Project PXD010244

PRIDE Assigned Tags:
Biological Dataset Biomedical Dataset

Summary

Title

Decreased antibiotic susceptibility driven by global remodeling of the Klebsiella pneumoniae proteome

Description

Bacteria can circumvent the effect of antibiotics by transitioning to a poorly understood physiological state that does not involve conventional genetic elements of resistance. Here we examine antibiotic susceptibility with a Class A β-lactamase+ invasive strain of Klebsiella pneumoniae that was isolated from a lethal outbreak within laboratory colonies of Chlorocebus aethiops sabaeus monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic. Drug-specific changes to proteomes included proteins for receptor-mediated membrane transport and sugar utilization, central metabolism, and capsule production, while mechanisms common to both antibiotics included elevated scavenging of reactive oxygen species and turnover of misfolded proteins. Resistance to combined antibiotics presented integrated adjustments to protein levels as well as unique drug-specific proteomic features. Our results demonstrate that dampening of Klebsiella pneumoniae susceptibility involves global remodeling of the bacterial proteome to counter the effects of antibiotics and stabilize growth.

Sample Processing Protocol

KpV513 cell pellets were suspended and sonicated for 200s (10s on, 10s off, 10 runs at 20W) in an ice bath of 1 ml ice-cold 25 mM Tris-HCl (pH 7.4, added lysozyme, DNAse I, Protease Inhibitor). Sonicated bacterial suspensions were centrifuged at 5000×g at 10°C for 20 minutes. Supernatants were analyzed by SDS-PAGE (Bio-Rad, Hercules, CA) followed by Coomassie stain, and total protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, CA). 100 µg of total cell lysate was used to generate tryptic digest using the ‘filter-aided sample preparation’ (FASP) protein digestion method. KpV513 proteins in buffer (50 mM ammonium bicarbonate, 8 M urea) were reduced by dithiothretiol and alkylated with 20 mM iodoacetamide in the dark for 1 hr. After diluting into 1 M urea, alkylated proteins were digested with trypsin (1:100 w/w, Promega, Madison, WI) at 37 °C for 12 h and cleaned with Stage-Tip prior to 1D-LC-MS/MS.

Data Processing Protocol

Protein identication and quantification. Raw MS data were collected and analyzed with the Proteome Discoverer software tool (version 2.2, Thermo Scientific) to obtain protein identification data with protein false discovery (FDR) rates at 1%. Two unique peptides were selected for a protein identification. This approach was used in initial analysis stages to ensure that raw datasets had sufficient proteome depth. Sequest was set up to search the Klebsiella pneumonia database, (downloaded on 9 July 2016, 5126 proteins, Taxonomy 272620) with trypsin as the digestion enzyme and up to two missed cleavage sites. Sequest was searched with a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine and N-terminal protein acetylation were specified as variable modifications and then Carbamidomethylation of cysteine specified as fixed modification in the analysis.Protein identication and quantification. Raw MS data were collected and analyzed with the Proteome Discoverer software tool (version 2.2, Thermo Scientific) to obtain protein identification data with protein false discovery (FDR) rates at 1%. Two unique peptides were selected for a protein identification. This approach was used in initial analysis stages to ensure that raw datasets had sufficient proteome depth. Sequest was set up to search the Klebsiella pneumonia database, (downloaded on 9 July 2016, 5126 proteins, Taxonomy 272620) with trypsin as the digestion enzyme and up to two missed cleavage sites. Sequest was searched with a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine and N-terminal protein acetylation were specified as variable modifications and then Carbamidomethylation of cysteine specified as fixed modification in the analysis. Protein identication and quantification. Raw MS data were collected and analyzed with the Proteome Discoverer software tool (version 2.2, Thermo Scientific) to obtain protein identification data with protein false discovery (FDR) rates at 1%. Two unique peptides were selected for a protein identification. This approach was used in initial analysis stages to ensure that raw datasets had sufficient proteome depth. Sequest was set up to search the Klebsiella pneumonia database, (downloaded on 9 July 2016, 5126 proteins, Taxonomy 272620) with trypsin as the digestion enzyme and up to two missed cleavage sites. Sequest was searched with a fragment ion mass tolerance of 0.6 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine and N-terminal protein acetylation were specified as variable modifications and then Carbamidomethylation of cysteine specified as fixed modification in the analysis.

Contact

Moo-Jin Suh, USAMRIID
Suh, Moo-Jin, US Army Medical Research Institute of Infectiouos Diseases, Frederick, MD ( lab head )

Submission Date

27/06/2018

Publication Date

14/03/2019

Tissue

Not available

Instrument

LTQ Orbitrap Elite

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Keasey SL, Suh MJ, Das S, Blancett CD, Zeng X, Andresson T, Sun MG, Ulrich RG. Decreased antibiotic susceptibility driven by global remodeling of the Klebsiella pneumoniae proteome. Mol Cell Proteomics. 2019 PubMed: 30617156