MassIVE MSV000086971

Complete Public PXD024470

Succinylome-controlled antibiotic resistance in MRSA - Succinylome

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Description

A sucC and a sucD mutant from the Nebraska Transposon Mutant Library (NTML, Strain NE569 and NE1770) were found to be susceptible to cefoxitin and oxacillin when other mutations in TCA cycle genes did not affect their resistance to these antibiotics. The SucC strain (Strain NE569) accumulated Succinyl CoA, which is known to result in higher succinylation rate. We compared the proteome of the sucC mutant in exponential and stationary stages to the one of the WT strain JE2. The global proteome of the following strains were analyzed by TMT-labelled proteomics: Early exponential JE2 (3Hrs), Exponential JE2 (4Hrs), Exponential SucC (4Hrs), Stationary JE2 (10Hrs), Stationary SucC (12Hrs). For each condition, the bacterial pellet was collected and washed twice with PBS. The proteins were extracted by bead beating, 1000 ug of proteins were reduced with DTT, alkylated with IAA and digested with 1:50 (trypsin:protein, w:w) for 3 Hr at 37 C. 100ug of peptides were labelled with TMT11Plex reagents. The samples were pooled and enriched for Succinyl-lysine containing peptides using the PTMScan Succinyl-Lysine Motif Enrichment Kit following the supplier's instructions. The retained fraction was cleaned-up by C18 solid phase extraction. The peptides were concentrated in 15 ul and 5 ul were analyzed by reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Waters nanoEquity UPLC system coupled with a QExactive HF-X mass spectrometer. [doi:10.25345/C5D800] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: proteomics ; antibiotic resistance ; MRSA ; SucC ; succinyl-lysine ; succinylome

Contact

Principal Investigators:
(in alphabetical order) 		Geremy Clair, Pacific Northwest National Laboratory, United States
James P. O'Gara, National University of Ireland, Galway, Ireland
Submitting User: alchemistmatt

Publications

Campbell C, Fingleton C, Zeden MS, Bueno E, Gallagher LA, Shinde D, Ahn J, Olson HM, Fillmore TL, Adkins JN, Razvi F, Bayles KW, Fey PD, Thomas VC, Cava F, Clair GC, O'Gara JP.
Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics.
mBio. 2021 Jun 29;12(3):e0053021. Epub 2021 Jun 29.

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