Naturally occurring polymorphisms in the virulence regulator Rsp modulate Staphylococcus aureus survival in blood and antibiotic susceptibility

This work was funded by the Imperial College President’s Scholarship awarded to A. K; M. T. G.H acknowledges funding from Chief Scientists Office (Grant Number SIRN10); S. J. P thanks the UKCRC Translational Infection Research Initiative and the Medical Research Council (Grant Number G1000803) for funding to cover the original isolate collection; A. M. E acknowledges funding from the Royal Society, Department of Medicine (Imperial College), and from the Imperial NIHR Biomedical Research Centre, Imperial College London; S.W is a recipient of a Wellcome Trust Investigator Award.Nasal colonization by the pathogen Staphylococcus aureus is a risk factor for subsequent infection. Loss of function mutations in the gene encoding the virulence regulator Rsp are associated with the transition of S. aureus from a colonizing isolate to one that causes bacteraemia. Here, we report the identification of several novel activity-altering mutations in rsp detected in clinical isolates, including for the first time, mutations that enhance agr operon activity. We assessed how these mutations affected infection-relevant phenotypes and found loss and enhancement of function mutations to have contrasting effects on S. aureus survival in blood and antibiotic susceptibility. These findings add to the growing body of evidence that suggests S. aureus 'trades off' virulence for the acquisition of traits that benefit survival in the host, and indicates that infection severity and treatment options can be significantly affected by mutations in the virulence regulator rsp.Publisher PDFPeer reviewe

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

Staphylococcus aureus (S. aureus) is an aggressive opportunistic pathogen of prominent virulence and antibiotic resistance. These characteristics are due in part to the accessory gene regulator (agr) quorum-sensing system, which allows for the rapid adaptation of S. aureus to environmental changes and thus promotes virulence and the development of pathogenesis. AgrA is the agr system response regulator that binds to the P2 and P3 promoters and upregulates agr expression. In this study, we reveal that S. aureus AgrA is modified by covalent binding of CoA (CoAlation) in response to oxidative or metabolic stress. The sites of CoAlation were mapped by liquid chromatography tandem mass spectrometry (LC-MS/MS) and revealed that oxidation-sensing Cys199 is modified by CoA. Surface plasmon resonance (SPR) analysis showed an inhibitory effect of CoAlation on the DNA-binding activity, as CoAlated AgrA had significantly lower affinity towards the P2 and P3 promoters than non-CoAlated AgrA. Overall, this study provides novel insights into the mode of transcriptional regulation in S. aureus and further elucidates the link between the quorum-sensing and oxidation-sensing roles of the agr system

Molecular insights into the control of transcription initiation at the Staphylococcus aureus agr operon

Central to the regulation of the diverse array of virulence factors encoded by the opportunistic human pathogen Staphylococcus aureus is the accessory gene regulator (agr) operon. The agr operon contains two transcription units; RNAII, encoding a quorum-sensing system including the master transcriptional activator AgrA, and RNAIII, the effector RNA molecule that regulates virulence gene expression. RNAII and RNAIII are transcribed from the divergent promoters P2 and P3, respectively. Due to the sub-optimal spacer length of the P3 promoter, it is widely believed that transcription from P3 is dependent on AgrA. A fully native S. aureus in vitro transcription system was setup to provide the first insight into the molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, results revealed that, in the absence of AgrA, RNA polymerase (RNAp) can interact with P2 and P3 equally well, however the transcription-competent open promoter complex (RPo) forms more readily at P2 than P3. AgrA was demonstrated to increase the occupancy of P2 and P3 by RNAp, to increase transcription initiation at both promoters, with a more pronounced effect at P3. The P3 promoter is unusual as it has a genuine extended -10 motif and a near to consensus -35 promoter element. "Scrambling" of the -35 element or shortening of the sub-optimal spacer to the optimal 17 nucleotides both significantly increased transcription initiation at P3, by facilitating the rate of isomerisation of the initial RNAp-P3 complexes to the RPo, in a strictly extended -10 promoter element dependent manner. Therefore, two possible mutually inclusive mechanisms by which AgrA activates transcription at the agr operon P3 promoter are proposed. In addition, the involvement of the transcription factor SarA on transcription from p2 and P3 was investigated. Overall, this study provides the first molecular insights into how transcription is regulated at the agr operon in S. aureus.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Sensing DNA Opening in Transcription Using Quenchable Förster Resonance Energy Transfer

Many biological processes, such as gene transcription and replication, involve opening and closing of short regions of double-stranded DNA (dsDNA). Few techniques, however, can study these processes in real time or at the single-molecule level. Here, we present a Förster resonance energy transfer (FRET) assay that monitors the state of DNA (double- vs single-stranded) at a specific region within a DNA fragment, at both the ensemble level and the single-molecule level. The assay utilizes two closely spaced fluorophores: a FRET donor fluorophore (Cy3B) on the first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Because our assay is based on quenching and dequenching FRET processes, i.e., the presence or absence of contact-induced fluorescence quenching, we have named it a “quenchable FRET” assay or “quFRET”. Using lac promoter DNA fragments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation by bacterial RNA polymerase. We also used quFRET to confirm the mode of action of gp2 (a phage-encoded protein that acts as a potent inhibitor of Escherichia coli transcription) and rifampicin (an antibiotic that blocks transcription initiation). Our results demonstrate that quFRET should find numerous applications in many processes involving DNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.

RsbW_Ct associates with σ⁶⁶, but not σ⁵⁴ or σ²⁸.

<p>Interactions between RsbW_Ct and the chlamydial σ-factors were screened by BACTH (A). Mean β-galactosidase activity is displayed in Miller Units (MU). Each box represents the upper/lower quartiles transected by the arithmetic mean (N = 9). Whiskers represent the 5–95% confidence interval (** represents p<0.01, **** represents p<0.0001; One-way ANOVA with Bonferroni’s post-test against the negative control). SPR analysis confirmed interaction of RsbW_Ct and σ⁶⁶. Overlay of sensorgrams from a single flow-cell in which σ⁶⁶ had been immobilized prior to injection of increasing concentrations of RsbW_Ct show a dose-dependent response (B). Flowcells in which σ⁵⁴ and σ²⁸ were immobilized were assessed in parallel. Equilibrium rates were calculated by curve fitting and these values were transformed by the theoretical maximum binding for each ligand (Rmax). Percent of Rmax was plotted as a function of RsbW_Ct concentration (C). Error bars (typically miniscule) represent the 95% confidence interval of the Req derived from curve fitting analysis. The overlay and Req vs. [RsbW_Ct] plots are representative of 3 experiments.</p