Investigating the mechanism by which prophage alter mycobacterial antibiotic resistance

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 bacteria. Resistant isolates commonly display increased expression of intrinsic antibiotic resistance genes, making drug treatment challenging. A major driver of bacterial antibiotic resistance are the bacteriophages (phages) present in the population, yet the mechanisms are unknown. M. abscessus isolates are typically lysogens, meaning their genomes carry one or more integrated phage genomes (prophages) that have the potential to regulate resistance genes. The goal of the Molloy lab is to understand how prophage regulate mycobacterial antibiotic resistance in the pathogen, M. chelonae. The conserved transcriptional regulator whiB7 is a central regulator of stress tolerance and antibiotic resistance in all mycobacteria.  whiB7 expression increases in response to stresses such as sub-inhibitory concentrations of antibiotics and plays a critical role in mycobacterial intrinsic antibiotic resistance and survival in macrophage. Our preliminary data reveal for the first time that prophage contribute to intrinsic antibiotic resistance and increased expression of the conserved mycobacterial antibiotic resistance gene, whiB7, a critical regulator of intrinsic antibiotic resistance genes. Mycobacteria carrying a single prophage have increased resistance to amikacin. We have established that mycobacteria carrying prophage have an enhanced whiB7 response to sub-lethal concentrations of antibiotics relative to non-lysogens (cells lacking a prophage). We have also found that superinfection by a second phage (a double lysogen) enhances the whiB7 response and intrinsic drug resistance, even in the absence of other stresses known to induce whiB7 expression. Finally, exposure to sub-lethal concentrations of antibiotics combined with superinfection by phage further enhances whiB7 expression and amikacin resistance. We hypothesize that prophage regulate antibiotic resistance and stress tolerance in mycobacteria. Our data suggest that a major component of this regulation is through the induction of the transcriptional activator, whiB7. To understand the mechanisms of whiB7 regulation in mycobacterial lysogens we propose to 1) characterize the cellular stresses that trigger an enhanced whiB7 response in bacteria carrying the primary prophage and 2) identify which primary prophage genes contribute to whiB7 upregulation in response to stress. The outcomes of this project will help us better understand how populations of pathogenic mycobacteria carrying prophage regulate adaptation to the stresses they encounter during infection. treatments.