Alexey A. Mouzykantov

Extracellular Vesicles Derived from Acholeplasma laidlawii PG8

Extracellular vesicle production is believed to be a ubiquitous process in bacteria, but the data on such a process in Mollicutes are absent. We report the isolation of ultramicroforms – extracellular vesicles from supernatants of Acholeplasma laidlawii PG8 (ubiquitous mycoplasma; the main contaminant of cell culture). Considering sizes, morphology, and ultrastructural organization, the ultramicroforms of A. laidlawii PG8 are similar to membrane vesicles of Gram-positive and Gram-negative bacteria. We demonstrate that A. laidlawii PG8 vesicles contain genetic material and proteins, and are mutagenic to lymphocytes of human peripheral blood. We show that Mycoplasma gallisepticum S6, the other mycoplasma, also produce similar structures, which suggests that shedding of the vesicles might be the common phenomenon in Mollicutes. We found that the action of stress conditions results in the intensive formation of ultramicroforms in mycoplasmas. The role of vesicular formation in mycoplasmas remains to be studied.

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

Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis.

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8--ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase--a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles.

Extracellular Membrane Vesicles and Phytopathogenicity of Acholeplasma laidlawii PG8

For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses) in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing) to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria—invasivity, infectivity—and toxigenicity—and to favor to bacterial phytopathogenicity.

Adaptation of Mycoplasmas to Antimicrobial Agents: Acholeplasma laidlawii Extracellular Vesicles Mediate the Export of Ciprofloxacin and a Mutant Gene Related to the Antibiotic Target

This study demonstrated that extracellular membrane vesicles are involved with the development of resistance to fluoroquinolones by mycoplasmas (class Mollicutes). This study assessed the differences in susceptibility to ciprofloxacin among strains of Acholeplasma laidlawii PG8. The mechanisms of mycoplasma resistance to antibiotics may be associated with a mutation in a gene related to the target of quinolones, which could modulate the vesiculation level. A. laidlawii extracellular vesicles mediated the export of the nucleotide sequences of the antibiotic target gene as well as the traffic of ciprofloxacin. These results may facilitate the development of effective approaches to control mycoplasma infections, as well as the contamination of cell cultures and vaccine preparations.

Phytopathogenicity of avian mycoplasma Mycoplasma gallisepticum S6: Morphologic and ultracytostructural changes in plants infected with the vegetative forms and the viable but nonculturable forms of the bacterium

The data obtained in this study proved that Mycoplasma gallisepticum S6 known as avian pathogen had a phytopathogenic potential. The vegetative forms and the viable but nonculturable (VBNC) forms of this mycoplasma could infect the plants via an assemblage of rootlets, invade different tissues, persist there and cause destructive events characteristic to phytomycoplasmoses. In comparison with the vegetative forms, the VBNC forms induced more prominent destructive changes. This phenomenon might be connected to increasing expression of proteins responsible for virulence in the bacterial cells. The fact that M. gallisepticum S6 could demonstrate virulent features (infectivity, invasiveness, persistence and toxigenicity) in regard to plants seems to require a development of new ways for controlling phytomycoplasmoses taking into account the probable presence of asymptomatic carriers of this bacterium.

Genomic and proteomic profiles of Acholeplasma laidlawii strains differing in sensitivity to ciprofloxacin.

As a result of comparative analysis of complete genomes as well as cell and vesicular proteomes of A. laidlawii strains differing in sensitivity to ciprofloxacin, it was first shown that the mycoplasma resistance to the antibiotic is associated with the reorganization of genomic and proteomic profiles, which concerns many genes and proteins involved in fundamental cellular processes and realization of bacterial virulence.

Atomic Force Microscopy Investigation of DNA Extracted from the Vegetative Forms and the Viable but Nonculturable Forms of Mycoplasma gallisepticum S6

Recent studies show that mycoplasmas have various programs of life. This means that changes in morphology and genome expression may occur once the environment of these microorganisms becomes extremely altered. In this article, we report on changes in the DNA molecule obtained from the vegetative forms and the viable but nonculturable (VBNC) forms of Mycoplasma gallisepticum S6. Atomic force microscopy studies show that the above-mentioned forms of the mycoplasma have different values of DNA parameters (height: 0.461 ± 0.141 and 0.236 ± 0.069 nm; width: 2.221 ± 0.286 and 1.291 ± 0.705 nm for the vegetative and the VBNC forms, respectively). We suppose that the observed phenomenon may be connected with the process of adaptation of these bacteria to severe environments.

Exported mycoplasmal proteins: Proteome of extracellular membrane vesicles of Acholeplasma laidlawii PG8

43 Ample theoretical and experimental data for mycoo plasmas obtained in recent years determined substann tial progress in understanding the molecular and celluu lar biology of the smallest prokaryotes [1]. Genome– transcriptome–proteome profiling and nanoscopic analysis made it possible to identify stresssreactive genes and proteins in a number of mycoplasmas. It was shown that adaptation to environmental conditions, cell–cell interactions, and pathogenicity of these microorganisms are largely associated with the extraa cellular membrane vesicles of these bacteria [1, 2]. Extracellular vesicles (EVs) are the key component of the bacterial secretome. First discovered several decades ago in the Grammnegative bacteria, they have recently been identified in the Grammpositive bacteria [3], archaea [4], and mycoplasmas [1, 2] and became the focus of attention of researchers. It was found that extracellular membrane vesicles are spherical nanoo structures surrounded with a membrane; in addition to the membrane components, they may also contain cytoplasmic proteins, toxins, and DNA and RNA nucleotide sequences [1, 2, 5]. These organelles medii ate the traffic of a wide range of components, the transfer virulence determinants, and the development of resistance to antibiotics. They are involved in sigg naling, intercellular communications, and pathogenee sis and represent a new type of infectogenes, the study of which is required for the analysis of antagonistic relationships between bacteria in communities, interr action of human microflora with colonized cells, and correction of the pathogen control strategy [6]. To perr form relevant studies, a comprehensive characterizaa tion of vesicular structures with the determination of all EV components is required, which involves a comm prehensive approach based on modern physicochemii cal and molecular methods, including posttgenomic technologies. The results of proteomic studies of EVs of some bacteria are already introduced into dataa bases. Information about mycoplasmal EVs is missing. In this regard, the goal of this study was to perform global proteomic profiling of extracellular membrane vesicles A. laidlawii and inventory of proteins exported from mycoplasma cells in vesicular structures. As a result of our study, 97 proteins were identified for the first time in EVs of A. laidlawii PG8by by mass spectrometry (LCCESIIMS/MS). In this work we used the Acholeplasma laidlawii strain PG8, obtained from the Collection of Microorr ganisms of the Gamaley Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow. After museum storage, the A. laidlawii PG8 culture was grown at 37°C in a liquid Edward medium with some modifications [1]. …

Extracellular vesicles of mycoplasmas and development of resistance to quinolones in bacteria.

34 Mycoplasmas infect humans, animals, and plants, contaminate cell cultures and vaccine preparations and, hence, represent a serious problem [1]. Myco plasmas quickly become resistant to antibiotics. How ever, despite its low efficiency, antibiotic therapy is the primary tool used in the treatment of mycoplasma infections and decontamination of cell cultures. The most widely used agents are fluoroquinolones—syn thetic antibacterial drugs such as enrofloxacin, ciprof loxacin, and sparfloxacin [2]. The mechanisms under lying the rapid development of mycoplasmal resis tance to fluoroquinolones remain obscure. According to some researchers, the mechanisms of formation of resistance to quinolones known for other bacteria, which are associated with the mutations in the target protein genes and limitation of accumulation of anti microbial drugs in microbial cells, are not the main mechanisms in mycoplasmas [3]. The elucidation of the mechanisms of the rapid development of resis tance to antibiotics and solving the problem of control of mycoplasmal infection and contamination are asso ciated with the studies of the adaptation of mycoplas mas to stress [3–5]. Successful implementation of genome projects for a number of mycoplasmas deter mined the possibility of using postgenomic technolo gies for studying respective processes. A unique species of mycoplasmas in terms of adaptive properties is Acholeplasma laidlawii, the causative agent of phyto mycoplasmoses and the main contaminant of cell cul tures and vaccines. Using transcriptomic and pro teomic analysis and nanoscopy, we for the first time identified the stress reactive proteins and genes of Acholeplasma laidlawii and showed that the adaptation to mycoplasma to stressors is associated with the secretion of extracellular vesicles (EVs) [4, 5]. Bacteria EVs are spherical nanostructures 20–200 nm in diam eter surrounded with a membrane, which mediate the traffic of a many compounds involved in signaling, intercellular interactions, and pathogenesis of infec tions [6]. Recently, it was shown that bacterial EVs may be involved in the development of resistance to antimi crobial agents [7]; however, such studies have not been conducted for mycoplasmas. The purpose of this study was to elucidate the role of extracellular membrane vesicles in the formation of the resistance of mycoplas mas (Acholeplasma laidlawii) to fluoroquinolones (ciprofloxacin).