Rohde, K. H. | bioRxiv Science

bioRxiv2025

CRISPRi-mediated validation of candidate Myocobacterium abscessus drug targets during host infection

Mycobacterium abscessus (Mab) is a multidrug-resistant nontuberculous mycobacterium that causes debilitating TB-like pulmonary infections for which effective treatment options are lacking. Poor in vivo drug efficacy may stem from altered vulnerability of drug targets driven by host-specific environmental conditions. To enable validation and prioritization of candidate drug targets in vivo, we exploited CRISPRi (CRi) gene silencing in multiple mouse infection models. Inducible silencing of ftsZMab, a previously validated target, and three predicted targets (leuSMab, folPMab, fusAMab) confirmed their essentiality in vitro. We then assessed the in vivo vulnerability of these targets in both immunocompetent C57BL/6N and immunodeficient NSG mice by assessing the impact of CRi silencing on pulmonary mycobacterial burden. In NSG mice, silencing of all four genes led to comparable decreases in Mab burden. However, in C57BL/6N mice, the degree of Mab clearance varied among targets, suggesting that immune pressure may influence the outcome of CRi-mediated gene silencing. Notably, repression of fusAMab yielded a larger decline in mycobacterial burden in C57BL/6N mice despite a lower level of gene silencing in vitro, consistent with enhanced vulnerability of this target. Overall, this study demonstrated that ftsZMab, leuSMab, folPMab, and fusAMab are essential for Mab growth in vitro and, for the first time, validated their vulnerability to inhibition by CRi during infection. These data also identified potential context-dependent target vulnerabilities, which could inform the prioritization of bacterial drug targets and accelerate the development of effective therapeutics for Mab infections.

microbiology↗

bioRxiv2024

Mycobacterium abscessus persistence in the face of Pseudomonas aeruginosa antagonism

Chronic bacterial infections are responsible for significant mortality and morbidity in cystic fibrosis (CF) patients. Pseudomonas aeruginosa (Pa), the dominant CF pathogen, and Mycobacterium abscessus (Mab) both cause persistent pulmonary infections that are difficult to treat. Co-infection by both bacterial pathogens leads to severe disease and poor clinical outcomes. To explore understudied interactions between these two CF pathogens, we employed culture- and molecular-based approaches. In a planktonic co-culture model, growth of Pa continued unimpeded, and it exerted a bacteriostatic effect on Mab. Strikingly, exposure of Mab grown in monoculture to cell-free spent supernatant of Pa resulted in a dramatic, dose-dependent bactericidal effect. Initial characterization indicated that this potent Pa-derived anti-Mab cidal activity was mediated by a heat-sensitive, protease-insensitive soluble factor of >3kDa size. Further analysis demonstrated that expression of this mycobactericidal factor requires LasR, a central regulator of Pa quorum sensing (QS). In contrast, {Delta}LasR Pa was still able to exert a bacteriostatic effect on Mab during co-culture, pointing to additional LasR-independent factors able to antagonize Mab growth. However, the ability of Mab to adapt during co-culture to counter the cidal effects of a LasR regulated factor suggested complex interspecies dynamics. Dual RNAseq analysis of Mab-Pa co-cultures revealed significant transcriptional remodeling of Mab, with differential expression of 68% of Mab genes compared to minimal transcriptional changes in Pa. Transcriptome analysis reflected slowed Mab growth and remodeling of carbon and energy metabolism akin to a non-replicating persister-like phase. A tailored Mab response to Pa was evident by enhanced transcript levels of genes predicted to interfere with alkylquinolone QS signals or provide protection against respiratory toxins and hydrogen cyanide. This is the first study to provide a transcriptome-level view of genetic responses governing the interplay between two important CF pathogens. This will provide insights into interspecies interaction mechanisms which can be targeted to disrupt their communities in a CF lung to improve patient clinical performance. Moreover, identification of a novel antimicrobial natural product with potent cidal activity against Mab will provide a chemical biology tool for identifying new drug targets in Mab.

microbiology↗