Elena S. Medvedeva

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.

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.

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.

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).

Mycoplasma adaptation to stress conditions: Proteome shift in Mycoplasma hominis PG37 in response to starvation and low temperatures

Mycoplasma hominis is a widely spread mycoplasma (class Mollicutes), associated with socially important human diseases and contamination of cell cultures. Controlling infections caused by M. hominis depends on determining the molecular mechanisms responsible for the bacterium’s survival in unfavorable conditions. A proteome analysis employing 2-DIGE and MALDI TOF/TOF MS was applied to identify, for the first time, 53 proteins of M. hominis PG37 whose levels altered in bacteria cultivated in stress conditions (starvation and low temperature). According to the protein classification by functional category (clusters of orthologous groups of proteins, COG), 47 of the 53 mycoplasma proteins identified are involved in fundamental cellular and biochemical processes: translation (12; 22.64%), transcription (2; 3.77%), posttranslational modification (7; 13.20%), cell cycle control (2; 3.77%), energy production and conversion (6; 11.32%), carbohydrate transport and metabolism (3; 5.66%), amino acid transport and metabolism (8; 15.09%), nucleotide transport and metabolism (6; 11.32%), and inorganic ion transport and metabolism (1; 1.89%). For six proteins (11.32%), the function was not determined; 24 proteins (45.28%) were bacterial virulence factors. Those proteins of M. hominis PG37 whose expression is modulated in response to unfavorable environmental conditions are components of stress adaptation mechanisms in mycoplasma and potential targets for controlling infections caused by this bacterium.

Genome Sequences of Acholeplasma laidlawii Strains with Increased Resistance to Tetracycline and Melittin

ABSTRACT Acholeplasma laidlawii is a well-suited model for studying the molecular basis for adapting mollicutes to environmental conditions. Here, we present the whole-genome sequences of two strains of A. laidlawii with increased resistance to tetracycline and melittin.