Dataset Information

Tail-specific protease (Tsp) - mediated envelope remodeling and beta-lactam tolerance in Escherichia coli

ABSTRACT: Bacterial persisters are transiently drug-tolerant cells that survive antibiotic treatment without genetic resistance. While certain cytosolic proteases are known to regulate persistence through toxin-antitoxin and stress pathways, the role of envelope-associated proteolysis remains poorly understood. Here, our high-throughput promoter-reporter screen identified several candidate proteases and subsequent antibiotic treatment of knockout strains lacking these degradative genes revealed that Tsp plays a critical role in beta-lactam persistence. Deletion of tsp markedly reduced cell survival to multiple cell-wall-targeting antibiotics. This loss of tsp also compromised cell envelope integrity and caused the accumulation of periplasmic, membrane-associated, and cell-wall-related proteins, including the DD-endopeptidase MepS, as shown by quantitative proteomics. Comprehensive genetic deletion and overexpression analyses revealed that both loss and excessive activity of envelope-associated proteins, particularly MepS, compromise persistence. Moreover, cell envelope permeabilization and antibiotic sensitivity can be independent, as certain envelope perturbations increased permeability without reducing beta-lactam survival. Altogether, our findings support a model in which Tsp-mediated proteolysis helps prevent aberrant accumulation of peptidoglycan-remodeling proteins that would otherwise destabilize the envelope.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Jenet Narzary

LAB HEAD: Mehmet Orman

PROVIDER: PXD078436 | Pride | 2026-06-18

REPOSITORIES: Pride

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			Action	DRS
	Ecoli_MG1655_WT_1_S1-A5_1_4082.d.zip	Other
	Ecoli_MG1655_WT_2_S1-B5_1_4083.d.zip	Other
	Ecoli_MG1655_WT_3_S1-C5_1_4084.d.zip	Other
	Ecoli_MG1655_tsp_1_S1-D5_1_4085.d.zip	Other
	Ecoli_MG1655_tsp_2_S1-E5_1_4086.d.zip	Other

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Project description:Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Gram-positive bacteria by targeting their ribosomes. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of Escherichia coli cells. Given that aminoglycoside uptake relies on the energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells.

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Tail-specific protease (Tsp) - mediated envelope remodeling and beta-lactam tolerance in Escherichia coli

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