Upregulation of PBP1B and LpoB in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in Escherichia coli

Mecillinam (amdinocillin) is a β-lactam antibiotic that inhibits the essential penicillin-binding protein 2 (PBP2). In clinical isolates of Escherichia coli from urinary tract infections, inactivation of the cysB gene (which encodes the main regulator of cysteine biosynthesis, CysB) is the major cause of resistance. How a nonfunctional CysB protein confers resistance is unknown, however, and in this study we wanted to examine the mechanism of resistance.


DA5438
E. coli MG1655 Lab collection, Wild type

Cell Lysis
The samples were homogenized in 250 µl lysis buffer (50 mM Triethylammonium bicarbonate (TEAB; Fluka, Sigma Aldrich) and 2% Sodium dodecyl sulfate (SDS)) with 1 mm silica beads using FastPrep®-24 instrument (MP Biomedicals, OH). The beads were centrifuged at maximum speed for 15 min, the beads were washed with the lysis buffer and centrifuged at maximum speed again. The protein extracts were combined and protein concentration was determined using Pierce™ BCA Protein Assay (Thermo Scientific) and the Benchmark Plus microplate reader (BIO-RAD) with BSA solutions as standards.

Protein Digestion and Labeling
Aliquots containing 50 or 100 µg of each sample were digested with trypsin using the filter-aided sample preparation (FASP) method (3). Briefly, protein samples were reduced with 100 mM dithiothreitol at 60°C for 30 min, transferred on 10 kDa MWCO Nanosep centrifugal filters (Pall Life Sciences, Ann Arbor, USA), wahed with 8M urea solution and alkylated with 10 mM methyl methanethiosulfonate in 50 mM TEAB and 1% sodium deoxycholate. Digestion was performed in 50 mM TEAB, 1% sodium deoxycholate at 37°C in two stages: the samples were incubated with 500 or 1000 ng (trypsin:protein ratio 1:100) of Pierce MS-grade trypsin (Thermo Scientific) for 3h, then 500 or 1000 ng more of trypsin was added and the digestion was performed overnight. The peptides were collected by centrifugation, labelled using TMT 10-plex isobaric mass tagging reagents (Thermo Scientific) according to the manufacturer instructions. The labeled samples were mixed, sodium deoxycholate was removed by acidification with 10% TFA. The combined labeled sample was fractionated using either strong cation exchange chromatography (SCX) or basic reversed-phase chromatography (bRP-LC). SCX was performed on the AKTA chromatography system (GE Healthcare Life Sciences, Sweden) using PolySULFOETHYL A™ column (100x2.1mm, 5µm 300Å, PolyLC Inc., Columbia, USA) at 0.25 ml/min and a gradient from 0 to 20% B in 20 min, to 40% B in 10 min and to 100% B in 10 min; solvent A was 25 mM ammonium formate, pH 2.8 and solvent B was 500 mM ammonium formate, pH 2.8. Eighteen peptidecontaining fractions were desalted using PepClean C18 spin columns (Thermo Fisher Scientific) according to the manufacturer's guidelines, the purified eluates were dried on Speedvac, the dried fractions were reconstituted in 3% acetonitrile, 0.2% formic acid for analysis. The bRP-LC was performed on the Dionex Ultimate 3000 UPLC system (Thermo Fischer Scientific) using the Waters XBridge BEH C18 column (3.0x150 mm, 3.5µm, Waters Corporation, Milford, USA) and the gradient from 3% to 40% solvent B over 17 min, from 40% to 100% B over 5 min, 100% B for 5 min, all at the flowrate of 0.4 ml/min; solvent A was 10 mM ammonium formate in water at pH 10.00, solvent B was 90% acetonitrile, 10% 10 mM ammonium formate in water at pH 10.00. The initial 20 fraction were combined into 8 pooled fractions in the order 5+13, 6+14, 7+15 etc. The pooled fractions were dried on Speedvac and reconstituted in 3% acetonitrile, 0.2% formic acid for analysis.

LC-MS/MS Analysis
Each SCX fraction was analyzed on Q Exactive mass spectrometer interfaced with Easy-nLC 1000 nanoflow liquid chromatography system (both -Thermo Fisher Scientific). Peptides were trapped on the in-house packed trap column (75 µm X 4.5 cm) and separated on the in-house packed analytical column (75 µm X 20 cm) packed with 3 µm Reprosil-Pur C18-AQ particles (Dr. Maisch GmbH, Ammerbuch, Germany). The following gradient was run at 200 nl/min; 7-27 % B-solvent (98% acetonitrile with 0.2% formic acid) over 70 min, 27-40 % B over 5 min, 40-80 % B over 5 min with a final hold at 80 % B for 10 min, using 0.2% formic acid in water as solvent A. Precursor ion scans were performed in a scan range of m/z 400-1600 at 70 000 resolution setting. MS/MS analysis was performed in a data-dependent mode, with the top ten most abundant doubly or multiply charged precursor ions in each MS scan selected for fragmentation by stepped high energy collision dissociation (stepped HCD) of NCEvalue of 30, 40 and 50. The MS2 scans were performed at 35000 resolution (at m/z 200), AGC 1e5 with a maximal injection time of 110 ms, a fixed first mass of m/z 100, an isolation window of 1.6 Da, intensity threshold of 1.1e4 and a dynamic exclusion of 30 seconds. Each bRP-LC fraction was analyzed on Orbitrap Fusion Tribrid mass spectrometer interfaced with Easy-nLC 1000 nanoflow liquid chromatography system (both -Thermo Fisher Scientific). Peptides were trapped on the Acclaim Pepmap 100 C18 trap column (100 µm X 2 cm, particle size 5 µm, Thermo Fischer Scientific) and separated on the in-house packed C18 analytical column (75 µm X 30 cm, particle size 3 µm) using the gradient from 5% to 32% B in 75 min, from 32% to 100% B in 5 min, solvent A was 0.2% formic acid and solvent B was 80% acetonitrile, 0.2% formic acid. Precursor ion mass spectra were recorded at 120 000 resolution, the most intense precursor ions were selected ('top speed' setting with a duty cycle of 3s), fragmented using CID at collision energy setting of 35, spectra and the MS/MS spectra were recorded in ion trap with the maximum injection time of 40 ms and the isolation window of 0.7 Da. Charge states 2 to 7 were selected for fragmentation, dynamic exclusion was set to 45 s with 10 ppm tolerance. MS3 spectra for reporter ion quantitation were recorded at 50 000 resolution with HCD fragmentation at collision energy of 60 using the synchronous precursor selection of the 7 most abundant MS/MS fragments, with the maximum injection time of 100 ms.

Database Search and Quantification
Data analysis was performed using Proteome Discoverer version 1.4 or version 2.1 (Thermo Fisher Scientific). The SCX data was processed with Proteome Discoverer 1.4 using the Swissprot database (November 2014) with Escherichia coli taxonomy filter. Mascot 2.3.2 (Matrix Science) was used as a search engine with precursor mass tolerance of 5 ppm and fragment mass tolerance of 0.2 Da. No missed cleavages were accepted, mono-oxidation on methionine was set as a variable modification, methylthiolation on cysteine and TMT-6 reagent modification on lysine and peptide Nterminus were set as a fixed modification. The target-decoy approach was used for the filtering of the identifications, with the target false discovery rate of 1% as a threshold to filter confident peptide IDs. Reporter ion intensities were quantified in MS2 spectra at 0.02 Da mass tolerance. The resulting ratios were normalized in the Proteome Discoverer 1.4 on the median protein value of 1.0 in each sample. The bRP-LC data was processed with Proteome Discoverer 2.1 using Swissprot database for Escherichia coli K12 (May 2016), supplemented with the mutant sequences and common proteomic contaminants. Mascot 2.5.1 (Matrix Science) was used as a search engine with precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.5 Da. One missed cleavage was accepted, mono-oxidation on methionine was set as a variable modification, methylthiolation on cysteine and TMT-6 reagent modification on lysine and peptide N-terminus were set as a fixed modification. Percolator was used for the validation of identification results, target false discovery rate of 1% was used as a threshold to filter confident peptide identifications. Reporter ion intensities were quantified in MS3 spectra at 0.003 Da mass tolerance. The resulting ratios were normalized in the Proteome Discoverer 2.1 on the median protein value of 1.0 in each sample.