Project Summary/Abstract – Dr. Heather Miller, PI The kinase Stk1 controls genes involved in antibiotic resistance, biofilm formation, and toxin expression in virulent strains of Staphylococcus aureus. There is a critical need to determine its mechanism of action. Without this information, there are key details missing about an important tool in minimizing antibiotic resistance. Inhibition of Stk1 in vitro potentiates methicillin-resistant S. aureus (MRSA) to b-lactam antibiotics, making it an attractive target for the development of novel antibiotic adjuvants. However, studies of stk1 deletion mutants have produced contradictory results about this protein’s role in virulence. Considered a master regulator, it is difficult to predict the full consequences of Stk1 inhibition. Additionally, the strain-specific differences in downstream gene expression affected by Stk1 phosphorylation have prevented comprehensive understanding in a medically relevant context. It is not clear yet how Stk1 inhibitors elicit these antibiotic adjuvant effects. The long-term goal of this research is to develop novel anti-virulence treatments to combat persistent and antibiotic resistant bacterial infections. Our overall objectives are to confirm the molecular target of the lead compound in S. aureus, investigate this Stk1 inhibition in clinically relevant strains of MRSA, and develop genetic tools to decode mechanistic details of Stk-1 mediated b-lactam resistance. Our central hypothesis is that our lead compound represents a promising antibiotic adjuvant scaffold that targets the master regulator Stk1. To test this hypothesis, adjuvants will be used as chemical probes to interrogate the Stk1 domain(s) necessary for binding. Comparative transcriptomics will be used to investigate Stk1-mediated gene expression across several strains of MRSA to elucidate differences that could affect development of broadly active inhibitors. Finally, genetic tools will be developed that will systematically mutate phosphorylated substrates to trace Stk1-mediated b-lactam resistance pathways in these clinically relevant MRSA strains. Upon completion of the proposed work, we expect to contribute to the existing knowledge of virulence pathways and their regulatory mechanisms in S. aureus. This work is innovative because the experimental design does not adhere to the status quo. We will not only investigate transcriptome-wide changes across clinically relevant strains, but also with adjuvant molecules alone and in combination with antibiotics to provide a more medically relevant context. These results will have a significant impact as they will afford necessary information for evaluation of Stk1 as a therapeutic target, thereby providing crucial new information for the development of novel antibacterial therapies.