Background: The (p)ppGpp synthetase/guanosine-3’,5’-bis(diphosphate) 3’-pyrophosphohydrolase SpoT is a bifunctional bacterial enzyme that globally regulates physiological adaptation to various environmental stressors including antibiotics. We have previously demonstrated that multidrug-resistant (MDR) Acinetobacter baumannii upregulated SpoT in response to polymyxin treatment and disruption of the spoT gene resulted in enhanced polymyxin B susceptibility.
Aim: To elucidate the role of SpoT in rewiring the A. baumannii metabolome in response to polymyxin B treatment.
Methods: We performed comparative untargeted metabolomics of a spoT-disrupted A. baumannii AB5075 mutant versus the wild-type and compared the metabolome differences at 1 and 4 h post 2 mg/L polymyxin B treatment.
Results: The comparative metabolomics data revealed that following polymyxin B treatment the metabolome of the spoT mutant at 1 h was significantly depleted in guanine-based purines (i.e., guanosine triphosphate, guanosine diphosphate). This is reflective of impaired (p)ppGpp hydrolysis activity, possibly leading to lethal accumulation of the alarmone (p)ppGpp. Moreover, we observed a significant depletion of phosphoenolpyruvate, succinate, coenzyme A, nicotinamide adenine diphosphate, adenosine diphosphate, glutathione, gamma-L-glutamyl-L-cysteine and (R)-S-lactoylglutathione in the spoT mutant compared to wild-type at 1 h post treatment. Intriguingly, a distinctive time-dependent perturbation of fatty acids was evident following polymyxin B treatment in the spoT mutant relative to the wild-type, suggesting that the mutant maybe more reactive to the outer membrane disruption activity of polymyxin B.
Conclusions: Taken together, our findings highlighted the role of SpoT in coordinating nucleotide, carbon, energy, membrane homeostasis and antioxidant metabolism in A. baumannii in response to polymyxin B treatment.