Using genetics and multi-scale imaging to understand the mechanisms underlying mycobacteriophage host choice

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PROJECT SUMMARY The arms race between humans and bacterial pathogens is accelerating. There is a clear need for the development of new tools and strategies to confront antibiotic resistance. In recent years, phage have returned as a possible lifeboat when antibiotics fail. Recent successes, including the treatment of a disseminated drug- resistant Mycobacterium abscessus (Mab) infection with a personalized phage cocktail2, motivate the continued refinement of this innovative therapy. Advances in phage engineering have made it possible to modify phages so that they are constitutively bactericidal and with potentially tunable host ranges3. This is appealing as clinical isolates of the M. abscessus complex have variable phage susceptibility profiles – most likely because they are genetically very diverse, with oversized accessory genomes. Furthermore, the multitude of phage defense systems identified in the model organism Mycobacterium smegmatis suggests that orthogonal systems exist in non-tuberculous mycobacteria (NTMs). To be able to specify phage host choice for genetically diverse NTMs, shared phage receptors used for host recognition need to be identified, and conserved phage defense systems need to be characterized. Antibiotics are first-line therapies for bacterial infections. But phage therapies may be able to complement the current standard of care. Many studies have described increased sensitivities to antibiotics in bacteria that have acquired resistance to phage. Combination antibiotic/phage therapies take advantage of this phenomenon, but to formulate more effective combinations, a better understanding is needed of the interplay between drug and phage pharmacokinetics/pharmacodynamics and the underlying genetic relationships driving resistance and susceptibility. Very little is known about the mycobacteriophage determinants of specificity. Even more mysterious, are the host factors that regulate, and function directly as receptors for phage. This project presents an experimental workflow to identify and characterize phage resistance mechanisms in Mab clinical isolates due to 1) dedicated phage defense systems 2) spontaneous mutations and 3) attachment inhibition. In the first aim, forward genetic screens will be employed to identify genes of interest. In the second aim, function and mechanism will be characterized, and in the third aim it will be determined whether acquisition of resistance is associated with changes in antibiotic sensitivity.