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For the first time it is shown that the development of resistance to melittin in Acholeplasma laidlawii, a mycoplasma that is widely spread in nature and that is the main contaminant of cell cultures and vaccines, is associated with significant changes in the genomic profile, in cellular and vesicular proteomes, as well as in virulence.

Acholeplasma laidlawii is widespread hypermutable bacteria (class Mollicutes) capable of infecting humans, animals, plants, which is the main contaminant of cell cultures and vaccine preparations. The mechanisms of the development of antimicrobial resistance of this bacterium are associated with the secretion of extracellular vesicles, which can mediate the lateral transfer of antibiotic resistance determinants. We compared the genome profiles of ciprofloxacin-resistant A.laidlawii strains PG8r1 (MIC 10 µg/ml) and PG8r3 (MIC 10 µg/ml) selected under different in vitro conditions - when ciprofloxacin-sensitive (MIC 0.5 µg/ml) A.laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone, and with vesicles derived from the ciprofloxacin-resistant (MIC 20 µg/ml) A.laidlawii PG8R10c-2 strain, respectively. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R10c-2 were analyzed too. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC) containing in extracellular vesicles of PG8c-3, PG8R10c-2, PG8r1 and PG8r3 were determined. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. We also compared cellular proteomes of PG8c-3, PG8r1 and PG8r3. The cellular proteome profiles of the A. laidlawii strains were determined by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. This work presents data on single nucleotide polymorphisms (SNPs) found in the genomes of the ciprofloxacin-resistant strains selected under different in vitro conditions and proteins that were differentially expressed in the cells of ciprofloxacin-resistant strains selected under different conditions in vitro.

The present review discusses the problem of controlling mycoplasmas (class Mollicutes), the smallest of self-replicating prokaryotes, parasites of higher eukaryotes, and main contaminants of cell cultures and vaccines. Possible mechanisms for the rapid development of resistance to antimicrobial drugs in mycoplasmas have been analyzed. Omics technologies provide new opportunities for investigating the molecular basis of bacterial adaptation to stress factors and identifying resistomes, the total of all genes and their products contributing to antibiotic resistance in microbes. The data obtained using an integrated approach with post-genomics methods show that antibiotic resistance may be caused by more complex processes than has been believed heretofore. The development of antibiotic resistance in mycoplasmas is associated with essential changes in the genome, proteome, and secretome profiles, which involve many genes and proteins related to fundamental cellular processes and virulence.

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

Acholeplasma laidlawii is a well-suited model for study of the molecular basis of the adaptation of mollicutes to environmental conditions. Here we present the whole-genome sequences of four strains of A. laidlawii with differential sensitivity to ciprofloxacin.

Mycoplasmas are incapable of de novo synthesis of nucleotides and must therefore secrete nucleases in order to replenish the pool of nucleic acid precursors. The nucleolytic activity of mycoplasmas is an important factor in their pathogenicity. Bacterial ribonucleases (RNases) may produce a broad spectrum of biological effects, including antiviral and antitumor activity. Mycoplasma RNases are therefore of interest. In the present work, the capacity of Acholeplasma laidlawii and Mycoplasma hominis for RNase synthesis and secretion was studied. During the stationary growth phase, these organisms were found to synthesize Mg2+-dependent RNases, with their highest activity detected outside the cells. Localization of A. laidlawii RNases was determined: almost 90% of the RNase activity was found to be associated with the membrane vesicles. Bioinformational analysis revealed homology between the nucleotide sequences of 14 Bacillus subtilis genes encoding the products with RNase activity and the genes of the mycoplasmas under study. Amino acid sequences of 4 A. laidlawii proteins with ribonuclease activity and the Bsn RNase were also established.

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.

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.

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.

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.

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 ofAcholeplasma laidlawiiPG8. 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. laidlawiiextracellular 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.

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 ofAcholeplasma laidlawiiPG8 (ubiquitous mycoplasma; the main contaminant of cell culture). Considering sizes, morphology, and ultrastructural organization, the ultramicroforms ofA. laidlawiiPG8 are similar to membrane vesicles of Gram-positive and Gram-negative bacteria. We demonstrate thatA. laidlawiiPG8 vesicles contain genetic material and proteins, and are mutagenic to lymphocytes of human peripheral blood. We show thatMycoplasma gallisepticumS6, 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.

Refereed/Peer-reviewed