Mycobacterial Cell Wall Synthesis Inhibitors Cause Lethal ATP Burst
Annanya Shetty1, Thomas Dick2,3
1Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Abstract:
Mycobacterial cell wall inhibitors interfere with targets involved in synthesis of mycolic acids, arabinogalactan and peptidoglycan. These antibiotics corrupt structural integrity of the cell envelope and this is believed to be the cause of drug mediated cell death. Here, we show that treatment of Mycobacterium bovis BCG with these mechanistically different classes of cell wall inhibitors at MIC caused a 4 to 5-fold increase in intrabacterial ATP concentration. This effect on ATP homeostasis was specific to inhibitors of cell wall synthesis and not observed for other anti-tuberculosis drugs. Treating M. bovis BCG with sub-MIC concentrations of the ATP synthase inhibitor bedaquiline or the uncoupler carbonyl cyanide 3-chlorophenylhydrazone suppressed drug induced ATP surge, suggesting that the increase in ATP concentration was due to increased oxidative phosphorylation. Pharmacological suppression of the ATP burst attenuated bactericidal activity of the cell wall-targeting drugs up to 100-fold, suggesting that increased ATP levels are associated with the lethal effect of these antibiotics. Interestingly, inhibition of the ATP burst also suppressed induction of the promoter of the cell envelope stress response operon iniBAC by cell wall inhibitors suggesting a link between ATP surge and iniBAC expression. In conclusion, we show that treatment of M. bovis BCG with inhibitors of cell wall synthesis causes a burst of intrabacterial ATP by increasing oxidative phosphorylation. This ATP surge appears to be required for induction of the iniBAC cell envelope stress response operon and to contribute to drug induced cell death. Hence, this work revealed links between inhibition of cell wall synthesis, oxidative phosphorylation, iniBAC induction and cell death. The identification of the molecular mechanisms linking these processes may reveal novel targets for the discovery of bactericidal antibiotics.
Insights
Cell wall inhibitors increase bacterial ATP levels via oxidative phosphorylation, a process crucial for their lethal effect and stress response induction in Mycobacterium bovis BCG.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycobacterial cell wall inhibitors disrupt the synthesis of essential components like mycolic acids, arabinogalactan, and peptidoglycan.
- This disruption compromises the structural integrity of the mycobacterial cell envelope, leading to cell death.
Purpose of the Study:
- To investigate the effect of cell wall synthesis inhibitors on intracellular ATP levels in Mycobacterium bovis BCG.
- To elucidate the mechanisms underlying the observed changes in ATP homeostasis and their role in antibiotic efficacy.
Main Methods:
- Treatment of Mycobacterium bovis BCG with various cell wall synthesis inhibitors at different concentrations (MIC and sub-MIC).
- Measurement of intrabacterial ATP concentration.
- Assessment of oxidative phosphorylation using ATP synthase inhibitors (bedaquiline) and uncouplers (carbonyl cyanide 3-chlorophenylhydrazone).
- Evaluation of the impact of ATP surge suppression on bactericidal activity and iniBAC operon induction.
Main Results:
- Cell wall inhibitors caused a significant 4-5 fold increase in intrabacterial ATP concentration in M. bovis BCG.
- This ATP surge was specific to cell wall synthesis inhibitors and linked to increased oxidative phosphorylation.
- Pharmacological suppression of the ATP burst attenuated the bactericidal activity of cell wall-targeting drugs by up to 100-fold.
- Inhibition of the ATP burst also suppressed the induction of the iniBAC cell envelope stress response operon.
Conclusions:
- Inhibitors of cell wall synthesis induce a surge in intrabacterial ATP in M. bovis BCG, primarily through increased oxidative phosphorylation.
- This ATP surge is essential for the induction of the iniBAC operon and contributes significantly to the bactericidal effect of these antibiotics.
- The findings reveal a critical link between cell wall synthesis inhibition, oxidative phosphorylation, iniBAC induction, and cell death, highlighting potential new targets for anti-tubercular drug discovery.
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