Screening phage-host receptor interactions using thermal proteome profiling

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The long-term goal of this work is to correlate environmental reservoirs of pathogenic NTM, mine reservoirs for NTM-specific mycobacteriophages from correlated environments, and develop a high-throughput tool to interrogate mycobacteriophage-NTM interactions. The overall objective of this proposal is to increase the discovery rate of new NTM molecular targets of mycobacteriophages to advance novel therapeutics, including phage-based treatments. The focus in this proposal is on Mycobacterium abscessus (MABS). While MABS is the second most common cause of pulmonary infections in the U.S., MABS infections may have higher rates of adverse patient outcomes compared to more common NTM pathogens. The hypothesis of this study is MABS-specific mycobacteriophages sourced from MABS-rich samples can be used to identify novel, phage-based molecular targets for treatment or removal of MABS. The rationale for this project is that the development of MABS-specific mycobacteriophage tools will contribute to quantitative MABS risk assessments and promote alternative treatment strategies for antibiotic-resistant MABS. To achieve the objectives of this proposal and test our hypothesis, we will correlate MABS-rich environments based on abundance and genetic similarity to pathogenic MABS strains using high-throughput sequencing data of environmental, human sputum, and clinically isolated samples. Novel, high throughput sequencing tools are required to capture spatial and temporal variation in MABS subspecies resolution, presence, and abundance. We will achieve this by employing multilocus, metabarcoding of 16S rRNA, rpoB, hsp65, and erm(41). Once environmental MABS reservoirs have been correlated, highly ranked environments will be mined for MABS-specific bacteriophages. Interactions between these mycobacteriophages and NTM organisms will be screened to identify novel NTM-molecular targets using thermal proteome profiling. The corresponding mycobacteriophage proteins and previously published reports for mycobacteriophage proteins with NTM affinity will be used to quantify binding efficiency and determine lysis from without potential. Results from this study can inform immunocompromised patient behavior to avoid high-risk NTM reservoirs and determine if high-throughput sequencing techniques can be applied for species-level resolution of MABS in human sputum. The development of a high-throughput phage/host-protein screening platform would accelerate the identification of phage molecular targets, a potential source for the development of novel NTM antimicrobials.