Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo.

Caroline Deshayes,Ricardo Núñez Miguel,Robert J Cain,Aitor De Las Heras,Mariela Scortti,Magdalena K Bielecka,José A Vázquez‐Boland,Zbigniew Pietras,Ben F Luisi

doi:10.1111/j.1365-2958.2012.08121.x

Abstract

The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the β-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfAallo substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues.

Highlights

Virulence of the food-borne pathogen Listeria monocytogenes depends on its ability to proliferate intracellularly
We examined the possible involvement of the PrfA N-terminal domain in allosteric signalling based on its structural similarities with the cAMP receptor protein (Crp) cAMP-binding domain
The jelly-roll b-barrel fold that forms most of the PrfA N-terminal domain is a key structural component of the cyclic nucleotide monophosphate-binding domain (CNBD)

Summary

Introduction

Virulence of the food-borne pathogen Listeria monocytogenes depends on its ability to proliferate intracellularly This is mediated by nine bacterial genes encoding products that promote host cell invasion (internalins InlA and InlB), escape from the phagocytic vacuole [pore-forming toxin listeriolysin O (LLO), phospholipases C PlcA and PlcB, protease Mpl], rapid replication in the cytosol (sugar phosphate permease Hpt) and direct cell-to-cell spread (actin-polymerizing surface protein ActA, small internalin InlC) (Vazquez-Boland et al, 2001; Portnoy et al, 2002; Hamon et al, 2006; Cossart, 2011). Its three-dimensional structure is similar to that of Escherichia coli Crp (aka catabolite activator protein, CAP) (Eiting et al, 2005) Both PrfA and Crp are homodimers with protomers organized in two domains: N-terminal, with an eight-stranded antiparallel jelly-roll b-barrel, which in Crp accommodates the binding pocket for its allosteric activator, cAMP; and C-terminal, in which the DNA-binding helix–turn–helix (HTH) motif is located. PrfA differs from Crp in that it possesses a unique 25-residue C-terminal extension comprising three short a-helices (aGHI) wedged between the N- and C-terminal

Results

Discussion

Conclusion