step activation mechanism wherein
PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular
release and binds DNA, causing a slight bend in the promoter and small increase in
transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24
likely visualize this intermediate complex. Increasing concentrations of GSH shift
the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and
is hypothesized to resemble closely the transcriptionally active structure of the
cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient
in this second step, which explains the decrease in virulence and implicates the cysteine
residues as critical for transcription efficiency. Further structural and biochemical
studies are on-going to clarify this mechanism of activation.</p>
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