Conformational features of the Staphylococcus aureus AgrA-promoter interactions rationalize quorum-sensing triggered gene expression

Abstract

The intracellular trigger for the quorum sensing response mechanism in Staphylococcus aureus involves the phosphorylation of the response regulator AgrA by the membrane anchored histidine kinase AgrC. AgrA activates transcription from three promoter sequences (P1–P3). The promoter strength, conditional association of AgrA with these promoter elements and temporal delay in AgrA-mediated changes in gene expression contribute to the diversity of the quorum sensing response in different S. aureus strains. AgrA promoters comprise of imperfect direct repeats of DNA with a consensus sequence- [TA][AC][CA]GTTN[AG][TG]. Here we describe crystal structures of the DNA-binding (LytTR) domain of AgrA with different cognate DNA sequences that reveal a hitherto unanticipated feature of AgrA-DNA interactions. AgrA promoter interactions are asymmetric with fewer interactions at the binding site proximal to the −35 promoter element. Biochemical assays to evaluate AgrA-promoter interactions suggests that phosphorylation induced dimerization of AgrA can compensate for the asymmetry in AgrA-DNA interactions. The structures also provide a basis to rationalize mutations that were noted to alter AgrA activity without affecting protein-DNA interactions. Put together, the structural data, gene expression and mutational analysis reveal that promoter strength and AgrA phosphorylation enable quorum-sensing triggered transcriptional changes leading to a transition from the persistent to virulent phenotype.

Redox Regulation of the Quorum-sensing Transcription Factor AgrA by Coenzyme A

“…It is worth noting in this context that the P3 promoter was sub-optimal (20 bp spacing between the -35 and −10 promoter elements) when compared to the P2 promoter. Indeed, the transcription competent open promoter complex (RPo) occurrs more readily at P2 than at P3 [21]. The finding that CoAlation abrogates SaAgrA-P3 interactions is thus consistent with previous observations that expression of RNAIII, a pleiotropic effector involved in the upregulation of exotoxins such as alpha-haemolysin, is tightly regulated.…”

“…It is worth noting in this context that the P3 promoter was sub-optimal (20 bp spacing between the -35 and −10 promoter elements) when compared to the P2 promoter. Indeed, the transcription competent open promoter complex (RPo) occurrs more readily at P2 than at P3 [21]. The finding that CoAlation abrogates SaAgrA-P3 interactions is thus 1.…”

“…The S. aureus agrA coding sequence was cloned into the pET28b expression vector, and the recombinant protein was overexpressed in E. coli Rosetta (DE3) pLysS cells [21]. Briefly, cells were grown in LB at 37 • C to an optical density of 0.5 at 600 nm (OD 600 ).…”

Quorum Sensing and Toxin Production in Staphylococcus aureus Osteomyelitis: Pathogenesis and Paradox

“…Phosphorylated AgrA has a high affinity for the P2 promoter, which facilitates the continued transcription of agrA-D to produce extracellular AIP. AgrA also binds to the P3 promoter to initiate transcription of RNAIII, although the affinity for this promoter is lower, allowing for AIP production prior to RNAIII transcription [30,31]. Downstream transcriptional and translational regulation of toxin-related genes is mediated, in part, by the RNAIII transcript [32].…”

2-(2-Methyl-2-nitrovinyl)furan but Not Furvina Interfere with Staphylococcus aureus Agr Quorum-Sensing System and Potentiate the Action of Fusidic Acid against Biofilms

“…Docking best poses were then energy minimized to simulate the induced fit effect of binding pocket ligand surrounding residues. Finally, the complex AgrA- compound 2 was aligned with AgrA LytTR domain complexed with P2 (RNAII) (pdb code 3bs1) [ 29 ] and P3 (RNAIII) (pdb code 4xqq) [ 30 ]. In silico docking experiments showed that AgrA-compound 2 complex is stabilized by several interactions with the shallow groove formed by the β10−α2 loop, particularly by the interaction with positively charged residues Lys236, Lys237, Arg233 and polar Asn234.…”