Characterisation of SpoT-disruption induced metabolic shift in bacteria

ABSTRACT SpoT, a pivotal stringent response regulatory enzyme, serves a dual function of both synthesizing and hydrolyzing the stress alarmones ppGpp and pppGpp, collectively known as (p)ppGpp. These alarmones act as global regulators, governing bacterial metabolic and physiological adaptation to diverse environmental stresses. Our previous investigation revealed that multidrug-resistant Acinetobacter baumannii notably upregulates SpoT in response to polymyxin treatment. Intriguingly, disrupting spoT gene enhances polymyxin killing. To comprehend SpoT metabolic regulatory function in mediating polymyxin tolerance, we conducted untargeted metabolomics, comparing metabolic perturbations upon polymyxin B (PMB) treatment between a spoT -disrupted A. baumannii mutant and its wild-type counterpart. Depletion of guanine-based purines GTP and GDP in the PMB-treated spoT -disrupted mutant suggests impaired (p)ppGpp hydrolysis, potentially leading to lethal accumulation of these molecules. PMB also induced more pronounced depletion of carbon sources (i.e., phosphoenolpyruvate, succinate, coenzyme A), energy metabolites (i.e., NADH biosynthesis pathway, ADP), amino acids, and the antioxidative system (i.e., glutathione, gamma-L-glutamyl-L-cysteine, ( R )- S -lactoylglutathione) in the spoT- disrupted mutant. Interestingly, a distinctive time-dependent perturbation of fatty acyls was also observed following polymyxin treatment, with multiple fatty acyl conjugates significantly elevated in the spoT -disrupted mutant at 1 hour. However, this situation reversed at 4 hours, with more elevated fatty acyl groups in the wild-type compared to the spoT -disrupted mutant, indicating greater and more rapid PMB-induced membrane disruption in the spoT -disrupted mutant. Collectively, our findings suggest a potential role for SpoT in intricately coordinating A. baumannii energy expenditure and metabolite repertoire to ensure optimal functionality in stress tolerance and repair machineries (e.g., glutathione system, fatty acid regulation) following polymyxin treatment. IMPORTANCE Polymyxins, the last-resort antibiotics for multidrug-resistant Gram-negative bacteria, face increasing challenges due to growing tolerance. To tackle Acinetobacter baumannii polymyxin tolerance, we turned to SpoT, a (p)ppGpp synthetase/hydrolase protein. Our study focused on spoT -disrupted mutant, which exhibited heightened polymyxin antibacterial killing, yet the underlying mechanisms remained elusive. Through metabolomics, we unraveled key insights. Enhanced polymyxin killing in spoT -disrupted mutant likely resulted from lethal accumulation of stress alarmones (p)ppGpp, perturbations in carbon and energy metabolism, thiol-based antioxidant system depletion, and disrupted lipidic membrane repair. These findings illuminate the intricate metabolic signaling networks driving polymyxin tolerance in A. baumannii . By untangling pathogen-directed stress tolerance mechanisms, we deepen our understanding and identify potential therapeutic targets. Our discoveries offer new avenues to combat polymyxin resistance, paving the way for more effective treatments against superbugs. Through continued exploration, we can harness this knowledge to develop innovative strategies and overcome the challenges posed by polymyxin tolerance.