Klebsiella pneumoniae protomic response to antibiotics
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.
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
Raw data files acquired by the MS system were processed using the Proteome Discoverer (version 2.2, Thermo Scientific). A workflow using the algorithms Sequest HT was employed. The search database was encompassing the comprehensive KP bacteria protein sequences (downloaded on 9 July 2016, 5126 proteins, Taxonomy 272620) combined with common contaminants proteins. Also, raw data files were loaded into the 1.5.2 version of the MaxQuant software in combination with the Andromeda search engine with the MaxQuant default parameters for database search. For LFQ analysis, “multiplicity” was set to one. Feature matching between raw files was enabled, using a retention time window of 2 min. The false discovery rate (FDR) was set to 0.01 for proteins and peptides, which had to have a minimum length of 6 amino acids. Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Carbamidomethylation of cysteine was set as fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications. The search results from the two technical replicates of each biological replicate were combined and analyzed together.
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