PROJECT ABSTRACT Patients with chronic wound infections have a rich microbial community and several bacterial species have been identified as the main drivers of persistent disease. Two of the most prominent wound pathogens are Staphylococcus aureus (including methicillin-resistant S. aureus (MRSA)), and Pseudomonas aeruginosa. The SENTRY antimicrobial surveillance program identified S. three 11%, aureus P. aeruginosa and Enterococcus spp. as the main pathogens causing skin and soft tissue infections (SSTI) in the United States accounting for 44%, and 9% of all SSTI during the years 1998 and 2004. , , Chronic leg ulcers (CLUs) affect 1-2% of people worldwide, are a major cause of prolonged morbidity and have a high recurrence rate. S. aureus and P. aeruginosa are the most common agents isolated from CLUs usually as biofilm resistant to antibiotic therapy. Biofilms are notoriously difficult to eradicate, due to their recalcitrance to antibiotics and ability to evade clearance by the immune system. There is evidence that these two pathogens can exchange specialized metabolites that have the potential to alter cellular behavior and lead to community-wide antibiotic tolerance. Specifically, in vitro co-cultivation studies with S. aureus increased Pseudomonas quinolone gene expression that regulates the expression of several quorum-sensing dependent P. aeruginosa virulence factors and iron acquisition systems. As the prevalence of MRSA increases in the US, new strategies are necessary to treat chronic and recurrent infections. Therefore, the long-term goal is to develop alternative treatment options for chronic wound patients infected by S. aureus and P. aeruginosa. The objective of this proposal is to determine the biological consequences of MRSA modification of P. aeruginosa pyochelin. The rationale underlying this proposal is our preliminary findings that S. aureus and P. aeruginosa act synergistically, which may in part explain why co- infections with these two pathogens lead to worse outcomes. In preliminary imaging mass spectrometry studies, we determined that a novel methylated pyochelin derivative is produced during the interaction of MRSA and P. aeruginosa. We identified the MRSA gene responsible and we can detect this new metabolite in a mouse wound infection model. Our initial studies confirm that the pyochelin originates from P. aeruginosa and then is enzymatically converted by MRSA into a new methylated form. We hypothesize that MRSA methylates pyochelin to compete in a polymicrobial environment. In order to test this hypothesis we will (i) characterize methylated pyochelin generated by MRSA through biochemical, genetic and infection modeling methods and; (ii) determine the functional consequences of methylated pyochelin on P. aeruginosa physiology and pathogenesis. The expected outcomes of this work will increase our understanding the biosynthesis and biological activities of specialized metabolites that drive inter-species interaction...