Two Unique Phosphorylation-Driven Signaling Pathways Crosstalk in Staphylococcus aureus to Modulate the Cell-Wall Charge: Stk1/Stp1 Meets GraSR

Abstract

The Stk1/Stp1 and GraSR signal-transduction pathways are two distinct pathways in Staphylococcus aureus that rely on a reversible phosphorylation process in transducing external stimuli intracellularly. Stk1/Stp1 is an eukaryote-like Ser/Thr kinase phosphatase pair involved in purine biosynthesis, cell-wall metabolism, and autolysis. GraSR is a two-component system involved in resistance to cationic antimicrobial peptides. Both systems are implicated in S. aureus virulence and resistance to cell-wall inhibitors. Our study shows that the response regulator protein GraR undergoes phosphorylation by Stk1 at three threonine residues in the DNA-binding domain. Phosphorylation by Stk1 depends on the structural integrity of GraR as well as the amino acid sequences flanking the phosphorylation sites. Its homologue in Bacillus subtilis , BceR, which harbors two of the three phosphorylation sites in GraR, does not undergo Stk1-dependent phosphorylation. GraR is involved in regulation of the dltABCD operon, the gene products of which add the d-Ala on wall teichoic acid (WTA). Investigation of WTA isolated from the S. aureus RN6390 ΔgraR strain by NMR spectroscopy showed a clear negative effect that graR deletion has on the d-Ala content of WTA. Moreover, complementation of ΔgraR mutant with graR lacking the Stk1 phosphorylation sites mirrors this effect. These findings provide evidence that GraR is a target of Stk1 in vivo and suggest that modification of WTA by d-Ala is modulated by Stk1. The crosstalk between these two otherwise independent signaling pathways may facilitate S. aureus interaction with its environment to modulate processes such as cell growth and division and virulence.

Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

“…Cross-talk between Stk1/Stp1 and GraS/GraR signalling pathways was shown in S. aureus (Fridman et al, 2013). Stk1/Stp1 is a Hanks-type STK/phosphatase pair and GraS/GraR is a TCS which controls the resistance to cationic antimicrobial peptides.…”

Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

“…101 What is known is that GraR is another substrate of the S. aureus PASTA-containing eSTK Stk1. 102 By employing in vitro kinase reactions combined with tandem mass spectrometry analyses, Fridman et al show that GraR phosphorylation by Stk1 occurs on 3 threonine residues, namely Thr128, Thr130 and Thr149 102 (Fig. 5).…”

“…Importantly, experiments reveal that these residues are required for efficient incorporation of D-ala into the bacterial cell wall, which could directly affect the efficiency of vancomycin binding and explain the GraR connection with vancomycin resistance. Additionally, the phosphorylation of these residues enhances the binding of GraR to the vraFG CAMP ABC transporter promoter to control its synthesis 102 (Fig. 4).…”

“…Consistent with the interspecies kinase assays described for eSTK phosphorylation of CcpA (above), despite the similarity of these regulators and the conservation of serine-threonine residues at the same positions found in BceR, both were unable to accept a phosphate from the S. aureus eSTK in vitro. 102 Thus identification of conserved binding sites only indicates a potential for eSTK regulation, and therefore must be treated with caution. Future directions might include BceR and/or VraR variants from different strains to be tested, as sequences between clinical isolates can vary considerably and could therefore 'unmask' substrate specificity though subtle changes in amino acid composition.…”

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action

“…Other studies have suggested that GraX as well as distinct two-component regulatory systems, such as VraSR and NsaSR, are also involved in S. aureus responses to antibiotics (21,22). Likewise, Stk1/Stp1 signaling appears to cross talk with GraSR in modulating S. aureus cell wall charge (23). GraSR may have special relevance to sensing peptide-based agents, including vancomycin (glycopeptide), daptomycin (lipopeptide), and structurally or functionally similar host defense peptides.…”