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.

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.

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.

Antimicrobial drug resistance mechanisms among Mollicutes.

Representatives of the Mollicutes class are the smallest, wall-less bacteria capable of independent reproduction. They are widespread in nature, most are commensals, and some are pathogens of humans, animals and plants. They are also the main contaminants of cell cultures and vaccine preparations. Despite limited biosynthetic capabilities, they are highly adaptable and capable of surviving under various stress and extreme conditions, including antimicrobial selective pressure. This review describes current understanding of antibiotic resistance (ABR) mechanisms in Mollicutes. Protective mechanisms in these bacteria include point mutations, which may include non-target genes, and unique gene exchange mechanisms, contributing to transfer of ABR genes. Better understanding of the mechanisms of emergence and dissemination of ABR in Mollicutes is crucial to control these hypermutable bacteria and prevent the occurrence of highly ABR strains.

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.

Effects of Mycoplasmas on the Host Cell Signaling Pathways.

Mycoplasmas are the smallest free-living organisms. Reduced sizes of their genomes put constraints on the ability of these bacteria to live autonomously and make them highly dependent on the nutrients produced by host cells. Importantly, at the organism level, mycoplasmal infections may cause pathological changes to the host, including cancer and severe immunological reactions. At the molecular level, mycoplasmas often activate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) inflammatory response and concomitantly inhibit the p53-mediated response, which normally triggers the cell cycle and apoptosis. Thus, mycoplasmal infections may be considered as cancer-associated factors. At the same time, mycoplasmas through their membrane lipoproteins (LAMPs) along with lipoprotein derivatives (lipopeptide MALP-2, macrophage-activating lipopeptide-2) are able to modulate anti-inflammatory responses via nuclear translocation and activation of the Nrf2 (nuclear factor-E2-related anti-inflammatory transcription factor 2). Thus, interactions between mycoplasmas and host cells are multifaceted and depend on the cellular context. In this review, we summarize the current information on the role of mycoplasmas in affecting the host's intracellular signaling mediated by the interactions between transcriptional factors p53, Nrf2, and NF-κB. A better understanding of the mechanisms underlying pathologic processes associated with reprogramming eukaryotic cells that arise during the mycoplasma-host cell interaction should facilitate the development of new therapeutic approaches to treat oncogenic and inflammatory processes.

Omics of antimicrobials and antimicrobial resistance.

INTRODUCTION: The development of new antimicrobials has become an urgent priority because of a global challenge emerging from the rise of antimicrobial resistant pathogens. Areas covered: In this review, the authors discuss the opportunities offered by modern omics approaches to address the challenge and the use of this approach in antimicrobial development. Specifically, the authors focus on the role of omics technologies and bioinformatics for the revelation of the effects of antimicrobials in a variety of microbial cellular processes, as well as the identification of potential cellular targets, the mechanisms of antimicrobial resistance, and the development of new antimicrobials. Expert opinion: Prevention of antimicrobial resistance does not only depend on rational drug design such as narrow-spectrum antimicrobials but on several factors. It is the opinion of the authors that the use of a multi-omics bioinformatics approach should become an integral part of antimicrobial drug discovery as well as in the prevention of antimicrobial resistance.

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.

Antimicrobial resistance in mollicutes: known and newly emerging mechanisms.

This review is devoted to the mechanisms of antibiotic resistance in mollicutes (class Bacilli, subclass Mollicutes), the smallest self-replicating bacteria, that can cause diseases in plants, animals and humans, and also contaminate cell cultures and vaccine preparations. Research in this area has been mainly based on the ubiquitous mollicute and the main contaminant of cell cultures, Acholeplasma laidlawii. The omics technologies applied to this and other bacteria have yielded a complex picture of responses to antimicrobials, including their removal from the cell, the acquisition of antibiotic resistance genes and mutations that potentially allow global reprogramming of many cellular processes. This review provides a brief summary of well-known resistance mechanisms that have been demonstrated in several mollicutes species and, in more detail, novel mechanisms revealed in A. laidlawii, including the least explored vesicle-mediated transfer of short RNAs with a regulatory potency. We hope that this review highlights new avenues for further studies on antimicrobial resistance in these bacteria for both a basic science and an application perspective of infection control and management in clinical and research/production settings.

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

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.

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 microm) 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.

The pattern of acute myocardial infarction in people with opportunistic infections.

First comparative analysis for some hemocoagulation reactions, immune state of acute myocardial infarction has been done in two groups of patients - with mycoplasma infections and without one. Postinfarction complications in the groups were observed. Atherosclerotic plaques of the patients were also studied on a presence of mycoplasmas and a content of strontium and zinc. A tendency to a mild pattern of acute myocardial infarction was observed in patients with mycoplasma infections. The role of mycoplasmas in atherogenesis and acute myocardial infarction is discussed.

Genome Sequences of Acholeplasma laidlawii Strains Differing in Sensitivity to Ciprofloxacin.

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.

Variability of the Vaa cytoadhesin genes in clinical isolates of Mycoplasma hominis.

The Mycoplasma hominis vaa gene encodes a highly variable surface antigen involved in adhesion to host cells. We studied 15 clinical isolates of Mycoplasma hominis with three types of the vaa gene. These vaa versions determine various forms of Vaa protein, which are characterized by different quantity and structure of homologous replaceable cassettes. Each cassette contains heptad repeats and sites for adherence. The differences on single nucleotides were observed in the primary sequences of the homologous modules of the vaa gene. A high frequency of nucleotide replacements in V module of the vaa gene (first and/or second position in codon) was determined. This region with various clusters of direct and indirect repeats of nucleotide sequences is incorporated into the area of the vaa gene. Amino-acid sequences corresponding to the hyper-variable region of the vaa gene are associated with the sections of coiled-coils and loops of Vaa. These bacterial regions involved in interaction with the host cell membranes could yield useful indications for more insights into the mechanism of mycoplasma persistence in humans.

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.

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.