Han, M.-L. | bioRxiv Science

bioRxiv2025

Characterisation of SpoT-disruption induced metabolic shift in bacteria

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. IMPORTANCEPolymyxins, 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.

microbiology↗

bioRxiv2024

Transcriptomic interplay between Acinetobacter baumannii, human macrophage and polymyxin

Optimization of antibiotic therapy has been hindered by our dearth of understanding on the mechanism of the host-pathogen-drug interactions. Here, we employed dual RNA-sequencing to examine transcriptomic perturbations in response to polymyxin B in a co-culture infection model of Acinetobacter baumannii and human macrophages. Our findings revealed that polymyxin B treatment induced significant transcriptomic response in macrophage-interacting A. baumannii, exacerbating bacterial oxidative stress, disrupting metal homeostasis, affecting osmoadaptation, triggering stringent stress response, and influencing pathogenic factors. Moreover, infected macrophages adapt heme catabolism, coagulation cascade, and hypoxia-inducible signaling to confront bacterial invasion. Disrupting rcnB, ompW, and traR/dksA genes in A. baumannii impairs metal homeostasis, osmotic stress defense and stringent responses, thereby enhancing antibacterial killing by polymyxin. These findings shed light on the global stress adaptations at the network level during host-pathogen-drug interactions, revealing promising therapeutic targets for further investigation. IMPORTANCEIn the context of the development of bacterial resistance during the course of antibiotic therapy, the role of macrophages in shaping bacterial response to antibiotic killing remains enigmatic. Herein we employed dual RNA-sequencing and an in vitro tripartite model to delve into the unexplored transcriptional networks of the Acinetobacter baumannii-macrophage-polymyxin axis. Our findings uncovered the potential synergy between macrophages and polymyxin B which appear to act in co-operation to disrupt multiple stress tolerance mechanisms in A. baumannii. Notably, we discovered the critical roles of bacterial nickel/cobalt homeostasis (rcnB family), osmotic stress defense (ompW family), and stringent response regulator (traR/dksA C4-type zinc finger) in tolerating the last-line antibiotic polymyxin B. Our findings may lead to potential targets for the development of novel therapeutics against the problematic pathogen A. baumannii.

microbiology↗