Elucidation of the Molecular Mechanism of Staphylococcus aureus Response to Cell-Wall Damage

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The cell wall is the first line of the bacterial defense against environmental changes and antimicrobial agents. With its selective permeability, mechanic strength and flexibility, the cell wall is essential to the cell growth. As such, the cell wall integrity is the target of a number of antibiotics. The overarching goal of my research is to understand the Staphylococcus aureus response to cell-wall damage. The cell wall in S. aureus consists of a glycopeptide polymer, referred to as peptidoglycan, and a polyanionic polymer covalently attached to peptidoglycan, referred to as wall teichoic acid. The integrity of both polymers is indispensable for bacteria to establish host colonization, infection and initiate an immune response. Genome-based studies on profiling the bacterial response to cell-wall damage have revealed that bacteria in general, and S. aureus in particular, have signal transduction pathways in place to sense and respond to the damage. In particular, a two-component signal transduction system (TCS), VraSR, is involved with the S. aureus response to antibiotics that target peptidoglycan biosynthesis. Another TCS, GraSR, is involved with the modulation of the cell-wall charge in response to cationic antimicrobial peptides. Furthermore, a Ser/Thr kinase/phosphatase signaling pathway, once considered unique to eukaryotes, is implicated in regulation of the cell-wall biosynthesis and sensing cell wall damage. Recently, our group discovered that Stk1 of S. aureus phosphorylates a novel D-amino esterase, FmtA, which modulates the cell-wall charge. Despite the ample information on the involvement of these signal transduction pathways in response to the cell-wall damage, the mechanism(s) of transducing stress intracellularly and mounting the response to cell-wall damage is(are) not fully understood. Moreover, the networking among these signal transducing pathways is being recognized but not fully elucidated and its significance is not understood. The proposed research program aims to: 1) determine the molecular mechanism of signal transduction by VraSR, GraSR and Stk1/Stp; 2) determine the mechanism of networking among them; and 3) determine the molecular mechanism of sensing cell-wall damage by VraSR, GraSR and Stk1/Stp. My laboratory has the established expertise in protein chemistry, molecular biology and microbiology to achieve the above objectives. The target proteins will be isolated and characterized for their activity by gel electrophoresis, and interactions among them will be tested by pull-down assays and isothermal titration calorimetry (ITC). In addition, the target genes will be knocked out to investigate in vivo gene function, using among others Electron Microscopy, and Nuclear Magnetic Resonance. The knowledge anticipated to be gained in this program will be of great interest to the research communities involved with microbial stress response, cell wall biosynthesis and gene regulation.