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CRISPRi-mediated silencing of the ABC transporter Ms_5779 compromises cell envelope integrity and impairs
Yuhao Song1, Didi Ban1, Rui Zhang2
1College of Animal Science and Technology, Jilin Agricultural University, Changchun, China.
Background:
Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) poses a severe global health threat, yet the contribution of cell envelope barriers to intrinsic drug tolerance remains poorly understood. The ABC protein Rv0820 is conserved across mycobacteria but functionally uncharacterized, presenting a structural paradox: it lacks canonical transmembrane helices, making conventional efflux pump function mechanistically implausible.
Methods:
Using CRISPR interference (CRISPRi), we silenced Ms_5779-the Mycobacterium smegmatis (M. smegmatis) ortholog of Rv0820-to two graded depths (81% and 69% repression) and subjected the resulting strains to systematic phenotypic interrogation spanning drug susceptibility, cell envelope permeability, biofilm formation, multi-stress tolerance, and macrophage infection.
Results:
Ms_5779 silencing did not affect growth kinetics but markedly disrupted cell envelope integrity, as evidenced by impaired biofilm formation, dissipated membrane potential, and increased ethidium bromide permeability. Silenced strains exhibited broad-spectrum drug hypersusceptibility across six antibiotic classes, with the most striking effect being a 32-fold reduction in norfloxacin MIC, alongside heightened sensitivity to oxidative, acidic, and thermal stress. In a RAW264.7 macrophage infection model, Ms_5779 silencing attenuated intracellular survival, impaired host cholesterol exploitation and foamy macrophage induction, and reduced apoptosis induction.
Conclusion:
Collectively, these findings strongly suggest that Ms_5779/Rv0820 is a critical functional contributor indispensable for the maintenance of cell envelope integrity, presenting a distinct mechanism from a conventional efflux pump. Targeting this essential uncharacterized ABC protein would simultaneously compromise the drug permeability barrier and the intracellular niche, nominating Rv0820 as a mechanistically distinct therapeutic target for anti-tuberculosis drug discovery.
Insights
Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) relies on the uncharacterized ABC protein Rv0820 for cell envelope integrity and drug tolerance. Silencing this protein compromises the cell barrier, increasing susceptibility to antibiotics and stress, and reducing virulence.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Discovery
Background:
- Multidrug-resistant tuberculosis (MDR-TB) is a major global health challenge.
- The role of cell envelope barriers in intrinsic MDR-TB drug tolerance is not well understood.
- The conserved ABC protein Rv0820 in mycobacteria is functionally uncharacterized and structurally paradoxical, lacking typical efflux pump features.
Purpose of the Study:
- To investigate the function of the uncharacterized ABC protein Rv0820 in mycobacteria.
- To determine the contribution of Rv0820 to cell envelope integrity, drug tolerance, and virulence.
- To explore Rv0820 as a potential therapeutic target for tuberculosis.
Main Methods:
- CRISPR interference (CRISPRi) was used to silence the Mycobacterium smegmatis ortholog of Rv0820 (Ms_5779) at two different repression levels.
- Phenotypic analysis included drug susceptibility testing, cell envelope permeability assays, biofilm formation, multi-stress tolerance tests, and macrophage infection models.
- Systematic interrogation of silenced strains was performed to assess various cellular functions and virulence attributes.
Main Results:
- Ms_5779 silencing disrupted cell envelope integrity, leading to impaired biofilm formation, dissipated membrane potential, and increased permeability.
- Silenced strains showed broad-spectrum hypersusceptibility to antibiotics, including a significant increase in susceptibility to norfloxacin.
- Ms_5779 silencing attenuated intracellular survival in macrophages, impaired host cholesterol utilization, and reduced foamy macrophage induction and apoptosis.
Conclusions:
- The ABC protein Ms_5779/Rv0820 is essential for maintaining cell envelope integrity and drug tolerance in mycobacteria, operating via a mechanism distinct from conventional efflux pumps.
- Targeting Rv0820 could simultaneously disrupt the drug permeability barrier and the intracellular survival niche of Mycobacterium tuberculosis.
- Rv0820 represents a novel and mechanistically distinct therapeutic target for developing new anti-tuberculosis drugs.
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