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Fine-tuning regulation of (p)ppGpp-driven outer membrane vesicle formation in Acinetobacter baumannii

ABSTRACT: While the roles of (p)ppGpp on cytosolic proteins are well established, its effects on membrane remodeling remain elusive. The translocation of signal recognition particle (SRP)-dependent proteins can be regulated by binding (p)ppGpp to two key GTPase components: FtsY, which interacts with SecYEG, and Ffh, a homolog of SRP54. A (p)ppGpp-specific Broccoli RNA aptamer and the chemometer PyDPA were used to measure the (p)ppGpp levels in the ΔrelA and ΔrelA/ΔspoT strains of Acinetobacter baumannii, confirming a stepwise reduction in (p)ppGpp levels: wild type > ΔrelA > ΔrelA/ΔspoT. The overproduction of outer membrane vesicles (OMVs) in the ΔrelA strain with intermediate (p)ppGpp levels contrasts with reduced production in the ΔrelA/ΔspoT strain, highlighting an unexpected non-linear relationship between OMV production and (p)ppGpp levels. Scanning- and transmission- electron microscope (SEM and TEM) analyses revealed intriguing (p)ppGpp-dependent effects on cell envelope integrity, as the relA mutant showed outer membrane disruption leading to OMV formation, while the relA/spoT mutant maintained an intact outer membrane, suggesting a correlation between (p)ppGpp levels and membrane stability. Western blotting and proteomics analyses revealed significant OmpA accumulation in the inner membrane of the ΔrelA/ΔspoT strain and SRP-dependent inner membrane proteins such as NuoB, NuoL, and TolA in the ΔrelA strain, indicating that (p)ppGpp levels are crucial for regulating membrane protein incorporation in A. baumannii. The regulation of (p)ppGpp levels using the CRISPRi system revealed that outer membrane disruption and OMV formation were most significant at intermediate (p)ppGpp concentrations, highlighting the importance of fine-tuning (p)ppGpp for the regulation of bacterial phenotypes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Acinetobacter Baumannii (strain Atcc 17978 / Cip 53.77 / Lmg 1025 / Ncdc Kc755 / 5377)

SUBMITTER: sunyong Han

LAB HEAD: Woojun Park

PROVIDER: PXD057710 | Pride | 2025-03-15

REPOSITORIES: Pride

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Project description:This work investigates the molecular mechanisms that determine the requirement of (p)ppGpp-mediated GTP homeostasis for viability during prolonged nutritional stress. Initially, we observed that the absence of (p)ppGpp in S. aureus resulted in reduced culturability and elevated levels of guanosine nucleotides (GMP, GDP, and GTP) when cells experienced prolonged starvation during stationary phase. Analysis of metabolic activity and cell membrane function revealed, that (p)ppGpp-deficiency does not immediately lead to cell death but rather to a VBNC state where cell become division-incompetent and display reduced metabolic activity. The cell membrane stayed intact, however (p)ppGpp-deficient cells displayed reduced membrane potential and increased membrane fluidity. Since missing translation inhibition and elevated oxidative stress levels could not be identified as the cause for the VBNC state of the (p)ppGpp-deficient cells, we conducted transcriptomic analysis on S. aureus wildtype and a (p)ppGpp0 mutant, as well as isogenic guaBA-deficient strains, to gain a more thorough understanding of the genetic basis through which (p)ppGpp maintains cell homeostasis during nutritional stress in the stationary phase and the importance of cellular GTP homeostasis to ensure viability. RNAseq analysis revealed that the TCA cycle and electron transport chain, particularly complex IV/the terminal oxidase qoxABCD, were strongly downregulated in the (p)ppGpp-deficient starved cells. By mutating the initiation nucleotide (TSS +1) of the qoxABCD operon, we were able to increase qoxA expression and membrane potential in a (p)ppGpp0 strain during stationary phase starvation. This resulted in increased culturability during prolonged nutrient starvation, demonstrating the necessity of PMF maintenance. Furthermore , we could show that the decreased membrane potential in starved (p)ppGpp-deficient cells contributes to decreased antibiotic tolerance towards oxacillin and ciprofloxacin. In summary, our data suggests, that (p)ppGpp-dependent regulation of cellular GTP homeostasis during nutritional stress is crucial for maintenance of culturability and antibiotic tolerance.

			Action	DRS
	Proteomics_Report_ppGpp_deficient_strains.xlsx	Xlsx
	WT.mzML	Mzml
	relA.mzML	Mzml
	relAspoT.mzML	Mzml

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Fine-tuning regulation of (p)ppGpp-driven outer membrane vesicle formation in Acinetobacter baumannii

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