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Autophagy-related genes affect drug resistance of mycobacteria by regulating autophagy
Gao Chen1, Bibo Wu1, Mengzheng Wu1
1Department of Infection, Deyang People's Hospital Deyang, Sichuan, China.
Objective:
To investigate the effect of autophagy-related gene (ATG) on the drug resistance of mycobacteria by regulating autophagy.
Methods:
In the present study, primary macrophages were selected as objects of study. The cell lines with ATG13 and ATG6 interference and stable overexpression were constructed with Crisp/Case technique and verified by fluorescence quantitative polymerase chain reaction (PCR) and western blotting, and the qualified cells were used for subsequent experiments. Then the above different mutant and wild-type cells were cultured in Dulbecco's Modified Eagle medium (DMEM) containing fetal bovine serum for 5 h, and Mycobacterium tuberculosis H37Rv was added, followed by co-culture for 4 h. The cells were treated and co-cultured with isoniazid (INH, 0.05 mg/L), rifampicin (RFP, 0.4 mg/L) and ethambutol (EMB, 25 mg/L) for 3 d. Then the cells were sampled and stained with monodansylcadaverine (MDC), and autophagy was observed. Finally, an appropriate number of cells were taken and cultured in the modified L-G medium, and the bacteria were counted.
Results:
The results of fluorescence quantitative PCR and western blotting revealed that the messenger ribonucleic acid (mRNA) transcription levels and protein expression levels of ATG13 and ATG6 in cells significantly declined after using Crisp/Case. The MDC staining showed that ATG13 and ATG6 interference could significantly reduce the number of autophagosomes in cells, while ATG13 and ATG6 overexpression could significantly increase the number of autophagosomes in cells. Compared with wild-type cells, the number of mycobacteria was obviously increased in mycobacterium-infected cells with ATG13 and ATG6 interference after they were treated with INH, RFP and EMB, displaying a significant difference (P<0.05), while the number of mycobacteria was obviously decreased in mycobacterium-infected cells with ATG13 and ATG6 overexpression after they were treated with INH, RFP and EMB, also a significant difference (P<0.05).
Conclusion:
ATG and other autophagy-related genes can affect the drug resistance of mycobacteria through regulating autophagy.
Insights
Autophagy-related genes (ATG) influence mycobacterial drug resistance by modulating autophagy. Manipulating ATG13 and ATG6 levels altered mycobacterial drug susceptibility in macrophages, highlighting their therapeutic potential.
Area of Science:
- Cell Biology
- Microbiology
- Immunology
Background:
- Autophagy plays a crucial role in cellular defense against microbial infections.
- Mycobacterial infections, particularly tuberculosis, pose a significant global health challenge.
- Understanding the mechanisms of drug resistance in mycobacteria is essential for developing effective treatments.
Purpose of the Study:
- To investigate the role of autophagy-related genes (ATG) in modulating the drug resistance of mycobacteria.
- To determine how ATG influences autophagy and subsequent mycobacterial drug susceptibility.
Main Methods:
- Constructed and verified ATG13 and ATG6-manipulated (interference and overexpression) macrophage cell lines using CRISPR/Cas9.
- Infected macrophages with Mycobacterium tuberculosis H37Rv and treated with isoniazid, rifampicin, and ethambutol.
- Assessed autophagy levels via monodansylcadaverine (MDC) staining and quantified intracellular mycobacteria.
Main Results:
- CRISPR/Cas9-mediated manipulation significantly altered ATG13 and ATG6 mRNA and protein levels.
- ATG13 and ATG6 interference reduced autophagosome formation, while overexpression increased it.
- ATG interference led to increased mycobacterial load post-antibiotic treatment, whereas ATG overexpression decreased it.
Conclusions:
- Autophagy-related genes significantly impact mycobacterial drug resistance by regulating the autophagy process.
- Modulating ATG expression presents a potential strategy to enhance the efficacy of anti-mycobacterial drugs.
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