Olga A. Chernova

Acholeplasma laidlawii PG8 Culture Adapted to Unfavorable Growth Conditions Shows an Expressed Phytopathogenicity

Mycoplasmas are the smallest, self-replicating, prokaryotic organisms with avid biochemical potential and spreading in higher eukaryotes in nature. In this study, Acholeplasma laidlawii PG8 cells were cultivated on a deficient medium for 480 days resulting in a mycoplasma culture that was adapted in vitro to unfavorable growth conditions. Cells that survive this condition had decreased sizes (about 0.2 μm) and increased phytopathogenicity. This resulted in more frequent appearance of various morphological alterations when plants of vinca (Vinca minor L.) were infected by adapted mycoplasma cells. The increasing pathogenicity was accompanied by changes in genome expression in these adapted cells. Further studies are needed to explore the exact mechanisms that permit adaptation to unfavorable growth conditions and changes in phytopathogenic potential.

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.

Adaptation of mycoplasmas to adverse growth conditions: Morphology, ultrastructure, and genome expression of Mycoplasma gallisepticum S6 cells

A considerable amount of experimental and theoretical data recently obtained in various laboratories of the world, including successful genome projects for 17 mycoplasma species, has greatly expanded our knowledge of the biology of minimal cell, which is associated with mycoplasmas, representing the class Mollicutes. However, the molecular bases of mycoplasma adaptation to biogenic and abiogenic stresses are still vague [1]. The goal of this work was to clarify the specific features of morphology, ultrastructure, and genomic expression of Mycoplasma gallisepticum S6 cells under various growth conditions. We are the first to demonstrate that the adaptation of M. gallisepticum S6 to stresses is associated with a specific remodeling of genome expression, which determines the reprogramming of mycoplasma cell biology in a manner allowing for survival under new conditions. Acholeplasma laidlawii , an omnipresent mycoplasma capable of infecting human, animal, and plant species and the main contaminant of cell cultures, and M. gallisepticum , a mycoplasma highly pathogenic for poultry and capable of infecting plants, the main contaminant of viral vaccines produced using chick embryos [2], are unique from the standpoint of their adaptive abilities. The absence of the cell wall, a miniature genome, and a limited metabolic potential are in no way important barriers for these mycoplasmas to survive under adverse conditions, overcome various host defense systems, and persist in the tissues of higher eukaryotes [1, 3‐5].

Atomic Force Microscopy Analysis of DNA Extracted from the Vegetative Cells and the Viable, but Nonculturable, Cells of Two Mycoplasmas (Acholeplasma laidlawii PG8 and Mycoplasma hominis PG37)

This article reports on a study of some characteristics of DNA extracted from the vegetative and viable, but nonculturable (VBNC), cells of two mycoplasma species (Acholeplasma laidlawii PG8 and Mycoplasma hominis PG37) using atomic force microscopy (AFM). DNA images were obtained by operating the AFM microscope in the tapping mode. It was found that DNA from the VBNC forms of M. hominis PG37 has decreased sizes (height: 0.177 ± 0.026 nm vs. 0.391 ± 0.041 nm for the vegetative forms, and width: 1.92 ± 0.099 vs. 2.17 ± 0.156 nm for the vegetative forms) in comparison to DNA from the vegetative forms of the mycoplasma. In the case of DNA from the A. laidlawii PG8 VBNC forms, we detected a decrease in width (1.506 ± 0.076 nm vs. 1.898 ± 0.117 nm for the vegetative forms), but an increase in height (0.641 ± 0.068 nm vs. 0.255 ± 0.010 nm for the vegetative forms) of the molecule. Analyzing the obtained results, one can speculate on some similarities in the physical-chemical properties of DNA from M. hominis PG37 and M. gallisepticum S6. In turn, this implies some general mechanisms of adaptation to a severe environment.

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.