Molecular Mechanisms Regulating Bacterial Two-component Signaling Systems

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PROJECT SUMMARY/ABSTRACT Staphylococcus aureus infections are a major global health problem and remain a significant health burden to society. In the U.S. alone it is estimated that over three-hundred thousand cases of hospital-associated S. aureus infections occur yearly at the cost of $2 billion. These infections also contribute to pneumonia, sepsis, infective endocarditis, osteomyelitis, and other diseases. S. aureus infections and associated diseases result from secreted virulence factors and the ability of the bacterium to survive in a wide range of environmental niches, including hypoxic conditions. Importantly, growth and virulence are regulated by two-component systems (TCS), which are composed of a membrane-bound sensor histidine kinase (HK) and a cytoplasmic response regulator protein. The kinase senses the extracellular environment, and under the appropriate stimuli transmits a signal across the cell membrane to induce phosphorylation of the response regulator, resulting in changes in gene expression. The staphylococcus respiratory response AB (SrrAB) TCS is activated under hypoxic conditions or in the presence of nitrosative stress and coordinates the regulation of virulence factors, fermentation enzymes, nitric oxide detoxifying enzymes and biofilm formation. In this proposal, the PI will pursue three aims designed to reveal the regulatory mechanisms of the SrrB sensor histidine kinase. The SrrB HK is a transmembrane protein that contains an N-terminal extracellular Cache domain and a cytoplasmic catalytic region (HAMP-PAS-DHpCA) containing a PAS domain. The first aim is to determine the role of the SrrB PAS domain and how binding to heme impacts SrrB function. The second aim will identify ligands that bind to the Cache domain and elucidate its sensing mechanism and role in virulence. The third aim will determine the structural basis for SrrB enzymatic regulation using X-ray crystallography, SAXS and cryogenic electron microscopy of full-length SrrB reconstituted in nanometer-scale lipid discs. Successful completion of these studies will provide the molecular mechanism(s) by which SrrB senses extracellular ligands and cellular redox to regulate catalytic function, and the biological consequences for disrupting this regulation. Our results will have important implications for the design of novel therapeutic strategies targeting the SrrAB TCS to combat antibiotic resistant S. aureus strains.