Analysis of the Adhesive-Invasive Potential of Two Morphologically Different Types of Mycoplasma hominis Colonies.

Mycoplasma hominis is an opportunistic human pathogen that causes acute and chronic infections of the urogenital tract. A new form of M. hominis colonies (microcolonies) was isolated, that differed from typical colonies by morphology, size, growth rate, and resistance to unfavorable factors, in particular, to antibiotics. The formation of microcolonies is associated with a switch in energy metabolism towards nucleoside utilization, which leads to a decrease in energy production and a transition to a persistor-like state. Typical and microcolony cultures of M. hominis H-34 were obtained and a comparative analysis of their adhesive-invasive potential, morphology, and size was carried out. It was shown that both typical and microcolonies can effectively attach and penetrate into HeLa cells. Unlike microcolonies, the morphology and size of cells in typical colonies change significantly after HeLa infection. This indicates functional changes in cells of typical colonies during infection.

Comparative Proteomic Analysis of the Mycoplasma gallisepticum Nucleoid Fraction before and after Infection.

Mycoplasma gallisepticum belongs to the class Mollicutes and induces severe chronic respiratory disease in chickens. It lacks the cell wall and contains a very small genome and, accordingly, a reduced set of regulatory proteins. It is assumed that one of the regulatory mechanisms in mycoplasmas may be the dynamics of the spatial organization of the chromosome. M. gallisepticum has only two known nucleoid-associated (NAP) histone-like proteins (Hup_1 and Hup_2). To search for new potential NAP that may play a role in the infection process, we isolated nucleoid fractions from M. gallisepticum cells before and after infection of HD3 chicken erythroblast cell line and performed a comparative proteomic analysis of these fractions. We identified several potential NAP that included the components of the terminal organelle and adhesion, VlhA antigen, NADH oxidase, and PykF pyruvate kinase.

Isolation of Nucleoid Fraction from Mycoplasma gallisepticum Cells with Synchronized Cell Division.

It is assumed that unknown mechanisms can be involved in adaptation Mycoplasma gallisepticum to unfavorable factors, one of these can be local rearrangements of the structure and spatial organization of the chromosome. To study these mechanisms, we obtained a culture of M. gallisepticum with synchronized division and isolated the nucleoid fraction from this culture by the method of mild cell lysis and centrifugation in a sucrose gradient. Liquid chromatography-mass spectrometry analysis of the proteome showed that in comparison with the cell lysate, the nucleoid fraction was enriched with DNA-binding proteins. This analysis will help to find new nucleoid-associated proteins and to study their dynamics, distribution, and their role during infection and under stress conditions.

Data on nucleoid-associated proteins isolated from Mycoplasma gallisepticum after intracellular infection.

Mycoplasma gallisepticum (M. gallisepticum) belongs to the class of Mollicutes. It causes chronic respiratory disease in avian species. It is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, M. gallisepticum has a limited variety of DNA-binding proteins (DBP) and transcription factors. Consequently, the diversity of DNA-binding proteins and transcription factors (TF) in M. gallisepticum is limited in comparison with related bacteria such as Bacillus subtilis. Studies have shown, however, that mycoplasmas demonstrate a wide range of differential expression of genes in response to various stress factors, which promotes effective adaptation to unfavorable conditions. We assume that in the case of mycoplasmas, which are characterized by a combination of the reduction of known gene expression regulation systems and a high adaptive potential, the coordination of gene expression can be provided due to local changes in the structure and spatial organization of the chromosome. The study of the dynamic changes of the proteomic profile of M. gallisepticum nucleoid may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation including its changes upon invasion of the host cells.

Complete genome and proteome of Acholeplasma laidlawii.

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

[Comparative proteomic characteristic of Mycoplasmas (Mollycutes)].

Saturating proteome identification and the study of post-translational protein modifications of Acholeplasma laidlawii using combination of single- and two-dimension gel electrophoresis followed by mass-spectrometry analysis have been carried out. Results were compared to the earlier identified proteome of Mycoplasma gallisepticum. It was found that M. gallisepticum and A. laidlawii express 61 and 58% of the annotated ORFs respectively. All subunits of DNA-polymerase III were identified during our study which indicates that our methods can detect single copies of a given protein per cell. Metabolic pathways of the respective mycoplasmas were compared further in this work.

Proteomic characterization of Mycoplasma gallisepticum nanoforming.

The goal of this work was to create a model for the long persistence of Mycoplasma gallisepticum in depleted medium and under low growth temperature followed by proteomic study of the model. Nanoforms and revertants for M. gallisepticum were obtained. Proteomic maps were produced for different stages of the formation of nanoforms and revertants. It is shown that proteins responsible for essential cellular processes of glycolysis, translation elongation, and DnaK chaperone involved in the stabilization of newly synthesized proteins are crucial for the reversion of M. gallisepticum to a vegetative form. Based on the current data, it is assumed that changes in the metabolism of M. gallisepticum during nanoforming are not post-mortal, thus M. gallisepticum does not transform to uncultivable form, but remains in a reversible dormant state during prolonged unfavorable conditions.

Proteome of the bacterium Mycoplasma gallisepticum.

Using modern proteomic assays, we have identified the products of gene expression and posttranslational modifications of proteins of the bacterium Mycoplasma gallisepticum S6. Combinations of different technologies of protein separation by electrophoresis and mass-spectrometric analysis gave us a total of 446 proteins, i.e. 61% of the annotated proteins of this microorganism. The Pro-Q Diamond and Pro-Q Emerald dye technology was used for fluorescent detection of ten phosphoproteins and two glycoproteins. The acylation of proteins was studied by electrophoresis after in vivo labeling with different 14C-labeled fatty acids, followed by autoradiography. Sixteen acylated proteins were identified, with a quarter of them involved in plasma membrane construction and another quarter involved in cell energy metabolism.

[Development of fluoroquinolone (ciprofloxacin) resistance in Mycoplasma hominis in the presence of Hela cells]. / Formirovanie ustoichivosti Mycoplasma hominis k ftorkhinolonam (tsiprofloksatsinu) v prisutstvii kletok Hela.

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonis and HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the mircoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 micrograms/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown for the first time that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.

[Analysis of regions determining resistence to fluoroquinolones in genes gyrA and parC in clinical isolates of Mycoplasma hominis]. / Analiz raionov, opredeliaiushchikh rezistentnost' k ftorkhinolonam, genov gyrA i parC v klinicheskikh izoliatakh Mycoplasma hominis.

Fifteen strains of M. hominis isolated from patients with urogenital inflammations were analyzed. Variations in the quinolone resistance-determining regions (QRDR) have been found in fluoroquinolone-resistant M. hominis clinical isolates in comparison with the reference PG21 strain. In one isolate, parC had Asn substitute at position 91.

[Role of mutations in parC and gyrA in forming resistance of Mycoplasma hominis to fluoroquinolones]. / Rol' mutatsii v parC i gyrA v formirovanii ustoichivosti Mycoplasma hominis k ftorkhinolonam.

The set of the laboratory strain M. hominis H-34 mutants resistant to fluoroquinolones (ciprofloxacin-Cfl, lomefloxacin-Lfl, ofloxacin-Ofl) was obtained by selection in broth medium. The mutation was found in the quinolone resistance-determining region (QRDR) of A subunit of topoisomerase IV gene (parC) and new mutations were found in QRDR of genes encoding the A subunit of DNA gyrase (gyrA) in M. hominis mutants resistant to various concentrations of the Cfl, Lfl and Ofl. After multistep selection of the obtained mutants at constant concentrations of Cfl additional mutation Ser83 to Trp was revealed. No mutations in parE and gyrB were found. Mutations in parC for laboratory strain M. hominis H34 appeared at lower antibiotic concentrations than in gyrA. All mutations in gyr A were associated with mutations in parC. This confirms the previous data that topoisomerase IV is the primary target of Cfl and Ofl and suggests that it is the primary target of Lfl. Some M. hominis mutants selected at Ofl without any substitution in QRDRs were shown to be insensitive to Cfl and of Lfl. Studies of cross-resistance of the selected M. hominis mutants showed that their resistance to various fluoroquinolone concentrations could not depend on any mutations in QRDR of topoisomerase IV and DNA gyrase genes and suggests involvement of other unknown molecular mechanisms specific for Mycoplasmas.

[Formation of M. hominis and A. laidlawii resistance to fluoroquinolones]. / O formirovanii rezistentnosti k ftorkhinolonam u M. hominis i A. laidlawii.

Mycoplasma hominis and Acholeplasma laidlawii cultures resistant to antibacterial fluoroquinolone drugs ciprofloxacin (Cpf), ofloxacin (Ofl), and lomefloxacin (Lmf) were prepared by selection in liquid nutrient medium with ascending concentrations of Cpf. Resistant mycoplasma clones contained point mutations in the gyrase. A gene region determining quinolone resistance (QRDR gyrA): M. hominis contained C-->T transition resulting in substitution of Ser(83) for Leu and A. laidlawii G-->A resulting in substitution of Asp (91) for Asn. The phenomena of mutation formation during mycoplasma culturing in the presence of fluoroquinolones is studied. In the presence of Cpf in culture medium in concentrations of up to 10 micrograms/ml (for M. hominis) and 1 microgram/ml (for A. laidlawii) the mycoplasma populations contained cells with both altered and wild genotype. Culturing in the presence of higher Cpf concentrations resulted in elimination of cells nonmutant for QRDR gyrA. Besides in vitro studies, we analyzed clinical strains of M. hominis in the presence of different Cpf concentrations. M. hominis clones resistant to Cpf varying in genotypes were detected. These data permit a conclusion that the mechanism of fluoroquinolone resistance formation in mycoplasma includes several stages.

[Comparative evaluation of the cytotoxic effect of hydrogen peroxide and tumor necrosis factor alpha on nonischemic and ischemic endothelial cells]. / Sravnitel'naia otsenka tsitotoksicheskogo éffekta perekisi vodoroda i faktora nekroza opukholi al'fa na neishemizirovannye i ishemizirovannye éndotelial'nye kletki.

We studied cytotoxic effects (CTE) induced in confluent cultures of human umbilical vein endothelial cells (HUVEC) by initiators of free-radical reactions (FRR): H2O2 (10(-6)-10(-9) M), recombinant human tumor necrosis factor-[symbol; see text] (TNF-alpha, 0.05-100 ng/ml), and a combination of TNF-alpha with low-density lipoproteins (LDL, 100 microgram/ml). HUVEC were incubated with these substances for 6 or 24 h in parallel tests performed under aerobic (CO2-incubator) and ischemic conditions (a mixture of 95% N2 + 5% CO2 in RPMI-1640 medium containing no substrate additives, growth factor or protein). HUVEC viability was determined by counting cells adherent to the bottom of wells after 24 h of reincubation under aerobic conditions in the growth medium (Plating Efficiency Index). The data showed that: 1) CTE of these compounds were dose-dependent (H2O2 and TNF-alpha) and time-dependent (TNF-alpha); 2) CTE of FRR initiators and CTE of ischemia were synergistic, that is, their combination produced a greater decrease HUVEC viability than any substance examined or ischemia alone; 3) CTE of TNF-alpha observed in experiments in substrate-deficient, protein-free medium was considerably stronger than in the growth medium; 4) a combination of TNF-a and LDL caused a stronger CTE on HUVEC than either factor alone, and this synergism was more pronounced during incubation under ischemic conditions. Thus, the data indicate that FRR initiators and TNF-alpha + LDL particularly increase the severity of ischemic injuries of EC and therefore they can be factors which in hypercholesterolemic patiens predispose vascular wall to atherosclerosis.

Cholesterol accumulation in plasma membrane and changes of membrane enzyme activity of Acholeplasma laidlawii cells during culture ageing.

The data presented in this report show that the microviscosity of the plasma membrane and the cholesterol/phospholipid molar ratio of its lipid bilayer increases during ageing of Acholeplasma laidawii cultures. At the same time the age changes of other lipid components content do not correlate with the change of membrane viscosity. It is also shown that membrane enzyme activities of mycoplasma cell decrease with age. These results confirm the membrane hypothesis of ageing according to which increased microviscosity is the essential factor of cell ageing.

[Dependence of membrane protein turnover in Mycoplasma cells on the age of the culture]. / Zavisimost' obnovleniia membrannykh belkov kletok mikoplazm ot vozrasta kul'tury.

To understand the molecular mechanisms of damages appearing in biological membranes in the process of cellular aging, changes in the rate of catabolic processes in Mycoplasma cells have been studied. This study has revealed that the aging of Acholeplasma laidlawii culture is accompanied by a decrease in the activity of such catabolic enzymes as DNA-ase, RNA-ase, cathepsin D and beta-glucosidase. A considerable increase in the duration of the half-life of membrane proteins has been registered, which is indicative of a decrease in their turnover rate. The electrophoretic separation of membrane proteins has revealed essential changes in their properties. Such decline in the functional activity of the plasma membrane of Mycoplasma cells at the stationary phase is probably due to the inactivation of membrane enzymes and to the decreased rate of their turnover.

Transport activity of mouse spleen lymphocytes after their interaction in vitro with Acholeplasma laidlawii cells.

Transport of two non-metabolized carbohydrates (3-O-methyl-D-glucose, 3-O-MG, and 2-deoxy-D-glucose, 2-DG) into mouse spleen lymphocytes after their interaction with Acholeplasma laidlawii cells has been studied. Incubation of A. laidlawii cells and particularly the liposomes prepared from A. laidlawii membrane lipids enhances the rates of the both carbohydrates transport. This treatment resulted in increasing of the Vmax values of 3-O-MG and 2-DG without changing the Km values. This stimulation can be explained by the increasing of the mobility of membrane carbohydrates carriers as a result of the exchange of lipid components between Acholeplasma and lymphocyte membranes. Actually, it has been shown that liposomes derived from A. laidlawii cells grown on the medium with great amount of unsaturated oleic acid stimulate the transport activity more actively than liposomes prepared from the cells grown on the medium with bovine serum or with oleic acid plus cholesterol. It should be suggested that an activation of carbohydrates transport into lymphocytes caused by alteration of the carriers lipid microenvironment.

[Accumulation of cholesterol in Acholeplasma laidlawii membranes in the steady-state phase of culture growth]. / Nakoplenie kholesterina v membranakh Acholeplasma laidlawii v statsionarnoi faze rosta kul'tury.

In early logarithmic phase of growth of the A. laidlawii cells the lipid composition of plasma membrane is changed: the total lipid, glycolipid and phospholipid contents are decreased, while that of cholesterol changes only insignificantly. In late logarithmic and steady state phases the cholesterol level in the membrane is increased in parallel with the decrease of the phospholipid content. Throughout the growth period a quantitative redistribution of membrane phospholipids in fatty acids and an increase of the molar content of saturated fatty acids are observed. Accumulation of cholesterol in the steady state phase is accompanied by an increase in the membrane viscosity which results in inhibition of membrane processes in the cell.

Immunological and functional activity of spleen lymphocytes from mice infected by Acholeplasma laidlawii cells and Rauscher virus.

The duration of Acholeplasma laidlawii antigen persistence in mice, resistant to Rausher leukemia virus, after infection with both A. laidlawii cells and Rausher virus has been studied. The antigen persistence was accompanied by marked depression of immune response which was especially severe in case of mixed acholeplasmavirus infection. Such immunosuppression and observed infiltration of the spleen with immature leukemic cells can be regarded as a preleukosis. Immunosuppression was accompanied by an increase of the transport of carbohydrates inthe lymphocytes. This stimulation an be explained by the exchange of lipid components between acholeplasma and lymphocyte membranes resulted in increase of lymphocyte membrane fluidity, or it may be due to the mitogenic effect of A. laidlawii cells and virus, accompanied by the same membrane effect.