(522) Random Mutagenesis Libraries to Define Interactions between M. abscessus and Therapeutic Phages

Background: Opportunistic infections with nontuberculous mycobacteria (NTM) such as Mycobacterium abscessus are common among people with cystic fibrosis (CF). These infections are a significant cause of morbidity and mortality and typically disqualify those infected from lung transplants. Effective treatments for NTM infections are limited due to intrinsic antibiotic resistance and poor patient tolerance of approved antibiotics. One promising alternative treatment is the use of bacteriophages (phages), viruses that infect and kill bacteria. In 2018, a cocktail of three phages was administered to a pediatric cystic fibrosis patient with a disseminated M. abscessus (strain GD01) infection who had received a bilateral lung transplant. The treatment resulted in resolution of skin nodules, wound closure and overall clinical improvement, supporting the use of phages as treatment. However, the three phages used to treat this patient do not infect and kill most other M. abscessus clinical isolates; over 80 M. abscessus strains analyzed thus far vary widely and unpredictably in their phage susceptibilities. Identifying the determinants of M. abscessus phage susceptibility will inform efforts to broaden the application of phage therapy to more people with CF. Here, we sought to identify M. abscessus factors required for phage infection and replication.

Methods: We generated random mutagenesis libraries of M. abscessus strain GD01 using the Himar1 transposon that inserts into TA dinucleotides throughout the bacterial genome. Each library contains a pool of mutants that are disrupted in typically one non-essential gene per cell. Each library was then challenged with phages used in the strain GD01 treatment cocktail; survivors of this challenge are disrupted in genes required for these phages to infect and replicate. Phage challenges were performed on phage-seeded solid media, followed by PCR to identify the disrupted gene, as well as in liquid culture, where the surviving mutants were identified via next generation sequencing (Tn-Seq).

Results: Phage resistant mutants of M. abscessus strain GD01 have mutations in members of a pathway for cell wall lipid synthesis, several enzymes of unknown function, and the virulence factor MgtC. For at least two phages, mutations in the putative tail spike protein can overcome the resistance of certain mutants, suggesting that resistance is conferred early in the phage life cycle at adsorption or DNA injection.

Conclusions: Challenge of random mutagenesis libraries of M. abscessus with therapeutic phages identified genes required for infection and replication of these phages. Future studies will determine the specific roles these gene products play in the phage replication cycle, and will help us to understand M. abscessus phage susceptibility patterns and better design phage therapies for people with CF.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation Postdoctoral Fellowship 00819F221-Wetzel.

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