PROJECT SUMMARY The goal of the proposed research is to understand how prophages regulate mycobacterial antibiotic resistance and alter gene expression in mycobacteria. Drug-resistant infections caused by clinically important mycobacteria continue to be a significant public health burden. Mycobacterium abscessus is an emerging pathogen in cystic fibrosis patients with a treatment success rate of only 45%, and is considered one of the most drug-resistant mycobacteria. Resistant isolates commonly display increased expression of intrinsic antibiotic resistance genes, making drug treatment challenging. M. abscessus isolates are typically lysogens, meaning their genomes carry one or more prophages, integrated viral genomes, that have the potential to regulate intrinsic antibiotic resistance. The mechanism by which prophage alter gene expression and antibiotic resistance in mycobacteria is not yet understood and is the focus of this grant. Expression of the transcriptional regulator whiB7 increases in response to stresses, such as sub- inhibitory concentrations of antibiotics and the intracellular environment of macrophages and plays a critical role in mycobacterial intrinsic antibiotic resistance and survival in macrophages (8). Our lab showed for the first time that prophage contribute to intrinsic antibiotic resistance and increased expression of whiB7, which in turn positively regulates a large set of intrinsic antibiotic resistance genes (8-10). We identified a novel group of prophages found in the genomes of M. abscessus and M. chelonae that belong to cluster MabR (7, 11) and determined that the MabR prophage, McProf, increases M. chelonae resistance to aminoglycosides, cornerstone antibiotic treatments for M. abscessus/chelonae infections (9). We found that mycobacteria carrying McProf have an enhanced whiB7 response particularly when superinfected by a second phage. It is not understood how the prophage McProf interacts with the second prophage to drive changes in whiB7 expression and intrinsic resistance. We hypothesize that genes expressed from the McProf genome detect stress and regulate antibiotic resistance in mycobacteria through the induction of whiB7. To understand the mechanisms of whiB7 regulation in mycobacterial lysogens, we propose characterizing the cellular stresses that enhance the whiB7 response in bacterial lysogens carrying the McProf prophage and identify and characterize McProf genes that upregulate whiB7 in response to stress.