Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins Vladislav M. Chernov, Alexey A. Mouzykantov, Natalia B. Baranova, Elena S. Medvedeva, Tatiana Yu. Grygorieva, Maxim V. Trushin, Innokentii E. Vishnyakov, Anton V. Sabantsev, Sergei N. Borchsenius, Olga A. Chernova PII: DOI: Reference:

S1874-3919(14)00381-9 doi: 10.1016/j.jprot.2014.07.020 JPROT 1897

To appear in:

Journal of Proteomics

Received date: Revised date: Accepted date:

2 April 2014 18 June 2014 12 July 2014

Please cite this article as: Chernov Vladislav M., Mouzykantov Alexey A., Baranova Natalia B., Medvedeva Elena S., Grygorieva Tatiana Yu., Trushin Maxim V., Vishnyakov Innokentii E., Sabantsev Anton V., Borchsenius Sergei N., Chernova Olga A., Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins, Journal of Proteomics (2014), doi: 10.1016/j.jprot.2014.07.020

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Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins

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Vladislav M. Chernov1,2, Alexey A. Mouzykantov1, Natalia B. Baranova1,

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Elena S. Medvedeva1, Tatiana Yu. Grygorieva1, Maxim V. Trushin1,2, Innokentii E.

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Vishnyakov3, Anton V. Sabantsev4, Sergei N. Borchsenius3, Olga A. Chernova1,2

Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Centre of

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the Russian Academy of Sciences, Kazan, Russia 2

Kazan (Volga region) Federal University, Kazan, Russia

3

Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

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Institute for Nanobiotechnologies, Saint Petersburg State Polytechnical

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University, St. Petersburg, Russia

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Abstract

Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-

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replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known

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regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from A. laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas.

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1. Introduction A. laidlawii (class Mollicutes) is a ubiquitous mycoplasma that is widespread

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in nature, found in tissues from humans, animals and plants. A. laidlawii is the

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causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures [1, 2, 3]. Control of mycoplasma infections and contaminations presents a

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serious concern for the biotechnology, cell biology, health care service and agricultural sectors [4]. Solving this problem is considered to involve the

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investigation of adaptation of mycoplasma to stressful environmental conditions, the mechanisms of their survival in various polymicrobial communities and the realisation of their virulence [5]. Recently, it became apparent that a significant contributor to the aforementioned processes may be bacterial extracellular membrane vesicles [6, 7]. Extracellular vesicles (EVs) present the most important

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component of the bacterial secretome. Identified a few decades ago in Gramnegative bacteria, EVs were recently detected in Gram-positive bacteria [8],

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Archaea [9] and the simplest wall-less bacteria, mycoplasmas [10], and became an

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object of serious attention among researchers. It was found that extracellular membrane vesicles are composed of lipopolysaccharide, lipids, soluble or

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membrane-associated proteins, genetic materials and other factors associated with virulence [11]. EVs have been proposed to play diverse roles, including the transfer of a broad range of components, participation in cell-to-cell communication, pathogenesis and to present a new class of infects. The investigation of EVs is necessary to elucidate the mechanisms of the antagonistic relationship between bacteria and the microbial community, the interaction between microflora in higher eukaryotes and colonised cells and the development of a strategy to control pathogens [7, 12]. To perform the corresponding investigations, comprehensive characterisation of EVs is required that may include the application of a complex approach based on modern physicochemical and molecular methods, including post-genomics technologies. The results of proteomics-based characterisation of EVs in a range of bacteria have already been

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documented in databases. However, data related to EVs derived from mycoplasmas are lacking. In this context, global proteomic profiling of extracellular membrane vesicles from A. laidlawii and identification of proteins

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exported from mycoplasma cells via vesicular structures were the aims of this

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study.

In the present study, global proteomic profiling of A. laidlawii PG8 EVs was

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performed for the first time. We identified a total of 97 proteins contained in bacterium-free EVs. Analysis of the identified proteins indicated that A. laidlawii-

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derived EVs are enriched in virulence proteins, which can play critical roles in mycoplasma-induced pathogenesis. We hope that this study opens the recently emerging field of mycoplasma EVs.

2. Materials and Methods

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2.1. Cell culture and purification of EVs A. laidlawii strain PG8, obtained from the N.F. Gamaleya Institute of

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Epidemiology and Microbiology (Moscow), was used in this study. The

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mycoplasma cells were cultured in liquid modified Edward’s medium (tryptose, 2% [w/v]; NaCl, 0.5% [w/v]; KCl, 0.13% [w/v]; Tris base, 0.3% [w/v]; horse

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serum, 10% [w/v]; yeast extract, 5% [w/v]; glucose solution, 1% [w/v]; benzylpenicillin [500,000 IE/mL], 0.2% [w/v]). Isolation of membrane vesicles from A. laidlawii PG8 cultures was performed according to Kolling and Matthews [13], with some modifications. The cells were precipitated via centrifugation at 5000 g for 20 min. The supernatant was filtered through a 0.10 µm filter (Sartorius Minisart, France), and the filtrate was concentrated 20-fold via ultrafiltration (Vivacell 100, 100,000 MWCO, Sartorius Stedim Biotech GmbH, Germany). Vesicles were collected via ultracentrifugation (100,000 g, 1 h, 8°C) using a MLA80 rotor (Beckman Coulter OptimaTM MAX-E), washed twice and resuspended in buffer (50 mM Tris-HCl, pH = 7.4; 150 mM NaCl; 2 mM MgCl2). The suspension was placed on a stepwise density gradient (10% - 30% - 40% Optiprep in 50 mM Tris-HCl supplemented with 150 mM NaCl, pH 7.4). After centrifugation at

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100,000 g for 3 h at 8°C, the fraction containing EVs was collected superiorly, diluted threefold in buffer and then centrifuged again. The pellet was resuspended in 50 mM Tris-HCl supplemented with 1mM PMSF (Fluka, Germany)". The

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protein concentrations of the samples were measured via the Bradford method

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using "Quick Start Bradford dye" (Bio-Rad). The isolation of vesicles from A. laidlawii PG8 cultures and the subsequent analyses were performed on 3 biological

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and 2 technical replicates.

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2.2. Transmission electron microscopy (TEM), atomic force microscopy (AFM) and scanning electron microscopy (SEM) analyses TEM was performed according to the method of Cole [14]. The samples were fixed using 2.5% glutaraldehyde (Fluka, Germany) in 0.1 M phosphate-buffered saline (PBS) (pH 7.2) for 2 h. The fixed samples were then dehydrated using an

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acetone, ethanol and propylene series prior to post-fixation in 0.1% OsO4 containing 25 mg/ml of saccharose. After treatment with epoxy resin (Serva,

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Switzerland), ultrathin sections were sliced using an LKB-III ultramicrotome

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(Sweden). The sections were stained with uranyl acetate for 10 min and lead citrate for 10 min. The stained samples were examined using a JEM-1200EX transmission

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electron microscope (Joel, Japan). To prepare samples for AFM analysis, aliquots of the A. laidlawii PG8 cells (1 mL) were centrifuged at 12000 rpm for 20 min at room temperature. The pellet was resuspended in 1 mL of PBS (pH 7.2). The prepared cells and EVs suspension were placed onto mica (Advanced Technologies Centre, Moscow, Russia) where the upper layer was removed. The cells were air-dried and washed twice with redistilled water. The samples were air-dried after each wash. AFM imaging was performed using a Solver P47H atomic force microscope (NT-MDT, Moscow, Russia) that was operated in the tapping mode using fpN11S cantilevers (r≤10 nm, Advanced Technologies Centre, Moscow, Russia). The height, the signal from the lock-in amplifier (Mag), the signal from the RMS detector (RMS), and the signal from the phase detector (phase) were set using

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Nova 1.0.26 RC1 software (NT-MDT). The scan rate was 1Hz, and the image resolution was 512 × 512 pixels. SEM analyses were performed as described by Pich et al. [15]. For this

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purpose, EVs from A. laidlawii PG8 were fixed using 1% glutaraldehyde for 1 h.

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The samples were washed with 50 mM Tris-HCl, dried and sputter coated with 15 nm of gold/palladium using a Quorum Q150T ES coating system. The samples

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were observed using a Merlin Carl Zeiss scanning electron microscope.

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2.3. SDS-PAGE

Protein samples (7 µg) from membrane, cytoplasm and purified EVs were prepared as described previously [16] and analyzed via SDS-PAGE (12% resolving gel). The gel was subsequently stained with CBB R-250.

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2.4. In-solution digestion

Purified EVs (150 µg) were resolved using a digestion solution consisting of 9

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M urea and 40 mM ammonium bicarbonate in HPLC-grade water. Protein

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reduction was performed using 25 mM DTT for 1 h, followed by alkylation using 25 mM iodoacetamide in the dark for 45 min at room temperature. The samples

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were diluted (1:10) in 200 mM NH4HCO3 and digested in-solution using 5 ng/mL of trypsin Gold (Promega, USA) for 18 h at 37°C. The samples were desalted using ZipTipC18 (Millipore).

2.5. LC-ESI-MS/MS analysis and identification of proteins Analysis was performed using a TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source (ABSciex, Canada) coupled with a NanoLC Ultra 2D+ nano-HPLC system (Eksigent, Dublin, CA). The HPLC system was configured in the trap-elute mode. For the sample loading buffer and buffer A, a mixture of 98.9% water, 1% methanol and 0.1% formic acid (v/v) was used. Buffer B was 99.9% acetonitrile and 0.1% formic acid (v/v). Aliquots of 70 µg protein were used for MS/MS analysis. The samples were loaded on a Chrom XP C18 trap column

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(Eksigent) at a flow rate of 3 μl/min for 10 min and eluted through the 3C18-CL120 separation column (Eksigent) at a flow rate of 300 nl/min. The gradient was from 5 to 40% of buffer B over 120 min. The column and the precolumn were

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regenerated between runs by washing with 95% buffer B for 7 min and

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equilibrated using 5% buffer B for 25 min.

The information-dependent mass spectrometry (MS) experiments included 1

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survey MS1 scan followed by 50 dependent MS2 scans. The MS1 acquisition parameters were as follows: the mass range for analysis and subsequent ion

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selection for MS2 analysis was 300-1250 m/z, and the signal accumulation time was 250 ms. The ions were selected for MS2 analysis based on their intensity using a threshold of 400 cps and a charge state from 2 to 5. The MS2 acquisition parameters were as follows: the resolution of the quadrupole was set as UNIT (0.7 Da), the mass range was 200-1800 m/z, the focus of the ion beam was optimised to

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attain maximal sensitivity, and the signal accumulation time was 50 ms for each parent ion. Collision-activated dissociation was performed using nitrogen gas and a

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collision energy ramping from 25 to 55 V within the 50 ms signal accumulation

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time. The analysed parent ions were sent to the dynamic exclusion list for 15 sec in order to obtain an MS2 spectra at the chromatographic peak apex (the minimum

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peak width throughout the gradient was approximately 30 sec). The MS/MS spectra were searched using MASCOT software (Matrix Science, Inc.),

the

genomic

data

(http://www.ncbi.nlm.nih.gov/)

of and

A. the

laidlawii

PG-8A

decoy

sequence

from

NCBInr

database.

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exponentially modified protein abundance index (emPAI) was generated using MASCOT software [17].

2.6. Subcellular localisation and function of the identified proteins The subcellular locations of the proteins were analysed using the Psortb v3.0.2 database [18]. The functions of each protein were categorised according to the clusters of orthologous groups of proteins (COG) method [19].

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2.7. Western blot analysis Proteins from EVs and whole cell lysates were separated via 12% SDS-PAGE and transferred to Hybond C nitrocellulose (GE Healthcare, USA) as described

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previously [20]. The membrane was blocked using 3% skim milk in PBS for 30

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min. The antibodies against Hsp20 were diluted 1:1000. Recombinant A. laidlawii PG8 Hsp20 protein and the Hsp20 antibodies were obtained as described

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previously [20].

The secondary antibodies (goat anti-rabbit antibodies conjugated with

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horseradish peroxidase, Sigma, USA) were diluted 1:10,000. The membrane was incubated in primary and secondary antibodies for 1 h, and after each step, the membrane was carefully washed with PBS. The membranes were stained with DAB (Sigma, USA). Lysates from M. gallisepticum A5969B cells were used as a

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negative control.

2.8. Immuno-electron microscopy

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A. laidlawii cells and EVs were fixed using formaldehyde and glutaraldehyde

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at final concentrations of 2% and 0.2%, respectively. The cells and the EVs were fixed at room temperature for 12 h and were collected via centrifugation (10,000 g

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for 10 min and 100,000 g for 1 h, respectively). The pellet was dehydrated using increasing concentrations of ethanol and embedded in LR-White resin (Polyscience,

Inc.,

USA).

LR-White

was

polymerised

at

50°C.

Immunocytochemistry was performed on ultrathin sections according to a standard protocol [20]. Hsp20-specific antibodies were used at 1:50 dilutions in PBS supplemented with 0.1% BSA. To visualise the distribution of the Hsp20 protein, goat anti-rabbit antibodies conjugated to 15-nm colloid gold particles (EY Laboratories, Inc., USA) were used. The sections were stained with uranyl acetate and lead citrate and examined using a JEM-100U electron microscope. Sections incubated in the absence of the polyclonal antibodies or incubated in rabbit preimmune serum served as negative controls.

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3. Results 3.1. Production of EVs from A. laidlawii We were the first to demonstrate that mycoplasma A. laidlawii PG8 naturally

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produces EVs during in vitro culture [10]. When thin sections of A. laidlawii PG8

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cells (log phase) cultured in Edward medium were analysed via TEM, EVs were apparent on the surface of the mycoplasma (Fig. 1A). Moreover, secreted EVs

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were observed in the surrounding milieu based on TEM and AFM analyses (Fig. 1A, B, F, G). Similar to native OMVs from Gram-negative bacteria and EVs from

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Gram-positive bacteria, EVs from A. laidlawii PG8 are spherical, bilayered, and closed membranous structures. The diameter of the mycoplasma EVs ranged from 70 to 120 nm; single vesicles whole diameter less than 70 nm and more than 120 nm were also revealed (about 5%). To obtain EVs for this study, we purified EVs from A. laidlawii PG8 as described previously [10] with some modifications

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(additional washing steps and density gradient ultracentrifugation using OptiPrep). Typically, we obtained 700-800 µg (wet weight) of EVs from 1 L of A. laidlawii

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PG8 culture broth. TEM, AFM and SEM analyses revealed that purified EVs were

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closed vesicular bodies, and no membrane whorls or cell fragments were detected (Fig. 1, C-E, H-J). The size of the purified EVs was similar to that of OMVs and

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MVs from Gram-negative and Gram-positive bacteria, respectively [8, 21], and was consistent with our previous data [10]. SDS-PAGE analysis revealed that A. laidlawii EVs contain distinct protein patterns compared to other subcellular locations (Fig. 2), suggesting that sorting mechanisms similar to those of Gramnegative bacterial OMVs and Gram-positive bacterial EVs are involved in the production of EVs [8, 21, 22].

3.2. Proteomic analysis of A. laidlawii-derived EVs We performed proteomic analysis of purified A. laidlawii-derived EVs using LC-ESI-MS/MS and identified a total of 97 vesicular proteins, which are listed in Table 1. Information regarding the protein properties and detailed peptide data corresponding to the identified proteins are presented in Table S1. DnaK,

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triosephosphate isomerase, phosphoglycerate kinase, heat shock protein Hsp20, glyceraldehyde-3-phosphate dehydrogenase, purine-nucleoside phosphorylase and enolase were the most abundant vesicular proteins. Fig. 3A contains a

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representative peptide mass fingerprinting spectrum of Hsp20. We further verified

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the presence of this protein in EVs via Western blot using a rabbit anti-Hsp20 antibody (Fig. 3B). The recombinant protein Hsp20 (19.65 kDa) is comprised of

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the full-length native Hsp20 sequence (15.2 kDa) fused in frame with the translated region (4 kDa) of pET-15b plasmid vector, which contains the "histidine tail" (for

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extraction from the cell lysate by affinity chromatography) [20]. Immuno-gold labelling of EVs using a goat anti-rabbit antibody also revealed positive staining (Fig. 3C).

The identified proteins were classified into four groups, as shown in Fig. 4, based on their localisation in the bacteria: cytoplasm (73), membrane (5),

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extracellular region (2) and unknown localisation (17). Many cytoplasmic proteins (53) were found in the A. laidlawii-derived EVs, most of which were metabolic or

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ribosomal proteins. The functions of the 97 vesicular proteins identified in EVs

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were categorised according to the COG method [19]. The vesicular proteins from A. laidlawii are involved in translation, ribosomal structure and biogenesis (22),

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transcription (3), replication, recombination and repair (2), defense mechanisms (1), cell wall/membrane/envelope biogenesis (1), intracellular trafficking, secretion and vesicular transport (1), posttranslational modification, protein turnover and chaperones (6), energy production and conversion (12), carbohydrate transport and metabolism (19), amino acid transport and metabolism (6), nucleotide transport and metabolism (3), inorganic ion transport and metabolism (1) and secondary metabolite biosynthesis, transport and catabolism (2), while the functions of 18 proteins are poorly characterised or unknown (Fig. 4). A. laidlawii-derived EVs contained 37 proteins associated with bacterial virulence (Table 1). The virulence factors include enzymes participating in the degradation of proteins and peptides (4), nucleic acids (2) and polysaccharides (6), adhesins (3) and some others (22)

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that may play pathological roles for plants, animals and humans when infected by mycoplasma.

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4. Discussion

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Taking a proteomics approach using LC-ESI-MS/MS, we have documented for the first time the proteins in EVs secreted by A. laidlawii PG8 mycoplasma

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cells (Table 1). We identified 97 proteins, representing 7% of the theoretical A. laidlawii PG8 proteome (1380 proteins). Protein identification was considered to

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be significant when two or more unique peptides were detected (pepscore ≥ 6). According to the data from the literature, the number of proteins secreted as contents

of

extracellular

membrane

vesicles

varies

among

different

microorganisms in a range of ~3-24% of the corresponding theoretical proteome, but this percentage does not exceed 8% among the majority of microbes [8, 21, 22,

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24]. To verify the obtained data, we performed Western blot analysis (Fig. 3B) as well as immuno-electron microscopy (Fig. 3C) to detect heat shock protein Hsp20

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in mycoplasma-free EVs. Hsp20 is a chaperone that participates in polypeptide

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folding and maintenance of cytoplasmic membrane integrity under stressful conditions [25, 26]. Previously, this protein of A. laidlawii was found to be

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localised to the cytoplasm, as well as plasma membrane and the extracellular space, based on immuno-electron microscopy [20]. The obtained data (Table 1, Fig. 3) demonstrated that Hsp20 protein is located on the membrane as well as inside of A. laidlawii EVs, and its abundance is greater than that of other proteins (Mol% = 7.24), which may indicate an important role of this protein in vesicles and/or target cells. The identified proteins of A. laidlawii EVs were classified according to Psortb. Based on this classification, the number of membrane proteins in mycoplasma EVs is significantly less than the number of cytoplasmic proteins. It is known that the predicted protein localisation according to this approach is not always correct, and falsehoods are possible when using this classification method [27]. According to the data reported by Serebryakova et al. [28], in which 17

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acylated and 192 membrane-associated proteins of A. laidlawii were identified, the number of membrane proteins identified in mycoplasma-derived EVs may reach 19 (19.6%). Nevertheless, the obtained results indicate the predominance of

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cytoplasmic proteins in A. laidlawii EVs. Similar results were also obtained for

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vesicles from some other bacteria [8, 21, 22] and microvesicles from mammals [23]. These results may highlight the sorting membrane and cytoplasmic proteins

in each corresponding situation.

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in EVs derived from corresponding organisms and indicate their principal function

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According to the COG classification method (Fig 4B), a majority of the identified proteins are involved in fundamental cell processes in A. laidlawii. In fact, some identified cytoplasmic proteins participating in fundamental cell processes, such as elongation factors EF-Tu and EF-G, chaperones GroEL and DnaK, subunits of DNA-dependent RNA-polymerase and ribosomal proteins, are

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also detected in EVs from many other microorganisms [8, 21, 22, 24], including both Gram-negative and Gram-positive bacteria (Fig. 5). This finding might

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indicate their particular role in the bacterial population as proteins exported via

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EVs. Clarification of their roles, as well the mechanisms of polypeptide sorting, remains to be elucidated.

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Many proteins (41%) detected in A. laidlawii EVs were previously identified in our studies [5] as stress-responsive mycoplasma proteins (Fig. 6). Significant modulation in the expression of these proteins in A. laidlawii cells occurred when these bacteria were cultivated under stressful conditions. Moreover, a significant increase in the vesiculation level was detected in A. laidlawii PG8 cultures. The alteration of the expression levels of RpoB, TufB, EF-G, IF-2, ProRS, GlyRS, GlyA, GAPDH, PdhA, PdhB, AckA, PepF and β-lac in A. laidlawii PG8 cells and the significant increase in the vesiculation level of the mycoplasma under stressful conditions may be interrelated processes. Similar data were also reported regarding a variety of bacteria [29, 30]. These data indicate that essential processes of bacterial adaptation to stressors may be mediated by proteins contained in EVs.

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We identified 37 proteins in A. laidlawii EVs that are considered as factors of bacterial virulence (Table 1). The relative abundance of these proteins (Mol%) was 59.5%. Moreover, 45.5% correspond to enolase, glyceraldehyde-3-phosphate triosephosphate

isomerase,

phosphoglyceromutase,

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dehydrogenase,

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phosphoglycerate kinase (catalyse reactions of glycolysis), heat shock protein Hsp20, molecular chaperone DnaK (participate in protein folding) and translation

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elongation factor EF-Ts (participates in protein synthesis). It is suggested that enolase and glyceraldehyde-3-phosphate dehydrogenase may facilitate the

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adhesion of vesicles to various surfaces [31]. The identified vesicular proteins of A. laidlawii that are associated with bacterial virulence may be involved in cell-tocell communication and pathogenesis.

Previously, it was reported in our studies [3, 10] that EVs from A. laidlawii PG8 exhibit infectious characteristics with respect to virulence and

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may contribute to the phytopathogenicity of this mycoplasma. A. laidlawii EVs penetrate plants via the root system faster than the mycoplasma cells – 20 min after

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growth in medium containing A. laidlawii PG8 - while the mycoplasma cells are

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detected in plants only after 7-8 days [3]. Metalloproteases detected within A. laidlawii-derived EVs might contribute to the invasion of vesicles and/or cells

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via the cell wall of plants. The participation of metalloproteases in the invasion of phytopathogens was reported [32]. However, the role of these enzymes contained in bacterial EVs in phytopathogenesis remains to be explored. A. laidlawii-derived

EVs

were

demonstrated

to

contain

enzymes

participating in the degradation of proteins and peptides (aminopeptidase T, peptidase M3, oligoendopeptidase F, Zn-dependent protease and others), nucleic acids (polyribonucleotide nucleotidyltransferase and

metallo-beta-lactamase

superfamily protein) and polysaccharides (pullulanase, glycogen phosphorylase and α-amylase) that may act on the corresponding structures of target cells. Some of the vesicular proteins of A. laidlawii PG8, such as enolase, glyceraldehyde-3phosphate dehydrogenase and the surface-linked protein containing a VWFA domain, may act as adhesins [33]. Moreover, the surface-linked protein containing

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a VWFA domain might be involved in the pathology of the hemostasis system routinely observed in patients suffering from persistent bacterial infections [34]. The presence of polyribonucleotide nucleotidyltransferase (PNPase),

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considered to function as a global regulator of bacterial virulence [35], in

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A. laidlawii EVs is of particular interest. It was demonstrated that bacterial PNPase may display RNase or DNase activity depending on the environmental conditions

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[36]. Because bacterial DNases may induce chromosomal mutations in eukaryotic cells [37], the clastogenic effect of A. laidlawii PG8 EVs, reported previously in

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our works [10], might be associated with this enzyme. The difficulty in suppressing mycoplasma infections and contaminations is well recognised [1]. The mechanisms by which mycoplasma rapidly adapt to antibacterial drugs are unclear. In our studies, we demonstrated the participation of EVs

from

A. laidlawii PG8

in

the

resistance

of

the

mycoplasma

to

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fluoroquinolones [38]. We revealed that A. laidlawii PG8 EVs mediate the efflux of ciprofloxacin and the export of the nucleotide sequence of an antibiotic target

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gene (parC) that contains a mutation in the QRDR locus. Recently, it was reported

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that Staphylococcus aureus EVs may participate in the transport of biologically active β-lactamase to confer bacterial resistance to antibiotics in microbial

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communities [39]. The authors demonstrated that S. aureus released β-lactamase protein via extracellular vesicles, and the release of these EVs enabled other ampicillin-susceptible Gram-negative and Gram-positive bacteria to survive in the presence of ampicillin. These results indicate that bacterial EVs might play an important role in intercellular communication to mediate the survival of microorganisms in bacterial communities. In the context of intercellular communication and the development of resistance to antibacterial drugs in microbial communities, the protein of the metallo-β-lactamase family identified in A. laidlawii EVs is of particular interest. Because Mollicutes lack a cell wall, that is target of β-lactam antibiotics, the corresponding enzyme of A. laidlawii PG8 may function to protect not the mycoplasma, but other bacteria in the polymicrobial environment that are important for its survival. In this context,

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investigation of the proteins transported by vesicles from mycoplasma and other bacteria in microbial communities may be of particular interest with respect to the regulation of pathogens. However, whether A. laidlawii-derived EVs transport

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biologically active enzymes remains to be elucidated.

Conclusion

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We were the first to demonstrate that mycoplasmas naturally produce EVs. As a result of this study, by characterising and establishing the first proteome of A.

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laidlawii-derived EVs, we found that many properties of vesicles are consistent between bacteria containing a rigid wall (Gram-negative and Gram-positive bacteria) and wall-less bacteria (Mollicutes). Growing evidence suggests that EVs produced by bacterial cells play diverse roles, including symbiosis and pathogenesis, in polymicrobial communities [40, 41]. Our results also indicate that

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many mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and

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pathogen-host interactions. These findings suggest that the secretion of EVs is an

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evolutionally conserved, universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms. It was revealed in this study that

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A. laidlawii-derived EVs are enriched in virulence proteins, which may play critical roles in mycoplasma-induced pathogenesis. These results indicate the necessity of developing new approaches to examine the smallest prokaryotes and to control infects. We hope that this study will stimulate further investigations, including proteomic studies, on mycoplasma EVs to elucidate their biogenesis, properties and functions to understand how very small cells function and to develop effective methods to suppress pathogens that are associated with socially significant infections in humans, animals and plants, as well as the contamination of cell cultures and vaccines.

Acknowledgments

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This work was supported by a grants from the Russian Foundation for Basic Research 12-04-01052a and 13-04-02070, a Grant from the President of the Russian Federation MK-3823.2013.4 and a Grant from a state supporting leading

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scientific schools of the Russian Federation (no. NSH-825.2012.4). The authors are

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grateful to V.V. Salnikov and G.F. Shaimardanova from the Kazan Institute of Biochemistry and Biophysics KSC RAS for their assistance with TEM, Y.N. Osin

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from Kazan (Volga region) Federal University for his assistance with SEM and I.A. Altukhov and S.I. Kovalchuk from the Research Institute for Physical

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Chemical Medicine of the Ministry of Public Health of the Russian Federation for their technical assistance with proteomics research. This work was funded by the subsidy of the Russian Government to support the Program of Competitive Growth

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of Kazan Federal University among World`s Leading Academic Centers.

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Figure 1. TEM (A-E), AFM (F-H) and SEM (I, J) of cells and EVs from A.

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laidlawii PG8. EVs of mycoplasma are indicated using arrows. Bar = 200 nm.

Figure 2. SDS-PAGE of proteins derived from the membrane (1), the cytoplasm (2), and EVs (3). Molecular weight standards are indicated on the left (kDa).

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Figure 3. Identification of the Hsp20 protein via mass-spectrometry (A) and

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verification of this result via Western blot (B) and immuno-electron microscopy (C). Bar = 50 nm. B. Electrophoresis gel image of proteins from A. laidlawii PG8

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lysates (2) and EVs (3) and M. gallisepticum A5969 lysates (negative control) (4). Lane 1 – recombinant A. laidlawii PG8 Hsp20 protein (that is comprised of the full-length native Hsp20 sequence fused in frame with the translated region of pET-15b plasmid vector [20]). Molecular weight standards are indicated on the left (kDa).

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Figure 4. Distribution of the A .laidlawii PG8 vesicular proteins according to

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their intracellular localisation (A) and function based on the COG method (B).

Figure 5. Venn diagram of vesicular proteins from A. laidlawii PG8, Grampositive (A) and Gram-negative (B) bacteria [8, 20, 21, 23]. Each protein is indicated by its term in the UniProt database.

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Figure 6. Venn diagram of the vesicular (I) and stress-responsive (II) proteins

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of A. laidlawii PG8. Each protein is indicated by its term in the UniProt database.

ACCEPTED MANUSCRIPT Graphical Abstract

A. laidlawii PG8 (the ubiquitous mycoplasma that is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures) was proved to produce extracellular vesicles (EVs). A.

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laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in

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Table 1. List of proteins identified in A. laidlawii-derived EVs №

Protein

1 2 3 4 5 6

acetate kinase bifunctional aldehyde/alcohol dehydrogenase dihydrolipoamide acetyltransferase dihydrolipoamide dehydrogenase F-type H+-transporting ATPase subunit alpha F-type H+-transporting ATPase subunit beta

A9NFI4 A9NEM3 A9NHS5 A9NHS4 A9NGW4 A9NGW2

ackA ACL_0496 ACL_0177 pdhC ACL_1310 pdhD ACL_1309 atpA ACL_0984 atpD ACL_0982

7

A9NGU7

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Na+-transporting NADH:ubiquinone oxidoreductase subunit C pyruvate dehydrogenase E1 component subunit alpha pyruvate dehydrogenase E1 component subunit beta pyruvate formate lyase V-type H+-transporting ATPase subunit A V-type H+-transporting ATPase subunit A ABC transporter substrat-binding component aminopeptidase T M3 peptidase family oligoendopeptidase oligoendopeptidase F oligoendopeptidase F peptidase M20B, peptidase T 2-deoxyribose-5-phosphate aldolase purine-nucleoside phosphorylase thymidine phosphorylase 6-phosphofructokinase 6-phosphofructokinase ABC transporter ligand-binding protein

25 26 27 28 29

0.45 1.58 0.75 0.38 0.06 0.66

0.57 1.98 0.94 0.48 0.08 0.83

72180

0.36

0.45

A9NHS7

pdhA ACL_1312

Cytoplasm

40737

0.48

0.60

A9NHS6

pdhB ACL_1311

Cytoplasm

35551

0.43

0.54

A9NE91 A9NGV3 A9NHD8 A9NHJ2 A9NET3 A9NG69 A9NHF0 A9NEG5 A9NGX8 A9NG35 A9NG34 A9NG19 A9NH72 A9NH71 A9NFZ7

pfl ACL_0028 ntpA1 ACL_0973 ntpA2 ACL_1169 ACL_1223 ampS ACL_0237 ACL_0733 pepB2 ACL_1181 ACL_0119 ACL_0998 deoC ACL_0699 deoD ACL_0698 deoA ACL_0683 pfk2 ACL_1092 pfk1 ACL_1091 ACL_0661

84590 65734 65088 87552 46150 69002 67000 65502 45348 23649 25967 46320 45496 33973 48385

0.46 0.1 0.05 0.94 0.51 0.15 0.47 0.22 0.15 0.48 6.74 0.23 1.16 0.2 0.14

0.58 0.13 0.06 1.18 0.64 0.19 0.59 0.28 0.19 0.60 8.47 0.29 1.46 0.25 0.18

ABC transporter substrate-binding protein alpha amylase alpha-amylase alpha-amylase enolase

A9NE41 A9NFD8 A9NFZ3 A9NG03 A9NF93

ACL_1410 ACL_0448 amyA3 ACL_0657 amyA1 ACL_0667 eno ACL_0402

54532 57462 71051 58510 46386

0.6 0.12 0.09 0.12 6.82

0.75 0.15 0.11 0.15 8.57

30 31 32 33 34 35 36 37 38 39

glucose-6-phosphate isomerase glyceraldehyde-3-phosphate dehydrogenase glycogen phosphorylase ketose bisphosphate aldolase, class-II phosphoglycerate kinase phosphoglyceromutase phosphopentomutase pullulanase, surface-anchored protein pyruvate kinase transketolase

A9NHX0 A9NHE5 A9NFL6 A9NE26 A9NHH8 A9NF91 A9NG16 A9NG01 A9NH70 A9NEC3

pgi ACL_1358 gap ACL_1176 glgP ACL_0528 ACL_1395 pgk ACL_1209 gpmI ACL_0400 deoB ACL_0680 ACL_0665 pyk ACL_1090 tktA ACL_0062

47793 35539 91733 30279 42636 55704 44374 110331 49156 71328

0.49 6.08 0.82 2.13 4.55 2.53 0.54 0.06 2.21 0.25

0.62 7.64 1.03 2.68 5.72 3.18 0.68 0.08 2.78 0.31

40 41 42 43 44 45 46 47

triosephosphate isomerase (TIM) 30S ribosomal protein S1 30S ribosomal protein S2 30S ribosomal protein S3 30S ribosomal protein S5 30S ribosomal protein S6 arginyl-tRNA synthetase asparaginyl-tRNA synthetase

A9NHH5 A9NGK8 A9NHC6 A9NED9 A9NEF0 A9NEN9 A9NFH4 A9NE58

tpiA ACL_1206 rpsA ACL_0875 rpsB ACL_1157 rpsC ACL_0093 rpsE ACL_0104 rpsF ACL_0193 argS ACL_0485 asnS ACL_1427

Cytoplasm Cytoplasm Cytoplasm Unknown Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasmic Membrane Unknown Cytoplasm Cytoplasm Unknown Cytoplasm Secreted. Cell surface. Cytoplasm Cytoplasm Cytoplasm Unknown Cytoplasm Cytoplasm Cytoplasm Unknown Cytoplasm Cytoplasmic Membrane Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm

27672 53971 38292 28629 17594 10652 64134 53257

4.48 0.34 0.18 0.25 1.01 0.74 0.1 0.13

5.63 0.43 0.23 0.31 1.27 0.93 0.13 0.16

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9

Gene name

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42802 94978 57130 48908 55362 50328

nqrC ACL_0967

Location in bacteria Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasmic Membrane Unknown

8

UniProt

MW

emPAI Mol %

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0.28 1.66 0.85 0.45 0.25 0.18 0.40 0.24 0.49 0.08 2.84 0.19 0.40 0.16 0.11 0.36 0.08 0.10 1.06 0.09 0.13 1.77 2.36 4.75 0.67 7.24 0.10 0.92 0.43 0.34 0.21 0.49 0.13

ACL_0309 ACL_0991 ACL_0992 ACL_0449

Cytoplasm Cytoplasm Unknown Unknown

62199 47765 50107 54151

0.23 0.59 0.37 0.13

0.29 0.74 0.46 0.16

A9NET4 A9NEV2 A9NG99 A9NF22

ACL_0238 ACL_0256 ACL_0763 secA ACL_0330

21163 61188 36389 93653

0.34 0.05 0.09 0.15

0.43 0.06 0.11 0.19

A9NFS6

hsdM1 ACL_0589

56702

0.12

0.15

90 91 92 93 94

type I site-specific restriction-modification system, M (modification) subunit hypothetical protein ACL_0210 hypothetical protein ACL_0662 hypothetical protein ACL_0672 hypothetical protein ACL_1034 hypothetical protein ACL_1409

Cytoplasm Cytoplasm Cytoplasm Cytoplasmic Membrane Cytoplasm

A9NEQ6 A9NFZ8 A9NG08 A9NH14 A9NE40

ACL_0210 ACL_0662 ACL_0672 ACL_1034 ACL_1409

120075 54346 35210 65395 97874

0.11 0.6 0.31 0.22 0.26

0.14 0.75 0.39 0.28 0.33

95 96 97

hypothetical protein ACL_0116 glycoside hydrolase family protein hypothetical protein ACL_1407

A9NEG2 A9NHG4 A9NE38

ACL_0116 ACL_1195 ACL_1407

Unknown Unknown Unknown Unknown Cytoplasmic Membrane Unknown Unknown Unknown

18251 71479 44832

0.41 0.05 0.07

0.52 0.06 0.09

85 86 87 88 89

aspS ACL_0823 fusA ACL_0187 tuf ACL_0188 glnS ACL_1352 glyS ACL_0946 hisS ACL_0824 ileS ACL_0702 leuS ACL_0242 pnp ACL_0808 proS ACL_0296 tsf ACL_1156 infB ACL_0320 tyrS ACL_1185 gltX ACL_0139 thrS ACL_0354 rpoA ACL_0114 rpoC ACL_0172 nusA ACL_0317 ACL_0872 gyrA ACL_0007 galU ACL_0839 groEL ACL_1247 tig ACL_0535 dnaK ACL_0550 grpE ACL_0549 ibpA ACL_0421 lon ACL_0536 sodA ACL_0302 pduL ACL_0013 fabG1 ACL_0463 ACL_1103 ACL_0387 ACL_0209

A9NF01 A9NGX1 A9NGX2 A9NFD9

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A9NGF7 A9NEN3 A9NEN4 A9NHW4 A9NGS6 A9NGF8 A9NG38 A9NET8 A9NGE2 A9NEY8 A9NHC5 A9NF12 A9NHF4 A9NEI5 A9NF46 A9NEG0 A9NEL8 A9NF09 A9NGK5 A9NE70 A9NGH3 A9NHL6 A9NFM3 A9NFN8 A9NFN7 A9NFB2 A9NFM4 A9NEZ4 A9NE76 A9NFF2 A9NH83 A9NF79 A9NEQ5

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Cytoplasm 65145 0.22 Cytoplasm 76204 1.32 Cytoplasm 42720 0.68 Cytoplasm 62748 0.36 Cytoplasm 53018 0.2 Cytoplasm 48039 0.14 Cytoplasm 103401 0.32 Cytoplasm 92679 0.19 Cytoplasm 78536 0.39 Cytoplasm 54122 0.06 Cytoplasm 31974 2.26 Cytoplasm 67403 0.15 Cytoplasm 46296 0.32 Cytoplasm 51565 0.13 Cytoplasm 75244 0.09 Cytoplasm 36891 0.29 Cytoplasm 155490 0.06 Cytoplasm 40350 0.08 Cytoplasm 9684 0.84 Cytoplasm 97411 0.07 Cytoplasm 31872 0.1 Cytoplasm 58136 1.41 Cytoplasm 48135 1.88 Cytoplasm 65552 3.78 Cytoplasm 21883 0.53 Unknown 15923 5.76 Cytoplasm 86562 0.08 Extracellular 22710 0.73 Cytoplasm 21181 0.34 Cytoplasm 26166 0.27 Cytoplasm 60166 0.17 Unknown 38560 0.39 Cytoplasm 34092 0.1

81 82 83 84

aspartyl-tRNA synthetase elongation factor G elongation factor Tu glutaminyl-tRNA synthetase glycyl-tRNA synthetase, class II histidyl-tRNA synthetase isoleucine amino-acyl tRNA synthetase leucyl-tRNA synthetase polyribonucleotide nucleotidyltransferase prolyl-tRNA synthetase translation elongation factor EF-Ts translation initiation factor IF-2 tyrosyl-tRNA synthetase glutamyl-tRNA synthetase threonyl-tRNA synthetase DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta' transcription termination factor DNA binding protein DNA gyrase subunit A UDP glucose pyrophosphorylase chaperonin GroEL FKBP-type peptidyl-prolyl cis-trans isomerase molecular chaperone DnaK molecular chaperone GrpE molecular chaperone, heat shock protein Hsp20 serine protease Lon, ATP-dependent Fe/Mn family superoxide dismutase propanediol utilization protein beta-ketoacyl-(acyl carrier protein) reductase ABC transporter ATPase hypothetical protein ACL_0387 metal-dependent phosphohydrolase, HD domain-containing protein metallo-beta-lactamase superfamily protein putative Zn-dependent protease putative Zn-dependent protease surface-anchored VWFA domain-containing protein NAD(P)H-dependent FMN reductase glycerone kinase meso-diaminopimelate dehydrogenase preprotein translocase subunit SecA

AC

48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80