Dissecting the molecular targets to overcome beta-lactam resistance in M. abscessus complex

Project Summary The prevalence of pulmonary non-tuberculous mycobacterial (NTM) diseases, primarily caused by the Mycobacterium abscessus complex (MABC), continues to increase in the United States and its success correlates with delayed diagnosis, high rates of antibiotic resistance, disease recurrence, and a vulnerable patient population, including cystic fibrosis and transplant patients, and other immunocompromised individuals. Complex regimens of different antibiotic classes have been used to treat chronic infections, but cure rates remain disappointingly low, and the pipeline for antibiotic development is stagnating. We and others have demonstrated that β-lactam combinations, with and without a β-lactam inhibitors, have good in vitro activity; however, understanding the biology this synergy remains a black box, and the redundancy of peptidoglycan modifying enzymes in MABC strains, the targets of β-lactams, adds to the complexity. Toward advancing the science associated with a rational approach to identify targets for effective dual-β-lactam treatment, we developed Himar1 transposon libraries, CRISPR editing tools, and a mouse infection model to identify and evaluate both in vitro and in vivo, synergistic targets that show reduced MICs or improved efficacy to imipenem and other β-lactams. The genetic screen identified three peptidoglycan synthesis genes, including the essential genes cwlM and pbpB and the non-essential ponA2, plus a secreted protein involved in replication (ripC) and a hypothetical gene MAB_0200. Each target showed synergy and reduced MICs to imipenem in vitro and in the mouse infection model. In this proposal, we will extend these approaches and methods to dissect the peptidoglycan synthesis gene-gene, gene-β-lactam and β-lactam-β-lactam interactions, first in the laboratory strain ATCC19977 (Aim 1), and in clinical isolates (Aim 2), to identify and characterize pairs of synergistic targets for their β-lactam response in vitro (Aims 1 and 2), including oral β-lactam compounds, and in both a hollow fiber infection model (HFIM) and mouse infection model (Aim 3). In Aim 1, we will also use biochemical tests to study the essential (cwlM and pbpB) and conditional essential (ponA2, ripC, MAB_0200) proteins and their binding to different β-lactams. Our dynamic HFIM model allows for precise control of drug concentration- time profiles to replicate in vivo exposures. We will use this model to investigate the effects of clinically relevant drug exposures on gene-β-lactam and β-lactam-β-lactam interactions. Additionally, it will help us identify compounds to assess in the novel mouse infection model and confirm the efficacy of dual-β-lactam combinations compared to a standard-of-care regimen (Aim 3). Ultimately, this proposal will provide insights into the molecular mechanisms driving β-lactam synergy, facilitating the rational design of optimal β-lactam combinations, identifying new drug targets, and improving patient care and treatment outcome.