Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer

Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer THERESA WAGNER, Sergi Torres-Puig, Thatcha Yimthin, Thomas Démoulins, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5564984/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Apr, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Extracellular vesicles (EVs) are central components of bacterial secretomes, including the small, cell wall-less Mollicutes . Although EV release in Mollicutes has been reported, EV proteomic composition and function have not been explored yet. We developed a protocol for isolating EVs of the pathogens Mycoplasma mycoides subsp. capri ( Mmc ) and Mycoplasma ( Mycoplasmopsis ) bovis and examined their functionality. Proteomic analysis demonstrated that EVs mirror the proteome of their homologous strain. EVs exhibited nuclease activity, effectively digesting both circular and linear DNA. Notably, EVs elicited immune responses in bovine primary blood cells, like those induced by live M. bovis . Our findings reveal that EVs can carry plasmids and enable their horizontal transfer, known as vesiduction. Specifically, the natural plasmid pKMK1, with an unknown transmission route, was detected in EVs of Mmc 152/93 and the tetM -containing pIV08 plasmid was associated with EVs released by an Mmc GM12 strain carrying this plasmid. pIVB08 could be transferred via homo- and heterologous vesiduction to Mmc , M. capricolum subsp . capricolum and M. leachii . Vesiduction was impeded by membrane disruption but resisted DNase and Proteinase K treatment, suggesting that EVs protect their cargo. These findings enhance our understanding of Mollicutes EVs, particularly in host interactions and horizontal gene transfer. Biological sciences/Microbiology/Bacteria/Bacterial host response Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Extracellular vesicles (EVs) are small membrane enclosed spheres shed by cells across all domains of life. EVs have been reported for many bacterial species, with their morphology and composition well-described. The cargo of bacterial EVs can vary, including membrane components, surface-structures, signalling molecules, periplasmic and cytoplasmic proteins, as well as nucleic acids 1 . Bacterial EVs have been linked to diverse functions, such as waste disposal, nutrient scavenging and growth, molecule export, phage interaction, antibiotic resistance, bactericidal activity, delivery of virulence factors and toxins to host cells, and modulation of host immune responses 1 , 2 . In Gram-negative bacteria, vesicles are shed from the outer membrane and thus called outer membrane vesicles (OMVs), while Gram-positive bacteria shed vesicles from their inner membrane, called membrane vesicles (MVs). The pleomorphic bacteria of the class Mollicutes originated from Gram-positive ancestors via reductive evolution. They are distinguished by their small genome, minute size ranging from 100 to 800 nm 3 and the absence of a cell wall. Mollicutes are only enclosed by a cytoplasmic membrane and a few species have been reported to produce EVs so far 4 . Mollicutes EVs were first observed in Acholeplasma laidlawii and Mycoplasma gallisepticum 5 , 6 , and later described in Mycoplasma mycoides subsp. mycoides, M. mycoides subsp. capri ( Mmc ), M. capricolum subsp. capricolum, M. agalactiae, M. fermentans , and M. bovis 4 . However, the proteome of these EVs relative to the homologous strain, as well as their functional and mechanistic significance, remains unexplored. M. bovis is the causative agent of bovine respiratory complex disease (BRD), characterized by enzootic pneumonia, pleuritis, and polyarthritis 7 , 8 . Given that EVs of other bacteria are known to influence pathophysiology and modulate immune responses 9 , and that the host immune response to M. bovis has been characterized 10 , M. bovis -derived EVs represent a relevant candidate for investigating the ability of EVs to trigger immune cell activation in bovine host cells. In addition to immune responses, EVs are increasingly reported as mediators for horizontal gene transfer (HGT) 11 . Vesicular transfer of plasmids was first described in Archaea and termed “vesiduction” 12 , 13 . This vesicle-mediated plasmid transfer has been described in a number of Gram-negative bacteria 14 – 16 , while in Gram-positive bacteria, it has only been observed in Enterococcus faecalis 17 . Within the Mollicutes , members of the M. mycoides cluster have been reported to harbour plasmids 18 and exchange genes horizontally 19 , 20 , prompting us to explore vesiduction in Mycoplasma . In this work we characterized EVs of two veterinary pathogens, M. bovis and Mmc , focusing on their composition and functional roles. Specifically, we investigated their ability to trigger host immune responses and mediate HGT. Methods Bacterial culture and strains. For standard liquid bacterial culture, strains were inoculated at a dilution of 1:1000 in SP5 medium 21 , supplemented with 5 µg/ml tetracycline when necessary, and grown in static culture at 37°C 5% CO 2 overnight for 16 ± 2 h or until color change from red to orange. Colonies were observed on SP5 agar plates or modified Hayflick agar plates 22 , supplemented with 5 µg/ml tetracycline when selection was necessary. Mycoplasma mycoides subsp. capri ( Mmc ) GM12, Mmc GM12 pIVB08 (carrying the tetracycline resistance gene on the 6.079 kbp oriC -plasmid pIVB08) 23 , and Mmc 152/93, which carries the small 1.875 kbp plasmid pKMK1 24,25 , as well as M. bovis Donetta PG45 (ATCC 25523) were used for EV isolation. The attenuated Mmc GM12::YCpMmyc1.1-Δ68 26 was used as a negative control for the MIB-MIP activity assay. Mycoplasma leachii PG50 ( M. leachii ), Mycoplasma capricolum subsp. capricolum California kid (ATCC27343) ( Mcap ), and its derivative mutant strain Mycoplasma capricolum subsp. capricolum ΔRE ( Mcap ΔRE), which lacks restriction enzyme activity due to inactivation of the CCATC-restriction enzyme gene 27 , were used as recipients in vesiduction experiments. EV Isolation. EVs were isolated from late exponential growth phase of bacterial liquid culture in 200 ml SP5. The EV isolation protocol is based on previous studies 4 , 28 , with few modifications. Bacterial cells were pelleted by centrifugation at 4.000 × g for 15 min at 4°C and the supernatant was subjected to serial filtration: first through a 0.45 µm, followed by a 0.22 µm and a 0.1 µm pore filter (Millipore Steritop vacuum bottle top filter, Sigma, Merck). The sterile supernatant was ultracentrifuged at 100.000 × g for 2 h at 4°C (SW32Ti rotor using six 38.5 ml Open-Top Thinwall Ultra-Clear Tube, 25 × 89mm, BeckmanCoulter) and then the supernatant was aspirated with a serological pipette until approximately 1 ml liquid was left in each of the six tubes. The EV-containing samples were resuspended in the remaining liquid, pooled and then PBS was added up to 13 ml and centrifuged at 100.000 × g for 2 h at 4°C (SW41Ti rotor, 13.2 ml Open-Top Thinwall Ultra-Clear Tube, 14 × 89mm, BeckmanCoulter). Again, the supernatant was aspirated with a serological pipette until approximately 400 µl containing the EVs were left. This EV containing sample was stored at -80°C until further use. EV characterisation. The protein content of the samples was quantified using a Qubit fluorometer (Thermo Fisher Scientific) and analysed using nanoparticle tracking. Samples were diluted in PBS to a final volume of 1 ml (10 8 – 10 9 particles/ml equal to 10–100 particles/frame). Then, they were recorded using a NanoSight NS300 instrument with a 405 nm laser (Malvern Panalytical, The Netherlands). Settings were adjusted according to manufacturer’s software manual (NanoSight NS300 User Manual, NanoSight 3.4): camera level was set to 11 then adjusted until particles were seen clearly and no more than 20% were saturated and the infusion rate was set to 1000. For each measurement, five videos were captured and after capture, the videos were analysed using the in-build NanoSight Software NTA 3.4. The detection threshold was set to include as many particles as possible with the restrictions that 10–100 red crosses were counted while blue cross count was limited to 5. Proteomic analysis. Three biological replicates were analysed regarding the proteomic content of the EVs and bacterial cells per strain. Per replicate, EVs were isolated from 80 ml of Mmc GM12 and M. bovis PG45, washed in PBS and pelleted as described above, but here the supernatant was aspirated as much as possible, and the dried pellets were stored at -80°C. For comparison of the proteomic content to bacterial cells, the latter were harvested from 1 ml cultures via centrifugation at 14.000 x g , washed with an equal volume of PBS, centrifuged at 7.000 x g and then stored at -80°C. Samples were subjected to proteomic analysis using comparative shot gun proteomics in an orbitrap LC-MS system (ThermoFisher Scientific). Mass spectrometry-derived proteomic data were analysed against the genomes CP001621.1 ( Mmc GM12) and CP002188.1 ( M. bovis PG45). Data were interpreted by the software Spectronaut, in the hybrid directDIA+ (Deep) mode and IBAQ 29 values (Intensity-Based Absolute Quantification, per protein) were reported, and relative abundance (rAbu) was calculated based on the IBAQ leading protein, so that the sum of rAbu is 1000000 for every individual sample. The subcellular localisation of the detected proteins was predicted based on their amino acid sequence using PsortB 30 . Proteins predicated as “CellWall” were grouped with “Unknown” and the percentage of proteins per subcellular localization, Cytoplasmic, CytoplasmicMembrane, Extracellular and Unknown was calculated based on the sum of their rAbu values. MIB-MIP activity of EV. IgG cleavage assays were performed as described earlier 23 , 31 , to test whether EVs display Mycoplasma immunoglobulin binding-protease (MIB-MIP) activity. EVs were isolated from 200 ml culture, washed once with SP5 w/o serum, and resuspended in 90 µl SP5 w/o serum. One ml bacterial culture of Mmc GM12 and Mmc GM12::YCpMmyc1.1-Δ68 were pelleted at 7000 × g for 10 min, washed once with SP5 w/o serum, and also resuspended in 90 µl SP5 w/o serum. Purified caprine IgG (H + L, Sigma-Aldrich) was added to a final concentration of 100 ng/µl and samples were incubated at 37°C for 60 min, or 120 min. Samples containing bacterial cell were pelleted, and only the supernatant was used further, while EV containing samples were used entirely. Samples were mixed with 6× Laemmli buffer before boiling at 100°C for 10 min. Samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore) using a Trans-Blot Turbo Transfer System (BioRad). The membrane was blocked in PBS with 5% skimmed milk (Becton Dickinson) and 0.05% Tween-20 (Sigma). IgG (H + L) was detected with polyclonal mouse anti-goat antibodies (AffiniPure, Jackson ImmunoResearch Laboratories) in 5% skim milk PBS-T 1:1000 followed by polyclonal Rabbit Anti-Mouse IgG HRP (Dako Agilent) in 5% skim milk PBS-T 1:2000. The membrane was developed using SuperSignal West Pico PLUS Chemiluminiscent substrate (ThermoFisher Scientific). Nuclease activity of EV. Nuclease activity assays were performed with a protocol based on a previous study 32 . Briefly, samples (bacterial pellet of 0.5 ml bacterial culture, or EVs pellet of 60 ml supernatant) were washed in SP5 w/o serum, and then incubated with 1 µg of the plasmid pIVB08 or Eco RI-digested pIVB08 (pIVB08 Eco RI) in 100 µl SP5 w/o serum at 37°C for 60 min. To test nuclease activity in Mmc GM12, CaCl 2 and MgCl 2 were added to a final concentration of 10 mM. Nuclease activity was inhibited by addition of 1 mM or 10 mM of EDTA in assays containing cells and EVs derived from Mmc GM12 or M. bovis PG45, respectively. The supernatant of the samples was visualized on a 2% agarose in TAE gel. Immune response of ruminant primary blood cells to M. bovis and its EV. Fresh bovine peripheral blood mononuclear cells (PBMCs) were isolated and stimulated as described earlier 10 , using an MOI of 0.1 of M. bovis bacterial cells or EVs shed by 2x10 8 CFUs of M. bovis . In brief, PBMCs were isolated from the blood of eight Holstein Frisian cows (age 1–3 years) and seeded at 2 million cells per ml and well. EVs were isolated from 200 ml of M. bovis in SP5 as described above and resuspended in 1 ml of Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) or diluted 100x in DMEM/10% FBS. 100 µl DMEM/10% FBS containing either bacteria or EVs were added to the PBMCs in 1 ml DMEM/10% FBS in flat-bottom 12-well plates, and finally incubated at 38.5°C (ruminant body temperature) and 5% CO 2 for 16 h. The different bovine immune cell subtypes were identified by flow cytometry using a 7-step, 12-color staining protocol. Combination staining analysed monocytes (classical, intermediate and non-classical), conventional type 1 and 2 dendritic cells (cDC1s and cDC2s) and plasmacytoid dendritic cells (pDCs). For the acquisitions, at least 100.000 events were recorded for each sample. For the fold-change induction of cellular surface marker following stimulation, the mean fluorescence intensity (MFI) measured in a stimulated sample for a given animal was normalized to the MFI measured in unstimulated sample from that same animal. FCM acquisitions were performed on a Cytek Aurora (Cytek Biosciences) using the SpectroFlo software with autofluorescence extraction, and further analysed with FlowJo 10.9.0. (TreeStar). Detection of plasmids in EVs. The plasmid content of the EVs was determined by quantitative PCR. First, extravesicular DNA was removed by adding 1 µl of DNase I at 10 U/µl (Roche) per ml of the EV sample and incubated at 37°C for 30 min, before the DNase was inactivated at 75°C for 10 min. The DNase treated EV sample was then resuspended in 12 ml PBS, pelleted as described above and finally lysed by resuspending it in 50 µl cell lysis solution (Wizard Genomic DNA Purification Kit, Promega). Bacterial cells of 8 ml culture in SP5 were pelleted, washed in PBS and resuspended in 500 µl cell lysis solution (Wizard Genomic DNA Purification Kit, Promega). qPCR was conducted in a 10 µl reaction volume with template DNA equalized to 600 ng (EV lysate or bacterial cell lysate) and primers detailed in Table S1 . SsoFast EvaGreen Supermix (Biorad) was used according to manufacturer’s instruction and the reaction was run for 40 cycles in a QuantStudio™ 5 System machine (ThermoFisher Scientific). Transformation of Mmc . An Mmc strain carrying a plasmid with a selectable marker was constructed for vesiduction experiments, as Mycoplasma lack natural antibiotic resistance plasmids. Mmc GM12 was transformed using a polyethylene glycol 8000 (PEG 8000 ) mediated-protocol described elsewhere 33 with few modifications. Briefly, 4 ml of Mmc GM12 culture grown to late exponential phase was spun down at 4000 x g 4°C for 15 min and the pellet was washed once in S/T buffer (250 mM Sucrose, 10 mM Tris-HCl pH 7). Then, the pellet was resuspended in 400 µl of 0.1 M CaCl 2 and cells were incubated on ice for 30 min. In a 50 ml Falcon tube, 10 µl of the pIVB08 plasmid 23 (approximately 10 µg) were added to 410 µl of 2 × Fusion buffer (500 mM Sucrose, 20 mM Tris-HCl pH 7, 40% PEG8000) and left at room temperature. After the incubation on ice, cells were added to the 2 × Fusion Buffer containing the DNA and swirled gently. The mixture was incubated for 25 min at 30°C and then the fusion reaction was stopped by addition of 9 ml SP5 medium. Cells were collected by centrifugation at 4000 x g for 15 min and the resulting pellet was resuspended in 1 ml of SP5. Cells were incubated at 37°C for 1 h and then plated onto selective Hayflick agar plates. Plasmid transfer via EVs. To assess whether the tetracycline resistance encoding plasmid pIVB08 can be transferred via vesiduction, EVs were isolated from Mmc GM12 pIVB08. EVs were DNase-treated (without heat-inactivation) to remove extravesicular DNA. Initially, vesiduction experiments were conducted by co-incubation (protocol A) of the recipient cells with DNase-treated EVs of Mmc GM12 pIVB08 in 400 µl PBS. Further, the genome transplantation protocol described elsewhere 23 was adapted (protocol B). Briefly, recipient cells were grown in SP5 medium until early stationary phase (pH 6.5), washed in Wash Buffer (10 mM Tris, 250 mM NaCl pH 6.5), and resuspended in cold 0.1 M CaCl 2 . Four hundred µl DNase-treated EVs were mixed with the recipient cells resuspended in 2 × Wash Buffer (20 mM Tris, 500 mM NaCl pH 6.5) with 20 mM MgCl 2 and centrifuged for 15 min at 10.000 x g . Mixtures were incubated statically for 90 min at 37°C, resuspended in 5 ml SP5, spun down for 15 min at 5.800 x g , resuspended in 500 µl SP5 then plated on selective Hayflick agar and incubated for 5–7 days at 37°C 5% CO 2 . To estimate the maximal vesiduction rate, 10% PEG 6000 was added to the 2 x Wash Buffer with 20 mM MgCl 2 (protocol C), as in the original genome transplantation protocol 23 . To investigate the potential of heterologous vesiduction, the phylogenetically related strains M. leachii , Mcap and its mutant Mcap ΔRE, were used as recipients. To screen for transformants obtained by vesiduction, colonies were picked in 1 mL selective SP5 and lysed using cell lysis solution (Wizard Genomic DNA Purification Kit, Promega). One µl of lysed cells was used as template in a PCR using the tet -specific primers for the plasmid pIVB08 and strain-specific primers (Table S1 ) in a 10 µl reaction in GoTaq G2 Green Master Mix (Promega, United States) according to manufacturer’s instruction and PCR products were visualized on a 2% agarose in TAE-buffer gel with RedSafe (iNtRON Biotechnology). EVs inactivation. EVs were subjected to different stresses, to assess the necessity of an intact membrane for vesiduction. For alkaline lysis, pH was raised to pH 10 by addition of 5 µl NaOH 1 M per 400 µl of bacterial or EV sample, incubated for 30 min at room temperature and the neutralized by adding 5 µl HCl 1 M per 400 µl EV sample. For heat stress, bacterial cells or EVs were incubated for 30 min at 50°C or 56°C. For digestion of extravesicular proteins, the EV sample was incubated with 1 µl of Proteinase K (10 µg/ml, Roche) at 37°C for 30 min, as reported elsewhere 14 . Vesiduction experiments were then conducted with the stress treated Mmc GM12 pIVB08 EVs as described above, with Mcap ΔRE as recipient and transformants were observed on selective Hayflick agar plates. Statistical analysis. Statistical analysis was done using the GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). A p value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). Results Mollicutes EVs represent the overall proteomic profile of the bacterial cell. EV isolation was first optimized with bacterial cultures of Mmc GM12. Isolation of Mycoplasma EV requires the use of a 0.1 µm filter, since Mycoplasma bacterial cells can pass a 0.22 µm filter (Supplementary Figure S1 ). Here, a serial filtration protocol was employed starting with a 0.45 µm filter, followed by a 0.22 µm filter and a final step using a 0.1 µm filter. Nanoparticle tracking analysis using NanoSight did not allow to distinguish between EVs and the background of the growth medium, even when FBS in standard SP5 medium was reduced from 17–2% (Supplementary Figure S2 ). To assess protein content of the isolated EVs from Mmc and M. bovis , proteome analysis of both EV and bacterial sample was conducted. Based on intensity-based absolute quantification (iBAQ) values, proteomics showed that 99.8 ± 0.05% were Mycoplasma protein in the bacterial cell samples of Mmc GM12 and 83.2 ± 0.53% were Mycoplasma protein in EV samples of Mmc GM12, while 94.5 ± 0.85% were M. bovis protein in the bacterial cell samples of M. bovis PG45 and 28.7 ± 2.13% were M. bovis protein in EV samples. A total of 691 Mycoplasma proteins were identified in Mmc GM12 samples (17 unique in bacterial cell; 3 unique in EV) and 565 M. bovis proteins were identified in M. bovis samples (89 unique in bacterial cell; 2 unique in EV). EV-associated proteins represent a subset of the proteomic profile of the producer strain (Fig. 1AB, Table S2 ). The relative abundance of proteins of subcellular localisation predicted with PSortB was similar in the EV samples compare to the whole cell lysates for Mmc GM12 (Fig. 1 C), while cytoplasmic membrane proteins had a slightly higher abundance in EVs (Fig. 1 D). Thirteen membrane proteins potentially involved in Mollicute -host interaction (lipoproteins p37 34 , LppB 35 , and LppA p72 36 , elongation factor TU 37 , chaperone protein DnaK (Hsp70) 38 , glyceraldehyde-3-phosphate dehydrogenase 39 , pyruvate kinase 39 , lactate dehydrogenase 39 , phosphoglycerate mutase 39 , transketolase 39 , PTS glucose permease ptsG 36 , pyruvate dehydrogenase E1 subunit α and β 39 ) were detected in both the bacterial and the EV samples of Mmc GM12. The elongation factor TU, which is a putative factor in Mycoplasma interaction with host extracellular matrix, and lactate and glyceraldehyde dehydrogenase were the most abundant ones, both in the bacterial cells and the EVs (Fig. 2 A). Adhesion is an essential attribute in virulence of M. bovis and several proteins mediating adhesion to fibronectin, plasminogen or epithelial cells have been described (NOX 40 , MbfN 41 , Fba 42 , TrmFO 43 , α-enolase 44 , MilA 45 , P27 46 , VpmaX 47 , variable surface lipoproteins Vps’ 48 , Mbov_0503 cytoadhesin 49 , MBOVJF4278_00255 and MBOVJF4278_00667 50 ). As shown in Fig. 2 B, the described adhesion related proteins were present in bacterial and EV samples of M. bovis . The variable surface lipoprotein VspA was the most abundant adhesion related protein in both bacteria and EVs of M. bovis . Functional proteins are associated with Mollicutes EVs. In the proteomic profile of Mmc GM12 and its EVs all components of the MIB-MIP gene cluster as well as three subunits of the adjacent ATPase were detected (Figure S3A). IgG cleavage could not be detected in EVs derived from 200 ml Mmc GM12 culture even when incubation was prolonged from 1 h up to 3 h (Figure S3B), which might be due to altered rations of binding proteins, proteases and ATPases in the EV sample or the detection limit. Since nucleases were not previously described in Mmc , putative nucleases were predicted based on conserved domains in NCBIs Conserved Domain Search 51 , and four putative nucleases were present in the proteome of both Mmc GM12 bacterial cells and EVs at comparable levels (Fig. 3 A). In Mmc GM12 bacterial samples nuclease activity was detected for the linearized plasmid pIVB08 Eco RI in presence of salts (10 mM CaCl 2 and 10 mM MgCl 2 ) and inhibited by addition of 1 mM EDTA. This exonuclease activity was also detected in the EVs (Fig. 3 B). Nucleases present in the proteome of M. bovis PG45 (MslA MBOVPG45_0311 52 , MbovNase MBOVPG45_0310 32,53 , MnuA MBOVPG45_0215 32,54 , 5’-nucleotidase MBOVPG45_0690 55 ) were also present in the EV samples (Fig. 3 C). Nuclease activity was detected in both the bacterial cells and EVs, and the reaction could be inhibited by addition of 10 mM EDTA. Both the circular plasmid pIVB08 and its linearized version pIVB08 Eco RI were degraded, while plasmid degradation was not observed in the SP5 w/o serum control (Fig. 3 D). EVs can elicit an ex vivo immune response. To investigate whether EVs elicit an immune response in the native host of M. bovis , bovine PBMCs were exposed to EVs. The response was compared to that induced by live M. bovis PG45, which has been recently characterized 10 . EVs stimulated bovine immune cells in a pattern similar to M. bovis bacterial cells, though the response was generally lower. Both bacterial cells and EVs induced a marked response of dendritic cells (DCs), known to play a crucial role in bridging innate and adaptive immunity, especially CD25, related to cellular activation, and to a lesser extent CCR7, related to cell migration towards draining lymph nodes (Fig. 4 ). The previously observed immune response diminished with EV dilution, as witnessed by lower CD25 and CCR7 induction on all DC subsets. Monocytes, key players of innate immunity, showed less stimulation in response to M. bovis , in line with our previous study 10 . As such, the specific effect of EVs was more difficult to demonstrate on this immune cell subset (Supplementary Figure S4, A-I). Lastly, both EVs and live bacterial cells induced comparable levels of primary cell death relative to corresponding unstimulated PBMCs (Supplementary Figure S4J). Mollicutes EVs can pack plasmid DNA. qPCR showed that EVs derived from Mmc 152/93 contain the small natural plasmid pKMK1 present in this Mmc strain 24 . In the EV lysate, the CT values of two plasmid regions were significantly lower than the CT values of two chromosomal genes (sigma factor rpoA and cell division factor ftsZ ), translating to 3 x 10 6 times more pKMK1 then chromosomal DNA in the EVs. In the whole cell lysate, the CT values of the plasmid regions were comparable to those of chromosomal genes (Fig. 5 a, CT values for individual targets are illustrated in Supplementary Figure S5). Similarly, in EVs derived from Mmc GM12 pIVB08 carrying the plasmid pIVB08 with the resistance markers for ampicillin and tetracycline, amp and tet , the CT values of the two plasmid markers were significantly lower than the CT values of the chromosomal genes, rpoA and ftsZ (Fig. 5 b, CT values for individual targets are illustrated in Supplementary Figure S5), meaning that 200 times more pIVB08 compared to chromosomal DNA was detected in the EVs. Of note, the CT values of the small natural plasmid pKMK1 were lower than of the larger oriC -plasmid pIVB08. Vesiduction: Plasmid transfer via EVs. To investigate whether EVs can transfer plasmids to other bacterial cells, vesiduction experiments were conducted. Since Mycoplasma lack natural antibiotic resistance plasmids, the oriC- plasmid pIVB08, a replicative plasmid based on the origin of replication ( oriC ) sequence of the chromosome 23 , 56 , was used. First, homologous vesiduction was tested between Mmc GM12 pIVB08 derived EVs and plasmid-free Mmc GM12. Using protocol A (co-incubation), six transformants per 10 11 CFU were obtained. With protocol B, seven transformants per 10 11 CFU were recovered (Table 1 , Supplementary Figure S6). Next, the capacity of EVs to transfer the plasmid to other species was assessed. Heterologous vesiduction experiments with Mcap and M. leachii as recipients resulted in successful plasmid transfer, with frequencies of four per 10 10 CFU for Mcap pIVB08 and six per 10 10 CFU for M. leachii pIVB08 (Table 1 ). PCR analysis confirmed vesiduction in at least five colonies per experimental setup, with a pIVB08 specific gene ( tet ) and a strain specific gene (Supplementary Figure S6, Table S1 ). The maximum vesiduction rate was established using protocol C, which includes the addition of 10% PEG 6000 in the 2x Wash Buffer. This yielded one per 5x10 7 CFUs, compared to four per 10 10 CFU using protocol B (without PEG 6000, Table 1 ). To investigate the impact of restriction-modification system on vesiduction, the strain Mcap ΔRE, which cannot restrict incoming DNA 27 , was used. Transformants, Mcap ΔRE pIVB08, were recovered at comparable rates (6 per 10 11 CFU, Table 1 ). To determine whether vesiduction requires intact EVs, they were subjected to three different conditions: alkaline lysis, heat stress or Proteinase K treatment. A pilot experiment on alkaline stress on bacterial cells showed that pH 10 causes a 10 10 -fold reduction in viable bacterial cells. When EVs were subjected to the same alkaline lysis prior to a vesiduction experiment, no transformants were recovered. In a heat stress pilot, bacterial cell viability remained unaffected by incubation at 45°C for 10 or 30 min. However, at 50°C for 10 minutes, a 10 2 -fold reduction was observed, and 50°C for 30 min caused a 10 5 -fold reduction. No live bacterial cells were recovered at 56°C for 10 or 30 min. To then subject EVs to intermediate and strong heat stress, they were heated for 30 min at 50 and 56°C. When heat-stressed EVs were used for vesiduction, no transformants were recovered. Finally, EVs were treated with Proteinase K (10 µg/ml at 37°C for 30 minutes) to digest extravesicular proteins. This treatment did not affect the vesiduction rate (2.5 per 10 10 CFU, Table 1 ). Table 1 Plasmid pIVB08 transfer frequencies in homo- and heterologous vesiduction. Recipient cell EV sample treatment Vesiduction protocol* Transfer frequency Mmc GM12 A 6 per 10 11 CFU Mmc GM12 B 7 per 10 11 CFU M. leachii B 4 per 10 10 CFU Mcap B 6 per 10 10 CFU Mcap ΔRE A 6 per 10 11 CFU Mcap ΔRE B 4 per 5x10 10 CFU Mcap ΔRE C 1 per 5x10 7 CFU Mcap ΔRE Alkaline lysis B 0 Mcap ΔRE 50°C B 0 Mcap ΔRE 56°C B 0 Mcap ΔRE Proteinase K B 2.5 per 10 10 CFU * A co-incubation, B standard protocol, C standard protocol with 10% PEG 6000 Discussion The pleomorphic, cell wall-less nature and small size of Mollicutes challenged EV isolation, detection and characterisation 4 . The minute size and lack of a cell wall of Mollicutes hinders distinguishing EVs from live bacterial cells, unlike in cell wall enclosed, larger bacteria. Here we present an isolation protocol employing serial filtration steps to remove all live bacterial cells from the EV containing sample. EVs of other bacteria are approximately one tenth of the size of the producing cell, with an EV size of 40 to 400 nm 1 , 57 . This would translate to an expected Mollicutes EV size of 10 to 100 nm, which brings current methods, such as Nanoparticle tracing, to a limit. Due to difficulties distinguishing vesicles from bacterial cells and background noise even after reducing FBS concentration, a reliable enumeration method remains elusive. Novel high resolution methods or innovative minimal growth media might overcome this issue in the future. Still, we could manage to identify a clear proteomic profile of Mollicutes EVs and to characterise them functionally. EVs of other bacteria generally reflect the proteomic content of their producing cell 1 , 58 , though certain species demonstrate selective enrichment of components, as seen in predatory MVs 59 – 61 . Our findings demonstrate that EVs from both Mmc and M. bovis reflect the overall proteomic profiles of their homologous strains. Several membrane proteins which are potentially involved in Mycoplasma -host interactions were found to be associated with Mmc GM12 EVs, which is in line with the findings of the proteomic analysis performed on the Triton X-114 enriched fractions of EVs of Mycoplasma mycoides subsp. mycoides strain Afadé 4 . Numerous previously characterized adhesins were also detected in the M. bovis bacterial and EV samples. Lipoproteins were abundant in both bacteria and EVs of M. bovis PG45, especially VspA and VspE. In related Mollicutes , lipoproteins have been described for their activating and evasive immunomodulatory effect 62 . We detected the components of the MIB-MIP gene cluster in the EV sample, but immunoglobulin cleavage levels of EVs were below detection limit, potentially due to low protein amounts in the EV sample or different protein ratios required for successful MIB-MIP activity. We illustrate that EVs contain functional proteins through their nuclease activity. M. bovis nucleases have a dual functionality, enabling nutritional acquisition by degradation of host nucleic acids required for nutritional supply 32 , 63 and immune evasion 55 . The major membrane nuclease MnuA and the nucleotidase 5’NT act in an enzymatic cascade to degrade and hydrolyse nucleotides, which can be transported through the cell membrane 55 . M. bovis also induces the release of neutrophil extracellular traps, which MnuA degrades to allow immune escape 54 . The nucleotidase 5’NT plays a role in virulence in bovine mastitis, particularly in the elicitation of inflammatory changes 55 . Here, we found that nucleases were present in the proteomic profile of M. bovis bacterial cells and their EVs. In line with previous findings 63 , 32 , 55 , bacterial cells of M. bovis were able to cleave the circular pIVB08 and the linearized pIVB08 Eco RI plasmids, indicating presence of endo- and exonuclease activity. For M. bovis PG45 EVs the same nuclease activity was detected. Nuclease activity remained uncharacterized in Mmc until now, even though bacterial cells of the closely related Mcap have been shown to digest extracellular DNA, with magnesium dependence and calcium inhibition 63 . The homolog to the Mcap CK predicted nuclease MCAP_0027 in Mmc GM12, MMCAP1_0024 (80.09% identity), was not detected in the EV proteome. However, based on conserved domains, we identified four putative nucleases which were present in the proteome of bacterial cells as well as their EVs and might exert oligonucleotide degradation. We confirmed nuclease activity by observing DNA degradation by Mmc bacterial cells and EVs in the dual presence of MgCl₂ and CaCl₂. These findings highlight the enzymatic functionality of EV-associated proteins and indicate that EVs could be involved in the nucleic acid breakdown required for bacterial growth and immune evasion. Using a recently described ruminant primary blood cell ex vivo platform 10 , we observed that EV stimulation mirrored bacterial cell stimulation, inducing both CD25 and CCR7 upregulation in DCs. Several factors challenge the comparison between live M. bovis and EVs, such as the lack of a reliable enumeration method for EVs. Additionally, unlike EVs, live cells replicate during experiments and can adjust protein expression in response to host factors. The use of the well-established M. bovis strain enabled us to directly compare our results to previous findings 10 . Isolated PBMCs have limitations in terms of timeframe and interplay with other parts of the immune system, as well as not being the first line of defence that M. bovis would meet during infection. Nonetheless, the experimental design offered the advantage of studying immune responses without the need for animal experiments and being time efficient yet data rich. Herein, this showed the immunomodulatory capacity of purified EVs, particularly their strong effect to stimulate all DC subsets, which suggest that EVs can interact directly with the host immune system during M. bovis infection. Given that some Mollicutes species reach in vivo concentrations of up to 10 9 to 10 10 CFU/ml in vivo 26 , 64 , 65 , such high bacterial loads are likely to result in substantial EV shedding, which could modulate the host immune responses. Beyond their immunomodulatory role, EVs may also serve as vehicles for HGT, emphasizing their multifaceted roles in bacterial-host and bacterial-bacterial interactions. This study provides first experimental evidence of EV-mediated plasmid transfer in Mollicutes . Vesiduction was first described in Archaea 12 , 66 , whose cell envelope consists of a single membrane, similar to the cell wall-less Mollicutes of the bacteria domain. In Gram-negative bacteria, vesiduction has been subject of a few studies. In Acinetobacter baylyi , OMVs were shown to transfer a plasmid to recipient cells at frequencies of 10 − 6 to 10 − 8 14 . Further, plasmid containing OMVs isolated from Aeromonas veronii , Enterobacter cloacae , and E. coli were used to induce the transformation of five different species of recipient strains 15 , and it was subsequently shown that the plasmid size and its copy number influenced the packaging efficiency into vesicles, while the origin of replication affected the absorption rate of the OMVs 16 . Also in Klebsiella , OMVs have been shown to play a role in interspecies transfer of plasmids containing resistance genes 67 . Enterococcus faecalis is the only Gram-positive species for which vesicle mediated gene transfer has been shown so far 17 , while in Enterococcus faecium resistance genes were associated with MVs but no resistance transfer was verifiable 68 . Our findings extend prior studies by demonstrating that EVs can transfer plasmids in minute bacteria belonging to the class Mollicutes . Since the characteristics of MGEs influence vesiduction 16 , it is likely that larger EVs – excluded by the filtration method used in this study - could potentially carry different or larger Mollicutes -related MGEs 69 , 70 . A 2012 study found plasmids in about 30% of Mollicutes belonging to the M. mycoides phylogenetic group and recombination event signatures suggest plasmid exchange between species sharing a host or niche 18 . The detection of the small plasmid pKMK1 in EVs, for which the mobilisation route is unknown 25 , suggests that vesiduction could be a putative transfer mechanism. Because of the absence of known natural Mycoplasma antibiotic resistance plasmids, we demonstrated EV-mediated plasmid transfer using the oriC- plasmid pIVB08 in this study. The larger size and artificial nature of pIVB08 likely contributed to its lower abundance in EVs compared to pKMK1. Comparted to other HGT machineries, vesiduction might occur at lower rates. However, it enables the mobilisation of non-replicative plasmids in absence of alternative pathways and adds to the complex interplay of MGEs and distribution of potential virulence and resistance genes. Environmental factors such as host factors or biofilms may enhance vesiduction, particularly in infection or antibiotic stress contexts. OMVs protect their cargo from enzymatic degradation, including resistance to proteinase treatment 71 , 72 . This aligns with our finding that proteinase treatment did not impact vesiduction. However, the experiments presented here could not differentiate between the digestion of EV-associated proteins and SP5-associated proteins. We also show that vesiduction depends on membrane integrity, as plasmid transfer was abolished following alkaline lysis and heat inactivation of EVs. Mycoplasma , which rely on a plasma membrane with associated lipids for structural integrity 73 , are particularly susceptible to lipid membrane disrupting lysis methods 74 , necessitating the use of alkaline lysis to disrupt EVs. The efficiency of vesiduction and its detection faces several bottlenecks. First, only part of the EV population carries the plasmid and second, transfer will depend on the fusion of membranes. Third, once the plasmid enters the recipient cell, it may be degraded by the restriction modification system targeting non-self-patterns, although this did not appear to impact vesiduction rates in Mcap , as observed in comparison to Mcap ΔRE. Lastly, oriC -plasmids like the pIVB08 used in this study, must replicate alongside the chromosome to avoid dilution and eventual loss. In summary, this study demonstrates that Mollicutes EVs carry a proteomic profile reflective of their parental cells, with functional proteins that may play a role in host interactions, including triggering immune responses, as illustrated by the stimulation of bovine PBMCs. Additionally, our findings reveal that Mycoplasma EVs can facilitate HGT, offering new insights into plasmid-mediated gene exchange within Mollicutes species. This work provides a foundation for further exploration of EV-mediated processes in microbial ecology and pathogenesis. Declarations Acknowledgements We thank the Proteomics and Mass Spectrometry Core Facility, Department for BioMedical Research, University of Bern, Switzerland for excellent technical support for the proteomic analysis, and the Flow Cytometry and Cell Sorting Core facility at the Department for BioMedical Research, University of Bern, Switzerland, for access to the NanoSight. We thank the Agroscope animal facility, Research Contracts Animals Group, Agroscope, Posieux, Switzerland, especially Lukas Eggerschwiler, for the excellent collaboration related to provision of bovine blood. We thank Jing Zhang and Marilou Bourgeon at the Institute of Veterinary Bacteriology for excellent assistance with data analysis and excellent technical support, respectively. The work was supported by the Swiss National Science Foundation (grant number 310030_201152, www.snf.ch). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions TMW Conceptualization, Project administration, Methodology, Investigation and Formal analysis, Data curation and visualization, Writing - original draft STP Conceptualization, Methodology, Investigation and Formal analysis (Functional Assays and Vesiduction), Writing - review & editing TY Investigation and Formal analysis (ex vivo platform), Writing - review TD Conceptualization, Methodology, Investigation and Formal analysis (ex vivo platform), Writing - review & editing JJ Funding acquisition, Resources, Conceptualization, Supervision, Writing - review & editing Ethical approval The collection of ruminant blood was performed in compliance with the Swiss animal protection law (TSchG SR 455; TSchV SR 455.1; TVV SR 455.163) under the cantonal license BE55/2022. 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Supplementary Files Supplement02122024.pdf Supplement for: Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer SupplTable2.xlsx Table S2 Proteomic Profile of Mmc GM12 and M. bovis PG45 bacterial cells and EVs GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 28 Apr, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5564984","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":391761095,"identity":"bfbede54-e2f3-482c-85a7-a7ce60441470","order_by":0,"name":"THERESA WAGNER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACCVSuDQMDMw+IcQCnDh40LWmkazkMEsOvxV66+diHD3/sGAyOH7/24OOe84nb2XkPPmCouYPbFpljyTNntiUzGJzJKTec8ex24s5mvmQDhmPP8Dgsx5iZt4GZwexATpo0z4HbiRsO85hJMDYcxqMl/zPznz/1DGbn36RJ/zlwDqTF/Ad+LTnMzAxshxnMbqQfk2Y4cABsCwNeLTfSjBl7247z2N94wybZcyDZeMNhvmSJhGO4tbDPSH7M8ONPtZxkf/oziR8H7GQ3nD978MOHGtxa4LYBkQGCm0BQA8TCB8SpGwWjYBSMghEHAFALWIvTCuppAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8605-6296","institution":"Institute of Veterinary Bacteriology, Vetsuisse Faculty – University of Bern, Switzerland","correspondingAuthor":true,"prefix":"","firstName":"THERESA","middleName":"","lastName":"WAGNER","suffix":""},{"id":391761096,"identity":"817e33e3-174f-4541-a5ab-925b51f9e952","order_by":1,"name":"Sergi Torres-Puig","email":"","orcid":"https://orcid.org/0000-0002-8976-6488","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Sergi","middleName":"","lastName":"Torres-Puig","suffix":""},{"id":391761097,"identity":"da3a621d-98bc-4110-aa39-82df61b4ceb7","order_by":2,"name":"Thatcha Yimthin","email":"","orcid":"https://orcid.org/0009-0004-4489-172X","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thatcha","middleName":"","lastName":"Yimthin","suffix":""},{"id":391761098,"identity":"f25d7957-c9f9-4f32-a83f-99902b7c9ac0","order_by":3,"name":"Thomas Démoulins","email":"","orcid":"https://orcid.org/0000-0003-0654-5813","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Démoulins","suffix":""},{"id":391761099,"identity":"58356408-39ca-441b-a22a-9a24e213a1eb","order_by":4,"name":"Jörg Jores","email":"","orcid":"https://orcid.org/0000-0003-3790-5746","institution":"University of Bern","correspondingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Jores","suffix":""}],"badges":[],"createdAt":"2024-12-02 13:26:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5564984/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5564984/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-08099-4","type":"published","date":"2025-04-29T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73726528,"identity":"0cc13951-0f04-4f69-90c4-9716ae9d8f40","added_by":"auto","created_at":"2025-01-14 04:14:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMmc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e GM12 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e PG45 bacterial cells and EVs. \u003c/strong\u003eVenn diagram showing the proteins identified in the bacterial (Bac) samples and EVs (by presence – absence, created in DeepVenn) in \u003cem\u003eMmc \u003c/em\u003eGM12 (A) and \u003cem\u003eM. bovis \u003c/em\u003ePG45 (B). Subcellular localisation of the identified proteins as predicted by PsortB in \u003cem\u003eMmc \u003c/em\u003eGM12 (C) and \u003cem\u003eM. bovis \u003c/em\u003ePG45 (D). Precent of \u003cem\u003eMmc \u003c/em\u003eor\u003cem\u003e M. bovis \u003c/em\u003eprotein is calculated based on rAbu values. Bars represent the media of three replicates and symbols individual values.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/6c4ccdc8ca95ea802870a796.png"},{"id":73726682,"identity":"a101d3c7-f2ef-42b2-91ea-cabe17dc4e9a","added_by":"auto","created_at":"2025-01-14 04:22:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePresence of proteins potentially involved in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMollicute\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-host interaction and adhesion related proteins. \u003c/strong\u003e(A) Relative abundance of membrane proteins potentially involved in \u003cem\u003eMycoplasma\u003c/em\u003e-host interaction in \u003cem\u003eMmc\u003c/em\u003e GM12 bacterial cells and its EVs. (B) Relative abundance of adhesion related proteins in bacterial and EV samples of \u003cem\u003eM. bovis\u003c/em\u003ePG45.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/6b6629a10ba9450e6ad0d2e6.png"},{"id":73726531,"identity":"e70815e5-6008-42ed-904e-d4c17f0dc552","added_by":"auto","created_at":"2025-01-14 04:14:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNuclease activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMmc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand their EVs. \u003c/strong\u003e(A) Relative abundance of putative nucleases in \u003cem\u003eMmc\u003c/em\u003e and its EVs in the proteome. (B) Cleavage of pIVB08 and pIVB08\u003cem\u003eEco\u003c/em\u003eRI by \u003cem\u003eMmc\u003c/em\u003e GM12 and its EVs, and bacteria of \u003cem\u003eM. bovis\u003c/em\u003e as positive control. (C) Relative abundance of nucleases in \u003cem\u003eM. bovis\u003c/em\u003e and its EVs in the proteome. (D) Cleavage of pIVB08 and pIVB08\u003cem\u003eEco\u003c/em\u003eRI by \u003cem\u003eM. bovis\u003c/em\u003e and its EVs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/7b2e0b3b517265563eb41139.png"},{"id":73726533,"identity":"55e4ae6e-9665-41d2-95cf-cfedca33d20d","added_by":"auto","created_at":"2025-01-14 04:14:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo response of dendritic cells (DCs) to bacteria or EVs of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eConventional type 1 DCs (A-C), conventional type 2 DCs (D-F), plasmacytoid DCs (G-I) with the markers MHC-II (A, D, G), CD25 (B, E, H), and CCR7 (C, F, I). Values are expressed as MFI of stimulated sample normalized to MFI of unstimulated blood cells from the same animal, and each coloured circle represents PBMCs of one animal (n=8) stimulated for 16 h at 38.5\u003cstrong\u003e°\u003c/strong\u003eC. (Significance based on \u003cem\u003ep\u003c/em\u003e values as calculated by Repeated measures paired ANOVA with Dunnett's multiple comparisons test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/d7c233e05f2bf057db637cbd.png"},{"id":73726684,"identity":"adbbc0ad-04e4-4d65-84e9-5cd783a1c459","added_by":"auto","created_at":"2025-01-14 04:22:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePresence of plasmids in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMmc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ederived EVs as measured by qPCR\u003c/strong\u003e. CT values for plasmid and chromosomal targets are shown in EVs, cell lysates and the non-template control, where symbols show the individual repeats and bars are median over n=12 over three biological replicates and two targets. (A) \u003cem\u003eMmc\u003c/em\u003e 152/93 with its plasmid pKMK1. (B) \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08 with its plasmid pIVB08. (Significance based on \u003cem\u003ep\u003c/em\u003e values as calculated by Kolmogorov-Smirnov test)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/fe23584cc1562d3d007cc38e.png"},{"id":93597462,"identity":"7a272415-a8c2-4d80-925d-3fc7240d67ee","added_by":"auto","created_at":"2025-10-15 14:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1585901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/5dca424b-95e9-4e15-895e-5f8cce4703dc.pdf"},{"id":73726683,"identity":"64e5cfe7-a376-40fb-859c-8ecd21da3eed","added_by":"auto","created_at":"2025-01-14 04:22:33","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":944283,"visible":true,"origin":"","legend":"\u003cp\u003eSupplement for: Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer\u003c/p\u003e","description":"","filename":"Supplement02122024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/f7bb229ded50b9e950d7dcfa.pdf"},{"id":73726534,"identity":"7a803421-d1b3-4a4c-a539-aad3dd847453","added_by":"auto","created_at":"2025-01-14 04:14:33","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":578257,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2 Proteomic Profile of Mmc GM12 and M. bovis PG45 bacterial cells and EVs\u003c/p\u003e","description":"","filename":"SupplTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/9893518571ac6e9780ada795.xlsx"},{"id":73726536,"identity":"54340aba-2014-4fee-9b8f-e5349da01e79","added_by":"auto","created_at":"2025-01-14 04:14:33","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":313371,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5564984/v1/8968a55f66ae1d876e5af502.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExtracellular vesicles (EVs) are small membrane enclosed spheres shed by cells across all domains of life. EVs have been reported for many bacterial species, with their morphology and composition well-described. The cargo of bacterial EVs can vary, including membrane components, surface-structures, signalling molecules, periplasmic and cytoplasmic proteins, as well as nucleic acids \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Bacterial EVs have been linked to diverse functions, such as waste disposal, nutrient scavenging and growth, molecule export, phage interaction, antibiotic resistance, bactericidal activity, delivery of virulence factors and toxins to host cells, and modulation of host immune responses \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn Gram-negative bacteria, vesicles are shed from the outer membrane and thus called outer membrane vesicles (OMVs), while Gram-positive bacteria shed vesicles from their inner membrane, called membrane vesicles (MVs). The pleomorphic bacteria of the class \u003cem\u003eMollicutes\u003c/em\u003e originated from Gram-positive ancestors via reductive evolution. They are distinguished by their small genome, minute size ranging from 100 to 800 nm \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and the absence of a cell wall. \u003cem\u003eMollicutes\u003c/em\u003e are only enclosed by a cytoplasmic membrane and a few species have been reported to produce EVs so far \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eMollicutes\u003c/em\u003e EVs were first observed in \u003cem\u003eAcholeplasma laidlawii\u003c/em\u003e and \u003cem\u003eMycoplasma gallisepticum\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and later described in \u003cem\u003eMycoplasma mycoides\u003c/em\u003e subsp. \u003cem\u003emycoides, M. mycoides\u003c/em\u003e subsp. \u003cem\u003ecapri\u003c/em\u003e (\u003cem\u003eMmc\u003c/em\u003e), \u003cem\u003eM. capricolum\u003c/em\u003e subsp. \u003cem\u003ecapricolum, M. agalactiae, M. fermentans\u003c/em\u003e, and \u003cem\u003eM. bovis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the proteome of these EVs relative to the homologous strain, as well as their functional and mechanistic significance, remains unexplored.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. bovis\u003c/em\u003e is the causative agent of bovine respiratory complex disease (BRD), characterized by enzootic pneumonia, pleuritis, and polyarthritis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Given that EVs of other bacteria are known to influence pathophysiology and modulate immune responses \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and that the host immune response to \u003cem\u003eM. bovis\u003c/em\u003e has been characterized \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eM. bovis\u003c/em\u003e-derived EVs represent a relevant candidate for investigating the ability of EVs to trigger immune cell activation in bovine host cells.\u003c/p\u003e \u003cp\u003eIn addition to immune responses, EVs are increasingly reported as mediators for horizontal gene transfer (HGT) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Vesicular transfer of plasmids was first described in Archaea and termed \u0026ldquo;vesiduction\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This vesicle-mediated plasmid transfer has been described in a number of Gram-negative bacteria \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, while in Gram-positive bacteria, it has only been observed in \u003cem\u003eEnterococcus faecalis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Within the \u003cem\u003eMollicutes\u003c/em\u003e, members of the \u003cem\u003eM. mycoides\u003c/em\u003e cluster have been reported to harbour plasmids \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and exchange genes horizontally \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, prompting us to explore vesiduction in \u003cem\u003eMycoplasma\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this work we characterized EVs of two veterinary pathogens, \u003cem\u003eM. bovis\u003c/em\u003e and \u003cem\u003eMmc\u003c/em\u003e, focusing on their composition and functional roles. Specifically, we investigated their ability to trigger host immune responses and mediate HGT.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eBacterial culture and strains.\u003c/b\u003e For standard liquid bacterial culture, strains were inoculated at a dilution of 1:1000 in SP5 medium \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, supplemented with 5 \u0026micro;g/ml tetracycline when necessary, and grown in static culture at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e overnight for 16\u0026thinsp;\u0026plusmn;\u0026thinsp;2 h or until color change from red to orange. Colonies were observed on SP5 agar plates or modified Hayflick agar plates \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, supplemented with 5 \u0026micro;g/ml tetracycline when selection was necessary.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycoplasma mycoides\u003c/em\u003e subsp. \u003cem\u003ecapri\u003c/em\u003e (\u003cem\u003eMmc\u003c/em\u003e) GM12, \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08 (carrying the tetracycline resistance gene on the 6.079 kbp \u003cem\u003eoriC\u003c/em\u003e-plasmid pIVB08) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eMmc\u003c/em\u003e 152/93, which carries the small 1.875 kbp plasmid pKMK1 \u003csup\u003e24,25\u003c/sup\u003e, as well as \u003cem\u003eM. bovis\u003c/em\u003e Donetta PG45 (ATCC 25523) were used for EV isolation.\u003c/p\u003e \u003cp\u003eThe attenuated \u003cem\u003eMmc\u003c/em\u003e GM12::YCpMmyc1.1-Δ68 \u003csup\u003e26\u003c/sup\u003e was used as a negative control for the MIB-MIP activity assay.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMycoplasma leachii\u003c/em\u003e PG50 (\u003cem\u003eM. leachii\u003c/em\u003e), \u003cem\u003eMycoplasma capricolum\u003c/em\u003e subsp. \u003cem\u003ecapricolum\u003c/em\u003e California kid (ATCC27343) (\u003cem\u003eMcap\u003c/em\u003e), and its derivative mutant strain \u003cem\u003eMycoplasma capricolum\u003c/em\u003e subsp. \u003cem\u003ecapricolum\u003c/em\u003e ΔRE (\u003cem\u003eMcap\u003c/em\u003e ΔRE), which lacks restriction enzyme activity due to inactivation of the CCATC-restriction enzyme gene \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, were used as recipients in vesiduction experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEV Isolation.\u003c/b\u003e EVs were isolated from late exponential growth phase of bacterial liquid culture in 200 ml SP5. The EV isolation protocol is based on previous studies \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, with few modifications. Bacterial cells were pelleted by centrifugation at 4.000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min at 4\u0026deg;C and the supernatant was subjected to serial filtration: first through a 0.45 \u0026micro;m, followed by a 0.22 \u0026micro;m and a 0.1 \u0026micro;m pore filter (Millipore Steritop vacuum bottle top filter, Sigma, Merck).\u003c/p\u003e \u003cp\u003eThe sterile supernatant was ultracentrifuged at 100.000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 2 h at 4\u0026deg;C (SW32Ti rotor using six 38.5 ml Open-Top Thinwall Ultra-Clear Tube, 25 \u0026times; 89mm, BeckmanCoulter) and then the supernatant was aspirated with a serological pipette until approximately 1 ml liquid was left in each of the six tubes. The EV-containing samples were resuspended in the remaining liquid, pooled and then PBS was added up to 13 ml and centrifuged at 100.000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 2 h at 4\u0026deg;C (SW41Ti rotor, 13.2 ml Open-Top Thinwall Ultra-Clear Tube, 14 \u0026times; 89mm, BeckmanCoulter). Again, the supernatant was aspirated with a serological pipette until approximately 400 \u0026micro;l containing the EVs were left. This EV containing sample was stored at -80\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEV characterisation.\u003c/b\u003e The protein content of the samples was quantified using a Qubit fluorometer (Thermo Fisher Scientific) and analysed using nanoparticle tracking. Samples were diluted in PBS to a final volume of 1 ml (10\u003csup\u003e8\u003c/sup\u003e \u0026ndash; 10\u003csup\u003e9\u003c/sup\u003e particles/ml equal to 10\u0026ndash;100 particles/frame). Then, they were recorded using a NanoSight NS300 instrument with a 405 nm laser (Malvern Panalytical, The Netherlands). Settings were adjusted according to manufacturer\u0026rsquo;s software manual (NanoSight NS300 User Manual, NanoSight 3.4): camera level was set to 11 then adjusted until particles were seen clearly and no more than 20% were saturated and the infusion rate was set to 1000. For each measurement, five videos were captured and after capture, the videos were analysed using the in-build NanoSight Software NTA 3.4. The detection threshold was set to include as many particles as possible with the restrictions that 10\u0026ndash;100 red crosses were counted while blue cross count was limited to 5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProteomic analysis.\u003c/b\u003e Three biological replicates were analysed regarding the proteomic content of the EVs and bacterial cells per strain. Per replicate, EVs were isolated from 80 ml of \u003cem\u003eMmc\u003c/em\u003e GM12 and \u003cem\u003eM. bovis\u003c/em\u003e PG45, washed in PBS and pelleted as described above, but here the supernatant was aspirated as much as possible, and the dried pellets were stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor comparison of the proteomic content to bacterial cells, the latter were harvested from 1 ml cultures via centrifugation at 14.000 x \u003cem\u003eg\u003c/em\u003e, washed with an equal volume of PBS, centrifuged at 7.000 x \u003cem\u003eg\u003c/em\u003e and then stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eSamples were subjected to proteomic analysis using comparative shot gun proteomics in an orbitrap LC-MS system (ThermoFisher Scientific). Mass spectrometry-derived proteomic data were analysed against the genomes CP001621.1 (\u003cem\u003eMmc\u003c/em\u003e GM12) and CP002188.1 (\u003cem\u003eM. bovis\u003c/em\u003e PG45). Data were interpreted by the software Spectronaut, in the hybrid directDIA+ (Deep) mode and IBAQ \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e values (Intensity-Based Absolute Quantification, per protein) were reported, and relative abundance (rAbu) was calculated based on the IBAQ leading protein, so that the sum of rAbu is 1000000 for every individual sample.\u003c/p\u003e \u003cp\u003eThe subcellular localisation of the detected proteins was predicted based on their amino acid sequence using PsortB \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Proteins predicated as \u0026ldquo;CellWall\u0026rdquo; were grouped with \u0026ldquo;Unknown\u0026rdquo; and the percentage of proteins per subcellular localization, Cytoplasmic, CytoplasmicMembrane, Extracellular and Unknown was calculated based on the sum of their rAbu values.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMIB-MIP activity of EV.\u003c/b\u003e IgG cleavage assays were performed as described earlier \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, to test whether EVs display \u003cem\u003eMycoplasma\u003c/em\u003e immunoglobulin binding-protease (MIB-MIP) activity. EVs were isolated from 200 ml culture, washed once with SP5 w/o serum, and resuspended in 90 \u0026micro;l SP5 w/o serum. One ml bacterial culture of \u003cem\u003eMmc\u003c/em\u003e GM12 and \u003cem\u003eMmc\u003c/em\u003e GM12::YCpMmyc1.1-Δ68 were pelleted at 7000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, washed once with SP5 w/o serum, and also resuspended in 90 \u0026micro;l SP5 w/o serum. Purified caprine IgG (H\u0026thinsp;+\u0026thinsp;L, Sigma-Aldrich) was added to a final concentration of 100 ng/\u0026micro;l and samples were incubated at 37\u0026deg;C for 60 min, or 120 min. Samples containing bacterial cell were pelleted, and only the supernatant was used further, while EV containing samples were used entirely. Samples were mixed with 6\u0026times; Laemmli buffer before boiling at 100\u0026deg;C for 10 min. Samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore) using a Trans-Blot Turbo Transfer System (BioRad). The membrane was blocked in PBS with 5% skimmed milk (Becton Dickinson) and 0.05% Tween-20 (Sigma). IgG (H\u0026thinsp;+\u0026thinsp;L) was detected with polyclonal mouse anti-goat antibodies (AffiniPure, Jackson ImmunoResearch Laboratories) in 5% skim milk PBS-T 1:1000 followed by polyclonal Rabbit Anti-Mouse IgG HRP (Dako Agilent) in 5% skim milk PBS-T 1:2000. The membrane was developed using SuperSignal West Pico PLUS Chemiluminiscent substrate (ThermoFisher Scientific).\u003c/p\u003e \u003cp\u003e \u003cb\u003eNuclease activity of EV.\u003c/b\u003e Nuclease activity assays were performed with a protocol based on a previous study \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Briefly, samples (bacterial pellet of 0.5 ml bacterial culture, or EVs pellet of 60 ml supernatant) were washed in SP5 w/o serum, and then incubated with 1 \u0026micro;g of the plasmid pIVB08 or \u003cem\u003eEco\u003c/em\u003eRI-digested pIVB08 (pIVB08\u003cem\u003eEco\u003c/em\u003eRI) in 100 \u0026micro;l SP5 w/o serum at 37\u0026deg;C for 60 min. To test nuclease activity in \u003cem\u003eMmc\u003c/em\u003e GM12, CaCl\u003csub\u003e2\u003c/sub\u003e and MgCl\u003csub\u003e2\u003c/sub\u003e were added to a final concentration of 10 mM. Nuclease activity was inhibited by addition of 1 mM or 10 mM of EDTA in assays containing cells and EVs derived from \u003cem\u003eMmc\u003c/em\u003e GM12 or \u003cem\u003eM. bovis\u003c/em\u003e PG45, respectively. The supernatant of the samples was visualized on a 2% agarose in TAE gel.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmune response of ruminant primary blood cells to\u003c/b\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003eand its EV.\u003c/b\u003e Fresh bovine peripheral blood mononuclear cells (PBMCs) were isolated and stimulated as described earlier \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, using an MOI of 0.1 of \u003cem\u003eM. bovis\u003c/em\u003e bacterial cells or EVs shed by 2x10\u003csup\u003e8\u003c/sup\u003e CFUs of \u003cem\u003eM. bovis\u003c/em\u003e. In brief, PBMCs were isolated from the blood of eight Holstein Frisian cows (age 1\u0026ndash;3 years) and seeded at 2\u0026nbsp;million cells per ml and well. EVs were isolated from 200 ml of \u003cem\u003eM. bovis\u003c/em\u003e in SP5 as described above and resuspended in 1 ml of Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) or diluted 100x in DMEM/10% FBS. 100 \u0026micro;l DMEM/10% FBS containing either bacteria or EVs were added to the PBMCs in 1 ml DMEM/10% FBS in flat-bottom 12-well plates, and finally incubated at 38.5\u0026deg;C (ruminant body temperature) and 5% CO\u003csub\u003e2\u003c/sub\u003e for 16 h.\u003c/p\u003e \u003cp\u003eThe different bovine immune cell subtypes were identified by flow cytometry using a 7-step, 12-color staining protocol. Combination staining analysed monocytes (classical, intermediate and non-classical), conventional type 1 and 2 dendritic cells (cDC1s and cDC2s) and plasmacytoid dendritic cells (pDCs). For the acquisitions, at least 100.000 events were recorded for each sample. For the fold-change induction of cellular surface marker following stimulation, the mean fluorescence intensity (MFI) measured in a stimulated sample for a given animal was normalized to the MFI measured in unstimulated sample from that same animal. FCM acquisitions were performed on a Cytek Aurora (Cytek Biosciences) using the SpectroFlo software with autofluorescence extraction, and further analysed with FlowJo 10.9.0. (TreeStar).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of plasmids in EVs.\u003c/b\u003e The plasmid content of the EVs was determined by quantitative PCR. First, extravesicular DNA was removed by adding 1 \u0026micro;l of DNase I at 10 U/\u0026micro;l (Roche) per ml of the EV sample and incubated at 37\u0026deg;C for 30 min, before the DNase was inactivated at 75\u0026deg;C for 10 min. The DNase treated EV sample was then resuspended in 12 ml PBS, pelleted as described above and finally lysed by resuspending it in 50 \u0026micro;l cell lysis solution (Wizard Genomic DNA Purification Kit, Promega). Bacterial cells of 8 ml culture in SP5 were pelleted, washed in PBS and resuspended in 500 \u0026micro;l cell lysis solution (Wizard Genomic DNA Purification Kit, Promega).\u003c/p\u003e \u003cp\u003eqPCR was conducted in a 10 \u0026micro;l reaction volume with template DNA equalized to 600 ng (EV lysate or bacterial cell lysate) and primers detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. SsoFast EvaGreen Supermix (Biorad) was used according to manufacturer\u0026rsquo;s instruction and the reaction was run for 40 cycles in a QuantStudio\u0026trade; 5 System machine (ThermoFisher Scientific).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransformation of\u003c/b\u003e \u003cb\u003eMmc\u003c/b\u003e. An \u003cem\u003eMmc\u003c/em\u003e strain carrying a plasmid with a selectable marker was constructed for vesiduction experiments, as \u003cem\u003eMycoplasma\u003c/em\u003e lack natural antibiotic resistance plasmids. \u003cem\u003eMmc\u003c/em\u003e GM12 was transformed using a polyethylene glycol 8000 (PEG\u003csub\u003e8000\u003c/sub\u003e) mediated-protocol described elsewhere \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e with few modifications. Briefly, 4 ml of \u003cem\u003eMmc\u003c/em\u003e GM12 culture grown to late exponential phase was spun down at 4000 x \u003cem\u003eg\u003c/em\u003e 4\u0026deg;C for 15 min and the pellet was washed once in S/T buffer (250 mM Sucrose, 10 mM Tris-HCl pH 7). Then, the pellet was resuspended in 400 \u0026micro;l of 0.1 M CaCl\u003csub\u003e2\u003c/sub\u003e and cells were incubated on ice for 30 min. In a 50 ml Falcon tube, 10 \u0026micro;l of the pIVB08 plasmid \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e (approximately 10 \u0026micro;g) were added to 410 \u0026micro;l of 2 \u0026times; Fusion buffer (500 mM Sucrose, 20 mM Tris-HCl pH 7, 40% PEG8000) and left at room temperature. After the incubation on ice, cells were added to the 2 \u0026times; Fusion Buffer containing the DNA and swirled gently. The mixture was incubated for 25 min at 30\u0026deg;C and then the fusion reaction was stopped by addition of 9 ml SP5 medium. Cells were collected by centrifugation at 4000 x \u003cem\u003eg\u003c/em\u003e for 15 min and the resulting pellet was resuspended in 1 ml of SP5. Cells were incubated at 37\u0026deg;C for 1 h and then plated onto selective Hayflick agar plates.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmid transfer via EVs.\u003c/b\u003e To assess whether the tetracycline resistance encoding plasmid pIVB08 can be transferred via vesiduction, EVs were isolated from \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08. EVs were DNase-treated (without heat-inactivation) to remove extravesicular DNA.\u003c/p\u003e \u003cp\u003eInitially, vesiduction experiments were conducted by co-incubation (protocol A) of the recipient cells with DNase-treated EVs of \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08 in 400 \u0026micro;l PBS.\u003c/p\u003e \u003cp\u003eFurther, the genome transplantation protocol described elsewhere \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e was adapted (protocol B). Briefly, recipient cells were grown in SP5 medium until early stationary phase (pH 6.5), washed in Wash Buffer (10 mM Tris, 250 mM NaCl pH 6.5), and resuspended in cold 0.1 M CaCl\u003csub\u003e2\u003c/sub\u003e. Four hundred \u0026micro;l DNase-treated EVs were mixed with the recipient cells resuspended in 2 \u0026times; Wash Buffer (20 mM Tris, 500 mM NaCl pH 6.5) with 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e and centrifuged for 15 min at 10.000 x \u003cem\u003eg\u003c/em\u003e. Mixtures were incubated statically for 90 min at 37\u0026deg;C, resuspended in 5 ml SP5, spun down for 15 min at 5.800 x \u003cem\u003eg\u003c/em\u003e, resuspended in 500 \u0026micro;l SP5 then plated on selective Hayflick agar and incubated for 5\u0026ndash;7 days at 37\u0026deg;C 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eTo estimate the maximal vesiduction rate, 10% PEG\u003csub\u003e6000\u003c/sub\u003e was added to the 2 x Wash Buffer with 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e (protocol C), as in the original genome transplantation protocol \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the potential of heterologous vesiduction, the phylogenetically related strains \u003cem\u003eM. leachii\u003c/em\u003e, \u003cem\u003eMcap\u003c/em\u003e and its mutant \u003cem\u003eMcap\u003c/em\u003e ΔRE, were used as recipients. To screen for transformants obtained by vesiduction, colonies were picked in 1 mL selective SP5 and lysed using cell lysis solution (Wizard Genomic DNA Purification Kit, Promega). One \u0026micro;l of lysed cells was used as template in a PCR using the \u003cem\u003etet\u003c/em\u003e -specific primers for the plasmid pIVB08 and strain-specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) in a 10 \u0026micro;l reaction in GoTaq G2 Green Master Mix (Promega, United States) according to manufacturer\u0026rsquo;s instruction and PCR products were visualized on a 2% agarose in TAE-buffer gel with RedSafe (iNtRON Biotechnology).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEVs inactivation.\u003c/b\u003e EVs were subjected to different stresses, to assess the necessity of an intact membrane for vesiduction. For alkaline lysis, pH was raised to pH 10 by addition of 5 \u0026micro;l NaOH 1 M per 400 \u0026micro;l of bacterial or EV sample, incubated for 30 min at room temperature and the neutralized by adding 5 \u0026micro;l HCl 1 M per 400 \u0026micro;l EV sample. For heat stress, bacterial cells or EVs were incubated for 30 min at 50\u0026deg;C or 56\u0026deg;C. For digestion of extravesicular proteins, the EV sample was incubated with 1 \u0026micro;l of Proteinase K (10 \u0026micro;g/ml, Roche) at 37\u0026deg;C for 30 min, as reported elsewhere \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Vesiduction experiments were then conducted with the stress treated \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08 EVs as described above, with \u003cem\u003eMcap\u003c/em\u003e ΔRE as recipient and transformants were observed on selective Hayflick agar plates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical analysis was done using the GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). A \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (* \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eMollicutes\u003c/b\u003e \u003cb\u003eEVs represent the overall proteomic profile of the bacterial cell.\u003c/b\u003e EV isolation was first optimized with bacterial cultures of \u003cem\u003eMmc\u003c/em\u003e GM12. Isolation of \u003cem\u003eMycoplasma\u003c/em\u003e EV requires the use of a 0.1 \u0026micro;m filter, since \u003cem\u003eMycoplasma\u003c/em\u003e bacterial cells can pass a 0.22 \u0026micro;m filter (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Here, a serial filtration protocol was employed starting with a 0.45 \u0026micro;m filter, followed by a 0.22 \u0026micro;m filter and a final step using a 0.1 \u0026micro;m filter. Nanoparticle tracking analysis using NanoSight did not allow to distinguish between EVs and the background of the growth medium, even when FBS in standard SP5 medium was reduced from 17\u0026ndash;2% (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo assess protein content of the isolated EVs from \u003cem\u003eMmc\u003c/em\u003e and \u003cem\u003eM. bovis\u003c/em\u003e, proteome analysis of both EV and bacterial sample was conducted. Based on intensity-based absolute quantification (iBAQ) values, proteomics showed that 99.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% were \u003cem\u003eMycoplasma\u003c/em\u003e protein in the bacterial cell samples of \u003cem\u003eMmc\u003c/em\u003e GM12 and 83.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53% were \u003cem\u003eMycoplasma\u003c/em\u003e protein in EV samples of \u003cem\u003eMmc\u003c/em\u003e GM12, while 94.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85% were \u003cem\u003eM. bovis\u003c/em\u003e protein in the bacterial cell samples of \u003cem\u003eM. bovis\u003c/em\u003e PG45 and 28.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13% were \u003cem\u003eM. bovis\u003c/em\u003e protein in EV samples. A total of 691 \u003cem\u003eMycoplasma\u003c/em\u003e proteins were identified in \u003cem\u003eMmc\u003c/em\u003e GM12 samples (17 unique in bacterial cell; 3 unique in EV) and 565 \u003cem\u003eM. bovis\u003c/em\u003e proteins were identified in \u003cem\u003eM. bovis\u003c/em\u003e samples (89 unique in bacterial cell; 2 unique in EV). EV-associated proteins represent a subset of the proteomic profile of the producer strain (Fig.\u0026nbsp;1AB, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The relative abundance of proteins of subcellular localisation predicted with PSortB was similar in the EV samples compare to the whole cell lysates for \u003cem\u003eMmc\u003c/em\u003e GM12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), while cytoplasmic membrane proteins had a slightly higher abundance in EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThirteen membrane proteins potentially involved in \u003cem\u003eMollicute\u003c/em\u003e-host interaction (lipoproteins p37 \u003csup\u003e34\u003c/sup\u003e, LppB \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and LppA p72 \u003csup\u003e36\u003c/sup\u003e, elongation factor TU \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, chaperone protein DnaK (Hsp70) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, glyceraldehyde-3-phosphate dehydrogenase \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, pyruvate kinase \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, lactate dehydrogenase \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, phosphoglycerate mutase \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, transketolase \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, PTS glucose permease ptsG \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, pyruvate dehydrogenase E1 subunit α and β \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e) were detected in both the bacterial and the EV samples of \u003cem\u003eMmc\u003c/em\u003e GM12. The elongation factor TU, which is a putative factor in \u003cem\u003eMycoplasma\u003c/em\u003e interaction with host extracellular matrix, and lactate and glyceraldehyde dehydrogenase were the most abundant ones, both in the bacterial cells and the EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAdhesion is an essential attribute in virulence of \u003cem\u003eM. bovis\u003c/em\u003e and several proteins mediating adhesion to fibronectin, plasminogen or epithelial cells have been described (NOX \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, MbfN \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, Fba \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, TrmFO \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, α-enolase \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, MilA \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, P27 \u003csup\u003e46\u003c/sup\u003e, VpmaX \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, variable surface lipoproteins Vps\u0026rsquo; \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, Mbov_0503 cytoadhesin \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, MBOVJF4278_00255 and MBOVJF4278_00667 \u003csup\u003e50\u003c/sup\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the described adhesion related proteins were present in bacterial and EV samples of \u003cem\u003eM. bovis\u003c/em\u003e. The variable surface lipoprotein VspA was the most abundant adhesion related protein in both bacteria and EVs of \u003cem\u003eM. bovis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFunctional proteins are associated with\u003c/b\u003e \u003cb\u003eMollicutes\u003c/b\u003e \u003cb\u003eEVs.\u003c/b\u003e In the proteomic profile of \u003cem\u003eMmc\u003c/em\u003e GM12 and its EVs all components of the MIB-MIP gene cluster as well as three subunits of the adjacent ATPase were detected (Figure S3A). IgG cleavage could not be detected in EVs derived from 200 ml \u003cem\u003eMmc\u003c/em\u003e GM12 culture even when incubation was prolonged from 1 h up to 3 h (Figure S3B), which might be due to altered rations of binding proteins, proteases and ATPases in the EV sample or the detection limit.\u003c/p\u003e \u003cp\u003eSince nucleases were not previously described in \u003cem\u003eMmc\u003c/em\u003e, putative nucleases were predicted based on conserved domains in NCBIs Conserved Domain Search \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and four putative nucleases were present in the proteome of both \u003cem\u003eMmc\u003c/em\u003e GM12 bacterial cells and EVs at comparable levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In \u003cem\u003eMmc\u003c/em\u003e GM12 bacterial samples nuclease activity was detected for the linearized plasmid pIVB08\u003cem\u003eEco\u003c/em\u003eRI in presence of salts (10 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) and inhibited by addition of 1 mM EDTA. This exonuclease activity was also detected in the EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eNucleases present in the proteome of \u003cem\u003eM. bovis\u003c/em\u003e PG45 (MslA MBOVPG45_0311 \u003csup\u003e52\u003c/sup\u003e, MbovNase MBOVPG45_0310 \u003csup\u003e32,53\u003c/sup\u003e, MnuA MBOVPG45_0215 \u003csup\u003e32,54\u003c/sup\u003e, 5\u0026rsquo;-nucleotidase MBOVPG45_0690 \u003csup\u003e55\u003c/sup\u003e) were also present in the EV samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Nuclease activity was detected in both the bacterial cells and EVs, and the reaction could be inhibited by addition of 10 mM EDTA. Both the circular plasmid pIVB08 and its linearized version pIVB08\u003cem\u003eEco\u003c/em\u003eRI were degraded, while plasmid degradation was not observed in the SP5 w/o serum control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEVs can elicit an\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003eimmune response.\u003c/b\u003e To investigate whether EVs elicit an immune response in the native host of \u003cem\u003eM. bovis\u003c/em\u003e, bovine PBMCs were exposed to EVs. The response was compared to that induced by live \u003cem\u003eM. bovis\u003c/em\u003e PG45, which has been recently characterized \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. EVs stimulated bovine immune cells in a pattern similar to \u003cem\u003eM. bovis\u003c/em\u003e bacterial cells, though the response was generally lower. Both bacterial cells and EVs induced a marked response of dendritic cells (DCs), known to play a crucial role in bridging innate and adaptive immunity, especially CD25, related to cellular activation, and to a lesser extent CCR7, related to cell migration towards draining lymph nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The previously observed immune response diminished with EV dilution, as witnessed by lower CD25 and CCR7 induction on all DC subsets. Monocytes, key players of innate immunity, showed less stimulation in response to \u003cem\u003eM. bovis\u003c/em\u003e, in line with our previous study \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As such, the specific effect of EVs was more difficult to demonstrate on this immune cell subset (Supplementary Figure S4, A-I). Lastly, both EVs and live bacterial cells induced comparable levels of primary cell death relative to corresponding unstimulated PBMCs (Supplementary Figure S4J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMollicutes\u003c/b\u003e \u003cb\u003eEVs can pack plasmid DNA.\u003c/b\u003e qPCR showed that EVs derived from \u003cem\u003eMmc\u003c/em\u003e 152/93 contain the small natural plasmid pKMK1 present in this \u003cem\u003eMmc\u003c/em\u003e strain \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In the EV lysate, the CT values of two plasmid regions were significantly lower than the CT values of two chromosomal genes (sigma factor \u003cem\u003erpoA\u003c/em\u003e and cell division factor \u003cem\u003eftsZ\u003c/em\u003e), translating to 3 x 10\u003csup\u003e6\u003c/sup\u003e times more pKMK1 then chromosomal DNA in the EVs. In the whole cell lysate, the CT values of the plasmid regions were comparable to those of chromosomal genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, CT values for individual targets are illustrated in Supplementary Figure S5).\u003c/p\u003e \u003cp\u003eSimilarly, in EVs derived from \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08 carrying the plasmid pIVB08 with the resistance markers for ampicillin and tetracycline, \u003cem\u003eamp\u003c/em\u003e and \u003cem\u003etet\u003c/em\u003e, the CT values of the two plasmid markers were significantly lower than the CT values of the chromosomal genes, \u003cem\u003erpoA\u003c/em\u003e and \u003cem\u003eftsZ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, CT values for individual targets are illustrated in Supplementary Figure S5), meaning that 200 times more pIVB08 compared to chromosomal DNA was detected in the EVs. Of note, the CT values of the small natural plasmid pKMK1 were lower than of the larger \u003cem\u003eoriC\u003c/em\u003e-plasmid pIVB08.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eVesiduction: Plasmid transfer via EVs.\u003c/b\u003e To investigate whether EVs can transfer plasmids to other bacterial cells, vesiduction experiments were conducted. Since \u003cem\u003eMycoplasma\u003c/em\u003e lack natural antibiotic resistance plasmids, the \u003cem\u003eoriC-\u003c/em\u003eplasmid pIVB08, a replicative plasmid based on the origin of replication (\u003cem\u003eoriC\u003c/em\u003e) sequence of the chromosome \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, was used.\u003c/p\u003e \u003cp\u003eFirst, homologous vesiduction was tested between \u003cem\u003eMmc\u003c/em\u003e GM12 pIVB08 derived EVs and plasmid-free \u003cem\u003eMmc\u003c/em\u003e GM12. Using protocol A (co-incubation), six transformants per 10\u003csup\u003e11\u003c/sup\u003e CFU were obtained. With protocol B, seven transformants per 10\u003csup\u003e11\u003c/sup\u003e CFU were recovered (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Figure S6).\u003c/p\u003e \u003cp\u003eNext, the capacity of EVs to transfer the plasmid to other species was assessed. Heterologous vesiduction experiments with \u003cem\u003eMcap\u003c/em\u003e and \u003cem\u003eM. leachii\u003c/em\u003e as recipients resulted in successful plasmid transfer, with frequencies of four per 10\u003csup\u003e10\u003c/sup\u003eCFU for \u003cem\u003eMcap\u003c/em\u003e pIVB08 and six per 10\u003csup\u003e10\u003c/sup\u003e CFU for \u003cem\u003eM. leachii\u003c/em\u003e pIVB08 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PCR analysis confirmed vesiduction in at least five colonies per experimental setup, with a pIVB08 specific gene (\u003cem\u003etet\u003c/em\u003e) and a strain specific gene (Supplementary Figure S6, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe maximum vesiduction rate was established using protocol C, which includes the addition of 10% PEG\u003csub\u003e6000\u003c/sub\u003e in the 2x Wash Buffer. This yielded one per 5x10\u003csup\u003e7\u003c/sup\u003eCFUs, compared to four per 10\u003csup\u003e10\u003c/sup\u003eCFU using protocol B (without PEG 6000, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To investigate the impact of restriction-modification system on vesiduction, the strain \u003cem\u003eMcap\u003c/em\u003e ΔRE, which cannot restrict incoming DNA \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, was used. Transformants, \u003cem\u003eMcap\u003c/em\u003e ΔRE pIVB08, were recovered at comparable rates (6 per 10\u003csup\u003e11\u003c/sup\u003eCFU, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine whether vesiduction requires intact EVs, they were subjected to three different conditions: alkaline lysis, heat stress or Proteinase K treatment. A pilot experiment on alkaline stress on bacterial cells showed that pH 10 causes a 10\u003csup\u003e10\u003c/sup\u003e-fold reduction in viable bacterial cells. When EVs were subjected to the same alkaline lysis prior to a vesiduction experiment, no transformants were recovered.\u003c/p\u003e \u003cp\u003eIn a heat stress pilot, bacterial cell viability remained unaffected by incubation at 45\u0026deg;C for 10 or 30 min. However, at 50\u0026deg;C for 10 minutes, a 10\u003csup\u003e2\u003c/sup\u003e-fold reduction was observed, and 50\u0026deg;C for 30 min caused a 10\u003csup\u003e5\u003c/sup\u003e-fold reduction. No live bacterial cells were recovered at 56\u0026deg;C for 10 or 30 min. To then subject EVs to intermediate and strong heat stress, they were heated for 30 min at 50 and 56\u0026deg;C. When heat-stressed EVs were used for vesiduction, no transformants were recovered.\u003c/p\u003e \u003cp\u003eFinally, EVs were treated with Proteinase K (10 \u0026micro;g/ml at 37\u0026deg;C for 30 minutes) to digest extravesicular proteins. This treatment did not affect the vesiduction rate (2.5 per 10\u003csup\u003e10\u003c/sup\u003eCFU, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePlasmid pIVB08 transfer frequencies in homo- and heterologous vesiduction.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecipient\u003c/p\u003e \u003cp\u003ecell\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEV sample\u003c/p\u003e \u003cp\u003etreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVesiduction\u003c/p\u003e \u003cp\u003eprotocol*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransfer\u003c/p\u003e \u003cp\u003efrequency\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMmc\u003c/em\u003e GM12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 per 10\u003csup\u003e11\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMmc\u003c/em\u003e GM12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 per 10\u003csup\u003e11\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM. leachii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 per 10\u003csup\u003e10\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 per 10\u003csup\u003e10\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 per 10\u003csup\u003e11\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 per 5x10\u003csup\u003e10\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 per 5x10\u003csup\u003e7\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlkaline lysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMcap\u003c/em\u003eΔRE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteinase K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5 per 10\u003csup\u003e10\u003c/sup\u003eCFU\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e*\u003c/sup\u003eA co-incubation, B standard protocol, C standard protocol with 10% PEG\u003csub\u003e6000\u003c/sub\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pleomorphic, cell wall-less nature and small size of \u003cem\u003eMollicutes\u003c/em\u003e challenged EV isolation, detection and characterisation \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The minute size and lack of a cell wall of \u003cem\u003eMollicutes\u003c/em\u003e hinders distinguishing EVs from live bacterial cells, unlike in cell wall enclosed, larger bacteria. Here we present an isolation protocol employing serial filtration steps to remove all live bacterial cells from the EV containing sample. EVs of other bacteria are approximately one tenth of the size of the producing cell, with an EV size of 40 to 400 nm \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This would translate to an expected \u003cem\u003eMollicutes\u003c/em\u003e EV size of 10 to 100 nm, which brings current methods, such as Nanoparticle tracing, to a limit. Due to difficulties distinguishing vesicles from bacterial cells and background noise even after reducing FBS concentration, a reliable enumeration method remains elusive. Novel high resolution methods or innovative minimal growth media might overcome this issue in the future. Still, we could manage to identify a clear proteomic profile of \u003cem\u003eMollicutes\u003c/em\u003e EVs and to characterise them functionally.\u003c/p\u003e \u003cp\u003eEVs of other bacteria generally reflect the proteomic content of their producing cell \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, though certain species demonstrate selective enrichment of components, as seen in predatory MVs \u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrate that EVs from both \u003cem\u003eMmc\u003c/em\u003e and \u003cem\u003eM. bovis\u003c/em\u003e reflect the overall proteomic profiles of their homologous strains.\u003c/p\u003e \u003cp\u003eSeveral membrane proteins which are potentially involved in \u003cem\u003eMycoplasma\u003c/em\u003e-host interactions were found to be associated with \u003cem\u003eMmc\u003c/em\u003e GM12 EVs, which is in line with the findings of the proteomic analysis performed on the Triton X-114 enriched fractions of EVs of \u003cem\u003eMycoplasma mycoides\u003c/em\u003e subsp. \u003cem\u003emycoides\u003c/em\u003e strain Afad\u0026eacute; \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Numerous previously characterized adhesins were also detected in the \u003cem\u003eM. bovis\u003c/em\u003e bacterial and EV samples. Lipoproteins were abundant in both bacteria and EVs of \u003cem\u003eM. bovis\u003c/em\u003e PG45, especially VspA and VspE. In related \u003cem\u003eMollicutes\u003c/em\u003e, lipoproteins have been described for their activating and evasive immunomodulatory effect \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe detected the components of the MIB-MIP gene cluster in the EV sample, but immunoglobulin cleavage levels of EVs were below detection limit, potentially due to low protein amounts in the EV sample or different protein ratios required for successful MIB-MIP activity.\u003c/p\u003e \u003cp\u003eWe illustrate that EVs contain functional proteins through their nuclease activity. \u003cem\u003eM. bovis\u003c/em\u003e nucleases have a dual functionality, enabling nutritional acquisition by degradation of host nucleic acids required for nutritional supply \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e and immune evasion \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The major membrane nuclease MnuA and the nucleotidase 5\u0026rsquo;NT act in an enzymatic cascade to degrade and hydrolyse nucleotides, which can be transported through the cell membrane \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eM. bovis\u003c/em\u003e also induces the release of neutrophil extracellular traps, which MnuA degrades to allow immune escape \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The nucleotidase 5\u0026rsquo;NT plays a role in virulence in bovine mastitis, particularly in the elicitation of inflammatory changes \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Here, we found that nucleases were present in the proteomic profile of \u003cem\u003eM. bovis\u003c/em\u003e bacterial cells and their EVs. In line with previous findings \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, bacterial cells of \u003cem\u003eM. bovis\u003c/em\u003e were able to cleave the circular pIVB08 and the linearized pIVB08\u003cem\u003eEco\u003c/em\u003eRI plasmids, indicating presence of endo- and exonuclease activity. For \u003cem\u003eM. bovis\u003c/em\u003e PG45 EVs the same nuclease activity was detected. Nuclease activity remained uncharacterized in \u003cem\u003eMmc\u003c/em\u003e until now, even though bacterial cells of the closely related \u003cem\u003eMcap\u003c/em\u003e have been shown to digest extracellular DNA, with magnesium dependence and calcium inhibition \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The homolog to the \u003cem\u003eMcap\u003c/em\u003e CK predicted nuclease MCAP_0027 in \u003cem\u003eMmc\u003c/em\u003e GM12, MMCAP1_0024 (80.09% identity), was not detected in the EV proteome. However, based on conserved domains, we identified four putative nucleases which were present in the proteome of bacterial cells as well as their EVs and might exert oligonucleotide degradation. We confirmed nuclease activity by observing DNA degradation by \u003cem\u003eMmc\u003c/em\u003e bacterial cells and EVs in the dual presence of MgCl₂ and CaCl₂. These findings highlight the enzymatic functionality of EV-associated proteins and indicate that EVs could be involved in the nucleic acid breakdown required for bacterial growth and immune evasion.\u003c/p\u003e \u003cp\u003eUsing a recently described ruminant primary blood cell ex vivo platform \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we observed that EV stimulation mirrored bacterial cell stimulation, inducing both CD25 and CCR7 upregulation in DCs. Several factors challenge the comparison between live \u003cem\u003eM. bovis\u003c/em\u003e and EVs, such as the lack of a reliable enumeration method for EVs. Additionally, unlike EVs, live cells replicate during experiments and can adjust protein expression in response to host factors. The use of the well-established \u003cem\u003eM. bovis\u003c/em\u003e strain enabled us to directly compare our results to previous findings \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Isolated PBMCs have limitations in terms of timeframe and interplay with other parts of the immune system, as well as not being the first line of defence that \u003cem\u003eM. bovis\u003c/em\u003e would meet during infection. Nonetheless, the experimental design offered the advantage of studying immune responses without the need for animal experiments and being time efficient yet data rich. Herein, this showed the immunomodulatory capacity of purified EVs, particularly their strong effect to stimulate all DC subsets, which suggest that EVs can interact directly with the host immune system during \u003cem\u003eM. bovis\u003c/em\u003e infection. Given that some \u003cem\u003eMollicutes\u003c/em\u003e species reach in vivo concentrations of up to 10 \u003csup\u003e9\u003c/sup\u003e to 10\u003csup\u003e10\u003c/sup\u003eCFU/ml in vivo \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, such high bacterial loads are likely to result in substantial EV shedding, which could modulate the host immune responses.\u003c/p\u003e \u003cp\u003eBeyond their immunomodulatory role, EVs may also serve as vehicles for HGT, emphasizing their multifaceted roles in bacterial-host and bacterial-bacterial interactions. This study provides first experimental evidence of EV-mediated plasmid transfer in \u003cem\u003eMollicutes\u003c/em\u003e. Vesiduction was first described in Archaea \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, whose cell envelope consists of a single membrane, similar to the cell wall-less \u003cem\u003eMollicutes\u003c/em\u003e of the bacteria domain. In Gram-negative bacteria, vesiduction has been subject of a few studies. In \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e, OMVs were shown to transfer a plasmid to recipient cells at frequencies of 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;8 14\u003c/sup\u003e. Further, plasmid containing OMVs isolated from \u003cem\u003eAeromonas veronii\u003c/em\u003e, \u003cem\u003eEnterobacter cloacae\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e were used to induce the transformation of five different species of recipient strains \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and it was subsequently shown that the plasmid size and its copy number influenced the packaging efficiency into vesicles, while the origin of replication affected the absorption rate of the OMVs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Also in \u003cem\u003eKlebsiella\u003c/em\u003e, OMVs have been shown to play a role in interspecies transfer of plasmids containing resistance genes \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e is the only Gram-positive species for which vesicle mediated gene transfer has been shown so far \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, while in \u003cem\u003eEnterococcus faecium\u003c/em\u003e resistance genes were associated with MVs but no resistance transfer was verifiable \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur findings extend prior studies by demonstrating that EVs can transfer plasmids in minute bacteria belonging to the class \u003cem\u003eMollicutes\u003c/em\u003e. Since the characteristics of MGEs influence vesiduction \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, it is likely that larger EVs \u0026ndash; excluded by the filtration method used in this study - could potentially carry different or larger \u003cem\u003eMollicutes\u003c/em\u003e-related MGEs \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. A 2012 study found plasmids in about 30% of \u003cem\u003eMollicutes\u003c/em\u003e belonging to the \u003cem\u003eM. mycoides\u003c/em\u003e phylogenetic group and recombination event signatures suggest plasmid exchange between species sharing a host or niche \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe detection of the small plasmid pKMK1 in EVs, for which the mobilisation route is unknown \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, suggests that vesiduction could be a putative transfer mechanism. Because of the absence of known natural \u003cem\u003eMycoplasma\u003c/em\u003e antibiotic resistance plasmids, we demonstrated EV-mediated plasmid transfer using the \u003cem\u003eoriC-\u003c/em\u003eplasmid pIVB08 in this study. The larger size and artificial nature of pIVB08 likely contributed to its lower abundance in EVs compared to pKMK1.\u003c/p\u003e \u003cp\u003eComparted to other HGT machineries, vesiduction might occur at lower rates. However, it enables the mobilisation of non-replicative plasmids in absence of alternative pathways and adds to the complex interplay of MGEs and distribution of potential virulence and resistance genes. Environmental factors such as host factors or biofilms may enhance vesiduction, particularly in infection or antibiotic stress contexts.\u003c/p\u003e \u003cp\u003eOMVs protect their cargo from enzymatic degradation, including resistance to proteinase treatment \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. This aligns with our finding that proteinase treatment did not impact vesiduction. However, the experiments presented here could not differentiate between the digestion of EV-associated proteins and SP5-associated proteins. We also show that vesiduction depends on membrane integrity, as plasmid transfer was abolished following alkaline lysis and heat inactivation of EVs. \u003cem\u003eMycoplasma\u003c/em\u003e, which rely on a plasma membrane with associated lipids for structural integrity \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, are particularly susceptible to lipid membrane disrupting lysis methods \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, necessitating the use of alkaline lysis to disrupt EVs.\u003c/p\u003e \u003cp\u003eThe efficiency of vesiduction and its detection faces several bottlenecks. First, only part of the EV population carries the plasmid and second, transfer will depend on the fusion of membranes. Third, once the plasmid enters the recipient cell, it may be degraded by the restriction modification system targeting non-self-patterns, although this did not appear to impact vesiduction rates in \u003cem\u003eMcap\u003c/em\u003e, as observed in comparison to \u003cem\u003eMcap\u003c/em\u003e ΔRE. Lastly, \u003cem\u003eoriC\u003c/em\u003e-plasmids like the pIVB08 used in this study, must replicate alongside the chromosome to avoid dilution and eventual loss.\u003c/p\u003e \u003cp\u003eIn summary, this study demonstrates that \u003cem\u003eMollicutes\u003c/em\u003e EVs carry a proteomic profile reflective of their parental cells, with functional proteins that may play a role in host interactions, including triggering immune responses, as illustrated by the stimulation of bovine PBMCs. Additionally, our findings reveal that \u003cem\u003eMycoplasma\u003c/em\u003e EVs can facilitate HGT, offering new insights into plasmid-mediated gene exchange within \u003cem\u003eMollicutes\u003c/em\u003e species. This work provides a foundation for further exploration of EV-mediated processes in microbial ecology and pathogenesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Proteomics and Mass Spectrometry Core Facility, Department for BioMedical Research, University of Bern, Switzerland for excellent technical support for the proteomic analysis, and the Flow Cytometry and Cell Sorting Core facility at the Department for BioMedical Research, University of Bern, Switzerland, for access to the NanoSight. We thank the Agroscope animal facility, Research Contracts Animals Group, Agroscope, Posieux, Switzerland, especially Lukas Eggerschwiler, for the excellent collaboration related to provision of bovine blood. We thank Jing Zhang and Marilou Bourgeon at the Institute of Veterinary Bacteriology for excellent assistance with data analysis and excellent technical support, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Swiss National Science Foundation (grant number 310030_201152, www.snf.ch). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTMW Conceptualization, Project administration, Methodology, Investigation and Formal analysis, Data curation and visualization, Writing - original draft\u003c/p\u003e\n\u003cp\u003eSTP Conceptualization, Methodology, Investigation and Formal analysis (Functional Assays and Vesiduction), Writing - review \u0026amp; editing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTY Investigation and Formal analysis (ex vivo platform), Writing - review\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTD Conceptualization, Methodology, Investigation and Formal analysis (ex vivo platform), Writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003eJJ Funding acquisition, Resources, Conceptualization, Supervision, Writing - review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collection of ruminant blood was performed in compliance with the Swiss animal protection law (TSchG SR 455; TSchV SR 455.1; TVV SR 455.163) under the cantonal license BE55/2022. The experiments were reviewed by the cantonal committee on animal experiments of the canton of Bern and the Fribourg Commission, Switzerland, and approved by the cantonal veterinary authority (Amt f\u0026uuml;r Landwirtschaft und Natur LANAT, Veterin\u0026auml;rdienst VeD, Bern, Switzerland).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eToyofuku, M., Schild, S., Kaparakis-Liaskos, M. \u0026amp; Eberl, L. Composition and functions of bacterial membrane vesicles. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 415\u0026ndash;430 (2023).\u003c/li\u003e\n \u003cli\u003eThapa, H. B., Ebenberger, S. P. \u0026amp; Schild, S. The Two Faces of Bacterial Membrane Vesicles: Pathophysiological Roles and Therapeutic Opportunities. \u003cem\u003eAntibiotics\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1045 (2023).\u003c/li\u003e\n \u003cli\u003eRideau, F. \u003cem\u003eet al.\u003c/em\u003e Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas. \u003cem\u003eMicrobiol. Spectr.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e00645-22 (2022).\u003c/li\u003e\n \u003cli\u003eGaurivaud, P. \u003cem\u003eet al.\u003c/em\u003e Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e0208160 (2018).\u003c/li\u003e\n \u003cli\u003eChernov, V. M. \u003cem\u003eet al.\u003c/em\u003e Extracellular vesicles derived from Acholeplasma laidlawii PG8. \u003cem\u003eScientificWorldJournal\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1120\u0026ndash;1130 (2011).\u003c/li\u003e\n \u003cli\u003eChernov, V. M. \u003cem\u003eet al.\u003c/em\u003e Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins. \u003cem\u003eJ. Proteomics\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 117\u0026ndash;128 (2014).\u003c/li\u003e\n \u003cli\u003eCalcutt, M. J. \u003cem\u003eet al.\u003c/em\u003e Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements. \u003cem\u003eTransbound. Emerg. Dis.\u003c/em\u003e \u003cstrong\u003e65 Suppl 1\u003c/strong\u003e, 91\u0026ndash;109 (2018).\u003c/li\u003e\n \u003cli\u003eMehinagic, K., Pilo, P., Vidondo, B. \u0026amp; Stokar-Regenscheit, N. Coinfection of Swiss cattle with bovine parainfluenza virus 3 and Mycoplasma bovis at acute and chronic stages of bovine respiratory disease complex. \u003cem\u003eJ. Vet. Diagn. Investig. Off. Publ. Am. Assoc. Vet. Lab. Diagn. Inc\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 674\u0026ndash;680 (2019).\u003c/li\u003e\n \u003cli\u003ePeregrino, E. S. \u003cem\u003eet al.\u003c/em\u003e The Role of Bacterial Extracellular Vesicles in the Immune Response to Pathogens, and Therapeutic Opportunities. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 6210 (2024).\u003c/li\u003e\n \u003cli\u003eD\u0026eacute;moulins, T. \u003cem\u003eet al.\u003c/em\u003e Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis. \u003cem\u003eVet. Res.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 18 (2024).\u003c/li\u003e\n \u003cli\u003eWen, A. X. \u0026amp; Herman, C. 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Microbiol.\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 150\u0026ndash;155 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5564984/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5564984/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtracellular vesicles (EVs) are central components of bacterial secretomes, including the small, cell wall-less \u003cem\u003eMollicutes\u003c/em\u003e. Although EV release in \u003cem\u003eMollicutes \u003c/em\u003ehas been reported, EV proteomic composition and function have not been explored yet.\u003c/p\u003e\n\u003cp\u003eWe developed a protocol for isolating EVs of the pathogens \u003cem\u003eMycoplasma mycoides\u003c/em\u003esubsp. \u003cem\u003ecapri\u003c/em\u003e (\u003cem\u003eMmc\u003c/em\u003e) and \u003cem\u003eMycoplasma \u003c/em\u003e(\u003cem\u003eMycoplasmopsis\u003c/em\u003e)\u003cem\u003ebovis\u003c/em\u003e and examined their functionality. Proteomic analysis demonstrated that EVs mirror the proteome of their homologous strain. EVs exhibited nuclease activity, effectively digesting both circular and linear DNA. Notably, EVs elicited immune responses in bovine primary blood cells, like those induced by live \u003cem\u003eM. bovis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eOur findings reveal that EVs can carry plasmids and enable their horizontal transfer, known as vesiduction. Specifically, the natural plasmid pKMK1, with an unknown transmission route, was detected in EVs of\u003cem\u003e Mmc\u003c/em\u003e 152/93 and the \u003cem\u003etetM\u003c/em\u003e-containing pIV08 plasmid was associated with EVs released by an \u003cem\u003eMmc \u003c/em\u003eGM12 strain carrying this plasmid. pIVB08 could be transferred via homo- and heterologous vesiduction to \u003cem\u003eMmc\u003c/em\u003e, \u003cem\u003eM. capricolum \u003c/em\u003esubsp\u003cem\u003e. capricolum \u003c/em\u003eand \u003cem\u003eM. leachii\u003c/em\u003e. Vesiduction was impeded by membrane disruption but resisted DNase and Proteinase K treatment, suggesting that EVs protect their cargo.\u003c/p\u003e\n\u003cp\u003eThese findings enhance our understanding of \u003cem\u003eMollicutes\u003c/em\u003e EVs, particularly in host interactions and horizontal gene transfer.\u003c/p\u003e","manuscriptTitle":"Extracellular Vesicles of Minimalistic Mollicutes as Mediators of Immune Modulation and Horizontal Gene Transfer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-14 04:14:28","doi":"10.21203/rs.3.rs-5564984/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f39a9644-63a8-4bde-87cf-00a335f5d281","owner":[],"postedDate":"January 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41757824,"name":"Biological sciences/Microbiology/Bacteria/Bacterial host response"},{"id":41757825,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2025-10-13T19:21:54+00:00","versionOfRecord":{"articleIdentity":"rs-5564984","link":"https://doi.org/10.1038/s42003-025-08099-4","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-04-29 00:00:00","publishedOnDateReadable":"April 29th, 2025"},"versionCreatedAt":"2025-01-14 04:14:28","video":"","vorDoi":"10.1038/s42003-025-08099-4","vorDoiUrl":"https://doi.org/10.1038/s42003-025-08099-4","workflowStages":[]},"version":"v1","identity":"rs-5564984","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5564984","identity":"rs-5564984","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}