Disk Diffusion Assays
A. baumannii 19606 was grown in nutrient broth at 37 oC overnight. The culture was diluted to 0.5 McFarland standard (1.5x108 CFU/mL) and 100 µL was spread onto Mueller-Hinton agar. Appropriate amounts on antibiotic were added to 6 mm disks in accordance to Clinical and Laboratory Standards Institute (CLSI). Plates were incubated at 37 oC for 18 h before zones of inhibition were determined. For subsequent exposure, bacteria were collected along the zone of inhibition of a disk, and re-cultured in nutrient broth. Cells were prepared identically, however, each disk diffusion assay plate only had antibiotic disks (3x) matching that from the disk creating the zone of inhibition the bacteria were collected from. Subsequent assays were carried out until mutations allowing for resistance to occur appeared, typically in 2–3 passages.
Culture Conditions for A. baumannii β-lactamase Expression
Acinetobacter baumannii (ATCC 19606) was grown at 37 oC in nutrient broth overnight and diluted to 0.5 McFarland standard (1.5x108 CFU/mL). To induce expression of β-lactamases, cultures of A. baumannii were spread on nutrient agar plates containing sub-inhibitory concentrations of antibiotic for colony isolation using the streak method and incubated at 37 oC for 24 h. Colonies were selected and suspended in 1 liter of nutrient broth with 5 µM of the same antibiotic it was selected for and grown for 72 h with shaking at 37 oC.
Supernatant Collection and Purification
The one-liter cultures previously described were used for analyzing the β-lactamase production induced by the antibiotic present in the media. After 72 h incubation, the media was centrifuged twice (8000 x g, 10 min). We filtered the clarified supernatant through a 0.2µm syringe filter to remove any remaining bacteria pathogens. The entire supernatant was then concentrated using Millipore Sigma Ultra-15 centrifugal filter units with 10 kDa cutoff (Catalog No. UFC901008).
β-lactamase Activity and Apparent Kinetic Assays
TEM-1 was expressed in Escherichia coli BL21 (DE3) with pET-TEM-1 vector, extracted by osmotic shock, and purified by Zn-chelating chromatography and gel filtration (Sephacryl-100). 50 mM Tris, pH 8.0, and 150 mM NaCl were used for storage.
The purified TEM-1 and β-lactamases in the supernatant activity were determined spectrophotometrically (spectramax-M5-reader) at room temperature in 50mM potassium phosphate buffer (pH 7.0) that contributes to enzyme stability at these volumes in a total volume of 100µl under the conditions with nitrocefin (ε486 nm = 20500 M-1.cm-1) as reporter substrate. Nitrocefin (0.001 to 100 µM) was freshly prepared in 50mM potassium buffer (pH 7.0). The apparent Km and kcat values were derived from at least four independent initial velocity measurements by applying a nonlinear regression fit with the Michaelis-Menten enzyme kinetics model in GraphPad Prism 9.
SDS Gel Electrophoresis and Staining
Concentrated supernatant samples (7.5 µL) were mixed in 1.5 mL microcentrifuge tubes with 2x Laemmli buffer stain, Bio-Rad (2.5 µL). The samples were heated in a water bath for 10 minutes at 100 oC and then centrifuged (12,000 rpm, 10 min). The proteins in antibiotic-selected bacterial pathogens supernatant were separated by SDS-PAGE 10% gradient Novex Tris-glycine resolving gel (Invitrogen, Carlsbad, CA, USA). Following electrophoresis separation at 130V for 1hr, the gel was fixed in 50% MeOH, 10% HoAC, 40% H2O for 20 min. The gels were placed in a plastic tray containing an appropriate volume (100-250mL) of staining solution (0.25% Coomassie Blue R-250) until the gel was a uniform blue color. Staining was completed when the gel was no longer visible in the dye solution. For destaining the gel, 5% MeOH, and 7.5% HoAC in 87.5% dH20 were used until the background was transparent. The gels were stored in 7% HoAC.
Proteomics Analysis
For the protein digestion, the bands were cut to minimize excess polyacrylamide, divided into a number of smaller pieces. The gel pieces washed with water and dehydrated in acetonitrile. The bands were then reduced with DTT and alkylated with iodoacetamide prior to the in-gel digestion. All bands were digested in-gel using trypsin, by adding 5 µL 10 ng/µL trypsin or chymotrypsin in 50 mM ammonium bicarbonate and incubating overnight digestion at room temperature to achieve complete digestion. The peptides that were formed were extracted from the polyacrylamide in two aliquots of 30µL 50% acetonitrile with 5% formic acid. These extracts were combined and evaporated to < 10 µL in Speedvac and then resuspended in 1% acetic acid to make up a final volume of ~ 30 µL for LC-MS analysis. The LC-MS system was a Bruker TimsTof Pro2 Q-Tof mass spectrometry system operating in positive ion mode, coupled with a CaptiveSpray ion source (both from Bruker Daltonik GmbH, Bremen). The HPLC column was a Bruker 15 cm x 75 µm id C18 ReproSil AQ, 1.9 µm, 120 Å reversed-phase capillary chromatography column. One µL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 µL/min were introduced into the source of the mass spectrometer on-line. The digests were analyzed using a Parallel Accumulation–Serial Fragmentation DDA method was used to select precursor ions for fragmentation with a TIMS-MS scan followed by 10 PASEF MS/MS scans. The TIMS-MS survey scan was acquired between 0.60 and 1.6 Vs/cm2 and 100–1,700 m/z with a ramp time of 166 ms. The total cycle time for the PASEF scans was 1.2 seconds and the MS/MS experiments were performed with a collision energies between 20 eV (0.6 Vs/cm2) to 59 eV (1.6 Vs/cm2). Precursors with 2–5 charges were selected with the target value set to 20,000 a.u and intensity threshold to 2,500 a.u. Precursors were dynamically excluded for 0.4 s. The data were analyzed by using all CID spectra collected in the experiment to search an Ab database compiled using Uniprot using the program MSFragger. The parameters for this search include a precursor mass accuracy of 20 ppm and fragment mass accuracy of 0.05 Da, fully tryptic peptides with 2 allowed missed cleavages, oxidized methionine and protein N-terminal acetylation as variable modifications, and carbamidomethylation as a static modification. Protein and peptide identification were validated to 1% FDR using a decoy database strategy.
Multiple-sequence analysis
Our sequence alignment method was used for database search in a straightforward manner. The multiple sequence alignment tools in Schrodinger package ver. 2019-3 based on classic Smith-Waterman algorithm were used. The comparing sequence data base were provided by UniProt and NCBI Protein Data Bank.