Chawla Meenal, Verma Jyoti, Kumari Shashi, Matta Tushar, Senapati Tarosi, Babele Prabhakar, Kumar Yashwant, Bhadra Rupak K, Das Bhabatosh
Functional Genomics Laboratory, Centre for Microbial Research, Translational Health Science and Technology Institute, Faridabad, India.
Non-communicable Diseases Division, Translational Health Science and Technology Institute, Faridabad, India.
Microbiol Spectr. 2025 Apr;13(4):e0116924. doi: 10.1128/spectrum.01169-24. Epub 2025 Feb 24.
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 , (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 or other bacteria. We engineered the genome of to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed that the N16:ΔΔΔΔ 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:ΔΔΔ, N16:Δ, and N16:ΔΔΔΔ strains. Furthermore, the mutant strains displayed enhanced inner and outer membrane permeabilities in comparison to the WT strains. These results correlate with 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:ΔΔΔ, N16:Δ, and N16:ΔΔΔΔ 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.
细菌应激感应途径中的关键信号分子——警报素（p）ppGpp和转录因子DksA，通过调节细胞代谢途径，在细菌营养剥夺和接触外源性物质期间的生存中发挥关键作用。在[具体细菌名称]中，（p）ppGpp代谢仅与三种蛋白质的功能相关：RelA、SpoT和RelV。在[具体细菌名称]或其他细菌中，尚未全面研究在有和没有DksA的情况下，（p）ppGpp阈值浓度或升高浓度对细胞代谢物和蛋白质的影响。我们对[具体细菌名称]的基因组进行改造，以在有和没有（p）ppGpp生物合成酶的情况下构建DksA缺失突变体。我们观察到，既缺乏（p）ppGpp又缺乏DksA的N16:ΔΔΔΔ突变体与野生型（WT）菌株相比，对不同的β-内酰胺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分析表明，N16:ΔΔΔ、N16:Δ和N16:ΔΔΔΔ菌株的细胞膜和肽聚糖生物合成途径发生了显著改变。此外，与WT菌株相比，突变菌株的内膜和外膜通透性增强。这些结果与[具体细菌名称]对β-内酰胺抗生素的耐受性和存活相关，并可能为开发抑制严格反应调节剂的佐剂提供信息。
重要性
（p）ppGpp生物合成途径在细菌中广泛保守。在存在抗生素和/或其他外源性物质的情况下，细胞内（p）ppGpp水平和转录因子DksA在细菌繁殖和生存能力中起关键作用。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β-内酰胺和其他抗生素的疗效。然而，对N16:ΔΔΔ、N16:Δ和N16:ΔΔΔΔ菌株的全细胞蛋白质组分析确定了由严格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。
