7. Project Summary/Abstract Bacterial pathogens encounter a multitude of chemical and biological insults during infection, including attack by host immune cells. These stimuli shape the evolution of the pathogen and prompt the development of response systems to swiftly counter these hostile environments. Two-component systems (TCSs) are the predominant sensory system for perceiving these stimuli and mediating transcriptional responses to adjust metabolism and virulence factor production to evade or subvert the host response. Not surprisingly, there is a great deal of interest in understanding mechanistically how these TCSs function, and how they regulate their gene targets. The PI’s laboratory studies intersections of metabolism and pathogenesis, with an interest in gene regulation. Several years ago, the PI’s laboratory discovered that the global regulatory protein CodY regulates the production of many of the virulence factors of Staphylococcus aureus indirectly through what is arguably one of the most important virulence regulators in S. aureus – the SaeRS TCS. The SaeRS system has emerged as one of the most heavily studied TCSs in S. aureus, mainly due to the non-canonical architecture of its membrane sensor kinase. In recent years, the PI’s group has reported that the effect of CodY on SaeRS function is multifaceted. CodY acts as a transcriptional repressor to prevent SaeRS activity from becoming unlimited. It also acts post- transcriptionally to upregulate SaeRS activity. Both functions occur when key amino acids are limiting, reducing CodY’s regulatory activity. The PI’s laboratory studies the nutritional regulation of pathogenesis in S. aureus. The long-term goal of the work supported by R01-AI137403 is to understand how metabolism fuels infection, and how metabolism and virulence are intertwined. The PI’s group will continue probe the mechanisms of SaeRS control using comprehensive genetic, molecular biological, biochemical and system-wide approaches. Herein, they propose to 1.) gain insights into the mechanism of SaeRS regulation by branched-chain fatty acids, 2.) to define the pathways used to synthesize these fatty acids that are an essential component of staphylococcal membranes, and 3.) to gain a better understanding of how branched-chain fatty acids are integrated into the overall virulence regulatory network of S. aureus during skin and soft tissue infections. In doing so, the PI’s group will provide the framework for potentially novel anti-virulence strategies.