(p)ppGpp and DksA play a crucial role in reducing the efficacy of β-lactam antibiotics by modulating bacterial membrane permeability.

IF 3.7 2区 生物学 Q2 MICROBIOLOGY
Microbiology spectrum Pub Date : 2025-04-01 Epub Date: 2025-02-24 DOI:10.1128/spectrum.01169-24
Meenal Chawla, Jyoti Verma, Shashi Kumari, Tushar Matta, Tarosi Senapati, Prabhakar Babele, Yashwant Kumar, Rupak K Bhadra, Bhabatosh Das
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引用次数: 0

Abstract

The key signaling molecules in the bacterial stress-sensing pathway, the alarmone (p)ppGpp and the transcription factor DksA, play a crucial role in bacterial survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. The effects of threshold or elevated concentrations of (p)ppGpp on cellular metabolites and proteins, both in the presence and absence of DksA, have not yet been comprehensively studied in V. cholerae or other bacteria. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed that the N16:ΔrelAΔrelVΔspoTΔdksA V. cholerae mutant, which lacks both (p)ppGpp and DksA, exhibits higher sensitivity to different ꞵ-lactam antibiotics compared with the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the N16:ΔrelAΔrelVΔspoT, N16:ΔdksA, and N16:ΔrelAΔrelVΔspoTΔdksA V. cholerae strains. Furthermore, the mutant strains displayed enhanced inner and outer membrane permeabilities in comparison to the WT strains. These results correlate with V. cholerae's tolerance and survival against β-lactam antibiotics and may inform the development of adjuvants that inhibit stringent response modulators.IMPORTANCEThe (p)ppGpp biosynthetic pathway is widely conserved in bacteria. Intracellular levels of (p)ppGpp and the transcription factor DksA play crucial roles in bacterial multiplication and viability in the presence of antibiotics and/or other xenobiotics. The present findings have shown that (p)ppGpp and DksA significantly reduce the efficacy of ꞵ-lactam and other antibiotics by modulating the availability of peptidoglycan and cell membrane-associated metabolites by reducing membrane permeability. Nevertheless, the whole-cell proteome analysis of N16:ΔrelAΔrelVΔspoT, N16:ΔdksA, and N16:ΔrelAΔrelVΔspoTΔdksA strains identified the biosynthetic pathways and associated enzymes that are directly modulated by the stringent response effector molecules. Thus, the (p)ppGpp metabolic pathways and DksA could be a potential target for increasing the efficacy of antibiotics and developing antibiotic adjuvants.

(p)ppGpp和DksA通过调节细菌膜通透性,在降低β-内酰胺类抗生素的疗效中起关键作用。
细菌应激感知途径中的关键信号分子,即警报酮(p)ppGpp和转录因子DksA,通过调节细胞代谢途径在营养剥夺和暴露于外源物时的细菌生存中起着至关重要的作用。在霍乱弧菌中,(p)ppGpp的代谢仅与三种蛋白的功能相关:RelA、SpoT和RelV。在存在和不存在DksA的情况下,阈值或(p)ppGpp浓度升高对细胞代谢物和蛋白质的影响尚未在霍乱弧菌或其他细菌中得到全面研究。我们设计了霍乱弧菌的基因组,在存在和不存在(p)ppGpp生物合成酶的情况下,开发了DksA无突变体。我们观察到N16:ΔrelAΔrelVΔspoTΔdksA霍乱弧菌突变体缺乏(p)ppGpp和DksA,与野生型(WT)菌株相比,对不同ꞵ-内酰胺类抗生素表现出更高的敏感性。我们的全细胞代谢组学和蛋白质组学分析显示,N16:ΔrelAΔrelVΔspoT, N16:ΔdksA和N16:ΔrelAΔrelVΔspoTΔdksA霍乱弧菌的细胞膜和肽聚糖生物合成途径显着改变。此外,与WT菌株相比,突变菌株表现出增强的内外膜通透性。这些结果与霍乱弧菌对β-内酰胺类抗生素的耐受性和存活有关,并可能为开发抑制严格反应调节剂的佐剂提供信息。(p)ppGpp生物合成途径在细菌中广泛保守。在抗生素和/或其他外源药物存在的情况下,细胞内ppGpp和转录因子DksA水平在细菌增殖和生存中起着至关重要的作用。目前的研究结果表明(p)ppGpp和DksA通过降低膜通透性来调节肽聚糖和细胞膜相关代谢物的可用性,从而显著降低ꞵ-内酰胺和其他抗生素的疗效。然而,通过对N16:ΔrelAΔrelVΔspoT、N16:ΔdksA和N16:ΔrelAΔrelVΔspoTΔdksA菌株的全细胞蛋白质组分析,我们发现了由严格的反应效应分子直接调节的生物合成途径和相关酶。因此,(p)ppGpp代谢途径和DksA可能是提高抗生素疗效和开发抗生素佐剂的潜在靶点。
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来源期刊
Microbiology spectrum
Microbiology spectrum Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
3.20
自引率
5.40%
发文量
1800
期刊介绍: Microbiology Spectrum publishes commissioned review articles on topics in microbiology representing ten content areas: Archaea; Food Microbiology; Bacterial Genetics, Cell Biology, and Physiology; Clinical Microbiology; Environmental Microbiology and Ecology; Eukaryotic Microbes; Genomics, Computational, and Synthetic Microbiology; Immunology; Pathogenesis; and Virology. Reviews are interrelated, with each review linking to other related content. A large board of Microbiology Spectrum editors aids in the development of topics for potential reviews and in the identification of an editor, or editors, who shepherd each collection.
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