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.
Highlights
Mycobacteria cause a range of infectious diseases
To determine whether any of the anti-mycobacterial drugs cause an increase in intrabacterial ATP concentration, we treated exponentially growing M. bovis BCG with 11 different drugs including inhibitors of cell wall, cofactor, nucleic acid- and protein-synthesis at their Minimum inhibitory concentrations (MIC) concentrations (Table 1)
All tested non-cell wall synthesis inhibitors had either no effect on ATP levels or, in the case of inhibitors of oxidative phosphorylation, reduced intrabacterial ATP concentration as expected (Figure 1). These results show that treatment of M. bovis BCG with different classes of cell wall synthesis inhibitors cause an increase in intrabacterial ATP concentration, not observed upon exposure to non-cell wall inhibitors
Summary
Mycobacteria cause a range of infectious diseases. Most prevalent is tuberculosis lung disease caused by Mycobacterium tuberculosis resulting in more than a million deaths per year (García-Basteiro et al, 2018). Lung disease caused by socalled non-tuberculous mycobacteria (NTM), including Mycobacterium abscesuss, is increasing. Death by ATP (Wu et al, 2018). Mycobacterial infections are difficult to eradicate due to intrinsic and acquired drug resistance (Wu et al, 2018). New drugs are urgently needed to improve the treatments of mycobacterial diseases. For the discovery of new drugs novel targets are needed. One approach to identify new targets is to determine the exact cellular mechanisms how our current antibiotics cause bacterial death
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