Data on proteomic profiling of extracellular vesicles of Acholeplasma laidlawii strains with increased resistance to antibiotics of different classes - ciprofloxacin and tetracycline

To elucidate the regularities of adaptation of the representatives of class Mollicutes to antimicrobials and to identify the promising targets for eradication of mycoplasma infections and contaminations the comparative analysis of the molecular basis of bacterial resistance to antibiotics of different classes is needed. Previously, we presented the data on the whole-genome sequences of Acholeplasma laidlawii strains with different susceptibility to ciprofloxacin (GenBank: LXYB00000000.1), tetracycline (GenBank: NELO00000000.2) and melittin (GenBank: NELN00000000.2) as well as the data on cell and extracellular vesicle proteomes of melittin-resistant A. laidlawii strain [1]. The lists of extracellular vesicle proteins secreted by A. laidlawii strains with the increased resistance to ciprofloxacin (PG8R10) and tetracycline (PG8RTet) are presented here. The vesicle proteome profiles were obtained by 1D SDS-PAGE and liquid chromatography-mass spectrometry.


a b s t r a c t
To elucidate the regularities of adaptation of the representatives of class Mollicutes to antimicrobials and to identify the promising targets for eradication of mycoplasma infections and contaminations the comparative analysis of the molecular basis of bacterial resistance to antibiotics of different classes is needed. Previously, we presented the data on the whole-genome sequences of Acholeplasma laidlawii strains with different susceptibility to ciprofloxacin (GenBank: LXYB0 0 0 0 0 0 0 0.1), tetracycline (GenBank: NELO0 0 0 0 0 0 0 0.2) and melittin (GenBank: NELN0 0 0 0 0 0 0 0.2) as well as the data on cell and extracellular vesicle proteomes of melittinresistant A. laidlawii strain [1] . The lists of extracellular vesicle proteins secreted by A. laidlawii strains with the increased resistance to ciprofloxacin (PG8R 10 ) and tetracycline (PG8R Tet ) are presented here. The vesicle proteome profiles were obtained by 1D SDS-PAGE and liquid chromatographymass spectrometry.
Specifications Table   Subject Microbiology Specific subject area Mollicute proteomics; vesiculome Type of data

Value of the Data
• These data show common and specific proteins transferred by extracellular vesicles of A. laidlawii strains with different susceptibility to tetracycline. • These data show common and specific proteins transferred by extracellular vesicles of A. laidlawii strains with different susceptibility to ciprofloxacin. • The presented data can be useful to the researchers studying molecular mechanisms of mycoplasma resistance to antimicrobials of different classes. • The presented data can be useful to identify the promising targets for eradication of mycoplasma infections and contaminations.

Data Description
Extracellular vesicles were isolated from A. laidlawii strains with different sucseptibility to ciprofloxacin and tetracycline (PG8R 10, PG8R Tet, PG8) ( Fig. 1 ). The absence of bacterial cells in the preparations of vesicles of the strains was confirmed by TEM and/or PCR analysis with  To characterize the vesicular proteome of the A. laidlawii strains with the increased resistance to ciprofloxacin (PG8R 10 ) and tetracycline (PG8R Tet ) 1D SDS-PAGE and LC-MS/MS were performed. As a result of the proteome analysis of extracellular vesicles secreted by tetracyclineand ciprofloxacin-resistant A. laidlawii strains (cultivation without/with antimicrobials), 20/21 and 17/19 proteins were identified, respectively. The proteins were isolated from pure vesicles and separated by one-dimensional electrophoresis. The protein bands were cleaved by trypsin and analyzed by mass-spectrometry. The lists of identified proteins of PG8R Tet and PG8R 10 , their annotation information and functional classification are presented in Supplementary Table 1 and 2, respectively. The lists of the specific vesicular proteins derived from the strains with the increased resistance to ciprofloxacin -PG8R 10 (MIC 20 mcg/ml) and tetracycline -PG8R Tet (MIC 1 mcg/ml) compared to those from their parent strain -PG8B (MIC 0.5 mcg/ml and 0.08 mcg/ml respectively) [2] are summarized in Supplementary Table 3. The common vesicular protein derived from the resistant strains to antimicrobials of different classes (ciprofloxacin, tetracycline, melittin) was indicated in the Supplementary Table 3.

Bacterial strains and culture conditions
The A. laidlawii cells were grown in a modified liquid Edward's medium (EM) at 37 °C [2] . The ciprofloxacin-(PG8R 10 ) and tetracycline-resistant (PG8R Tet ) strains of A. laidlawii were grown in EM with antimicrobials (10 μg ml −1 ciprofloxacin and 0.64 μg ml −1 tetracycline, respectively) and without (one passage in EM) the antimicrobials.

Isolation of extracellular vesicles
The isolation of the A. laidlawii extracellular vesicles was performed as described previously [ 1 , 3 ]. The cell-free vesicles were collected by ultracentrifugation (Beckman Coulter Optima TM MAX-E) and were purified by stepwise density gradient 20%-40% Optiprep ultracentrifugation. The resulting visual banding fraction was removed from the top, diluted and ultracentrifuged. The pellet obtained was dissolved in buffer supplemented with 1 mM PMSF. The absence of bacterial cells in the vesicle preparation was confirmed by transmission electron microscopy, plating on EM and PCR analysis with primers for marker nucleotide sequences of vesicle -16S-23S rRNA gene intergenic spacer region − , ftsZ − , pnp + , tufB + [2] . Transmission electron microscopy TEM was performed as described previously [1] . Mycoplasma cells were collected by centrifugation 7,0 0 0 × g for 15 min. The cell and EV pellets were fixed with 2.5% glutaraldehyde solution prepared on phosphate buffered saline (0.1 M, pH 7.2) for 1.5 h at room temperature. Then the sample was kept in 1% OsO 4 solution prepared in the same buffer supplemented with 25 mg / ml sucrose for 1.5 h. The pellets were washed with phosphate buffered saline (0.1 M, pH 7.2). Dehydration of the samples was carried out in increasing concentrations of alcohols as follows: rinsed in a 30% aqueous solution, then kept for 10 min 3 times at each stage in a 50%, 60%, 70%, 80% solution, in 90% -4 times for 15 min, in pure acetone -4 times for 15 min. After dehydration, the samples were kept in propylene oxide for 45 min after which epoxy resins (Epon 812, DDSA, MNA, DMP-30) and propylene oxide for 1 h at 37 °C was performed in the following ratios: 1:2; 1:1; 2:1; then the sample was placed in a clean resin for 1-1.5 h at 45 °C. Polymerization was carried out at temperatures of 37 °C, 45 °C and 60 °C for 12 h. Ultrathin sections were prepared on a microtome LKB-III (Sweden), mounted on nickel grids, stained with an aqueous solution of uranyl acetate and lead citrate. Samples were analyzed on a JEM-1200EX electron microscope (Japan).
The negative-staining TEM was performed as described in [4] . The extracellular vesicles were placed on the carbon coated grid and incubated at 25 °C for 10 s for adsorption of the proper amount of particles. Then excess solution was removed from the grid by blotting with a filter paper. Then to stain the particles a 2.5 μL of uranyl acetate solution was applied on the grid for 1 min. Excess of uranyl acetate solution was removed by blotting, and the grid was dried in air before imaging. A JEM-1200EX electron microscope was used to acquire a negative stain image.

Atomic force microscopy
Atomic force microscopy (AFM) was performed according to [5] . To prepare samples for AFM A. laidlawii cell cultures were pelleted by centrifugation (10,0 0 0 g for 20 min) at room temperature. The pellets were then resuspended in phosphate buffered saline (0.1 mM, pH 7.2) and centrifuged again (10,0 0 0 g for 15 min). The resulting pellets were resuspended in phosphate-buffered saline (0.1 mM, pH 7.2). Top layer of mica ("Center for Advanced Technologies", Russia) was removed by stationery tape. A cell suspension was applied to the surface of the mica in an amount of 5 μl; 5 min after application, the sample was washed three times with ddiH 2 O and dried. Examination of the samples was carried out at an atomic force microscope Solver P47H (NT-MDT, Russia). Cantilevers (fpN11S) with a needle radius of 10 nm ("Center for Advanced Technologies", Russia) were used. The measurements were carried out in a semi-contact manner. For data processing, the Nova 1.0.26 RC1 software (NT-MDT, Russia) was used.

SDS-PAGE and in-gel digestion
Preparation of vesicular proteins, their separation using 1D SDS-PAGE and tryptic digestion were performed according to [ 3 , 6 ]. The vesicles were mixed with 6x sample buffer (375 mM Tris-HCl pH 6.8, 60% glycerol, 10% SDS, 0.6 M DTT, and 0.01% bromophenol blue) and incubated at 95 °C for 5 min. Samples were run in 12% SDS-PAGE mini-gel until bromophenol blue front was 1 cm from the bottom. The gel was subsequently stained with Coomassie G-250. The protein fractions were cut out from the gel and washed for 15 min in ddiH 2 O and then in a solution containing a 1:1 mixture of acetonitrile and 200 mM NH 4 HCO 3 at 50 °С for 30 min. Protein reduction and alkylation was performed using a 10 mM DTT and 50 mM iodoacetamide respectively [6] . The gels were incubated in acetonitrile, dried and incubated in trypsin Gold (Promega, USA) solution for 60 min at 4 °C. The tryptic digestion was performed overnight at 37 °C. To extract the peptides from the gel, a 0.5% TFA solution was added to each tube and the tubes were incubated in an ultrasonic bath for 10 min. The extract was transferred to a clean tube and dried in a centrifugal evaporator. Samples were analyzed with mass-spectrometry.

LC-MS/MS analysis
LC-MS/MS analysis and identification of proteins were as described previously [2] . The tryptic digested samples were dissolved in a mixture of 98.9% water, 1% methanol, 0.1% formic acid (v/v), loaded to an Acclaim PepMap RSLC column (Thermo Fisher Scientific, USA) and eluted for 5 h, increasing the concentration of a mixture of 99.9% acetonitrile and 0.1% formic acid (v/v) from 2 to 60%. Mass spectra were obtained on a mass spectrometer Maxis Impact (Bruker, Germany) equipped with a HPLC system Dionex Ultimate 30 0 0 Series (Thermo Fisher Scientific, USA). The MS1 mass spectra were obtained as follows: detection of molecular ions was performed in the range was 30 0-20 0 0 m/z, with a signal accumulation time of 250 ms. To obtain MS2 spectra, ions with a signal-to-noise ratio of at least 400 and a charge from 2 to 5 were selected. Ion detection was performed in the range of 20 0-20 0 0 m/z, with a signal accumulation time of 50 ms for each parent ion. The measurement accuracy was 0.6 Da. The resulting MS/MS spectra were analyzed using the MASCOT program [7] . Protein identification was considered reliable when at least two peptides with different amino acid sequences with a pepscore value ≥ 15 were detected.

Analysis of amino acid sequences in silico
The functional annotation of each protein was performed according to EggNOG ( http:// eggnog5.embl.de/ ) [8] .

Ethics Statemen
The work involved bacteria, but did not involve the use of human subjects or animals.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.