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

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 Delta graR strain by NMR spectroscopy showed a clear negative effect that graR deletion has on the D-Ala content of WTA. Moreover, complementation of Delta graR 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.