Mycoplasma fermentans deacetylase promotes mammalian cell stress tolerance.

Mycoplasma fermentans is a pathogenic bacterium that infects humans and has potential pathogenic roles in respiratory, genital and rheumatoid diseases. NAD+-dependent deacetylase is involved in a wide range of pathophysiological processes and our studies have demonstrated that expression of mycoplasmal deacetylase in mammalian cells inhibits proliferation but promotes anti-starvation stress tolerance. Furthermore, mycoplasmal deacetylase is involved in cellular anti-oxidation, which correlates with changes in the proapoptotic proteins BIK, p21 and BIM. Mycoplasmal deacetylase binds to and deacetylates the FOXO3 protein, similar with mammalian SIRT2, and affects expression of the FOXO3 target gene BIM, resulting in inhibition of cell proliferation. Mycoplasmal deacetylase also alters the performance of cells under drug stress. This study expands our understanding of the potential molecular and cellular mechanisms of interaction between mycoplasmas and mammalian cells.

Enzymatic synthesis of Mycoplasma fermentans specific glycoglycerophospholipid from 1,2-dipalmitoylglycerol.

A gene, mf3, encoding glycosyltransferase in glycoglycerophospholipid (GGPL; GGPL-I and GGPL-III) biosynthesis in Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, modified codon usage, and expressed in Escherichia coli. The mf3 gene consists of an open reading frame of 1221 bp encoding 406 amino acids. The mf3 gene product, Mf3, has 27% amino acid homology with glycosyltransferase of Borrelia burgdorferi but no homology to genes of other Mycoplasma species in the GenBank database. The reaction product of Mf3 using 1,2-dipalmitoilglycerol and UDP-glucose as substrates showed a specific sodium adducted ion at m/z 753, which corresponded to glucopyranosyl dipalmitoilglycerol as determined by MALDI-TOF mass spectrometry. Furthermore, in the reaction product by Mf3 and Mf1 which was a cholinephosphotransferase and previously cloned from M. fermentans PG18, an ion at m/z 896 corresponding to GGPL-I was detected by mass spectrometry. The product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem MS analysis of protonated molecules at m/z 896. From these results, mf3 was identified as a glycosyltransferase. It was suggested that glucose transfer and phosphocholine transfer to 1,2-dipalmitoylglycerol are involved in the GGPL biosynthesis pathway of M. fermentans PG18.

Variation of genes encoding GGPLs syntheses among Mycoplasma fermentans strains.

The information of the biosynthesis pathways of Mycoplasma fermentans specific major lipid-antigen, named glycoglycerophospholipids (GGPLs), is expected to be some of help to understand the virulence of M. fermentans. We examined primary structure of cholinephosphotransferase (mf1) and glucosyltransferase (mf3) genes, which engage GGPL-I and GGPL-III synthesis, in 20 strains, and found four types of variations in the mf1 gene but the mf3 gene in two strains was not detected by PCR. These results may have important implications in virulence factor of M. fermentans.

Molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of Mycoplasma fermentans.

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.

Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen.

Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with epsilon-aminocaproic acid. The eluted approximately 50-kDa protein was identified by mass spectrometric techniques as alpha-enolase. The possibility that alpha-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-alpha-enolase antiserum, which showed that alpha-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-alpha-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface alpha-enolase is a major Plg binding protein of M. fermentans.

Characterization of DNA topoisomerase activity in two strains of Mycoplasma fermentans and in Mycoplasma pirum.

DNA topoisomerases (topos) are essential enzymes that participate in many cellular processes involving DNA. The presence of the DNA-gyrase genes in various mycoplasmas has been reported elsewhere. However, the characterization of DNA topo activity in mycoplasmas has not been previously undertaken. In this study, we characterized the topo activity in extracts of Mycoplasma fermentans K7 and incognitus and in Mycoplasma pirum, as well as in partially purified extract of M. fermentans K7. The topo activity in these microorganisms had the following properties. (i) The relaxation of supercoiled DNA was ATP dependent. (ii) ATP independent relaxation activity was not detected. (iii) Supercoiling of relaxed topoisomers was not observed. (iv) The relaxation activity was inhibited by DNA gyrase and topo IV antagonists (novobiocin and oxolinic acid) and by eukaryotic topo II (m-AMSA [4'-(9-acridylamino)methanesulfon-m-anisidide]) and topo I antagonists (camptothecin). Other eukaryotic topo II antagonists (teniposide and etoposide) did not affect the topo relaxation activity. (v) Two polypeptides of 66 and 180 kDa were found to be associated with the mycoplasma topo activity. These results suggest that the properties of the topo enzyme in these mycoplasma species resemble those of the bacterial topo IV and the eukaryotic and the bacteriophage T4 topo II. The findings that mycoplasma topo is inhibited by both eukaryotic topo II and topo I antagonists and that m-AMSA and camptothecin inhibited the growth of M. fermentans K7 in culture support our conclusion that these mycoplasma species have topo with unique properties.

[The modification of the internucleotide relations of antisignature oligodeoxyribonucleotides as a means of increasing their resistance to nuclease cleavage in mollicute cells]. / Modifikatsiia mezhnukleotidnykh sviazei antisignaturnykh oligodezoksiribonukleotidov kak sposob povysheniia ikh ustoichivosti k nukleaznomu rasshcheplenii v kletkakh mollikutov.

Modifications in 5'- and 3'-ends and internucleotide bondings of antisignature oligodesoxyribonucleotides have been studied for their resistance to nuclease cleavage in cells of Acholeplasma laidlawii PG-8 and Mycoplasma fermentans PG-18. It is shown that adding of benzylamide grouping to the 5'-end of oligonucleotides increases their half-life period in the studied mollicute cells to 15 h. S-methyl modification of 3'-end of antisignature oligonucleotides did not take essential effect on the process of degradation of the given compounds in the mollicute cells. Considerable increase of stability in the studied cels of thio- and dithiophosphate analogues of oligodesoxyribonucleotides has been detected.

Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.

The catalytic RNA moiety of (eu)bacterial RNase P is responsible for cleavage of the 5' leader sequence from precursor tRNAs. We report the sequence, the catalytic properties, and a phylogenetic-comparative structural analysis of the RNase P RNA from Mycoplasma fermentans, at 276 nt the smallest known RNase P RNA. This RNA is noteworthy in that it lacks a stem-loop structure (helix P12) that was thought previously to be universally present in bacterial RNase P RNAs. This finding suggests that helix P12 is not required for catalytic activity in vivo. In order to test this possibility in vitro, the kinetic properties of M. fermentans RNase P RNA and a mutant Escherichia coli RNase P RNA that was engineered to lack helix P12 were determined. These RNase P RNAs are catalytically active with efficiencies (Kcat/Km) comparable to that of native E. coli RNase P RNA. These results show that helix P12 is dispensable in vivo in some organisms, and therefore is unlikely to be essential for the mechanism of RNase P action. The notion that all phylogenetically volatile structures in RNase P RNA are dispensable for the catalytic mechanism was tested. A synthetic RNA representing the phylogenetic minimum RNase P RNA was constructed by deleting all evolutionarily variable structures from the M. fermentans RNA. This simplified RNA (Micro P RNA) was catalytically active in vitro with approximately 600-fold decrease in catalytic efficiency relative to the native RNA.

[The detection of proteolytic enzymes in the culture broth of Mollicutes]. / Vyiavlennia proteolitychnykh fermentov u kul'tural'nii ridyni deiakykh molikutiv.

Four strains of mollicutes different in their role in pathology of people, animals and plants have been studied to detect general proteolytic (caseinolytic) activity with results presented. Proteolytic activity was determined both in the deposited proteins and in supersediment liquids under suturation with ammonium sulphate of 55 and 80%, respectively. Maximum proteolytic activity was observed in supersediment liquids. It was 137.2 E per 1 mg of protein in M. fermentans PG-18; 106.2 in A. laidlawii PG-8; 61.8 in A. laidlawii var. granulum 118 and 11.5 E per 1 mg of protein in M. pneumoniae FH. In the sediments of studied mollicutes proteolytic (caseinolytic) activity was also found but it was considerably less. The relation of proteolytic enzymes found in the sediment being quantitatively expressed, the following proportion will be obtained: 118:FH:PG-18:PG-8=8.8:2.3:2.8:1. Sediments can serve initial material for isolation of these enzymes and for detailed study of their properties.

Purification and characterization of an acid phosphatase from Mycoplasma fermentans.

An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X-100, then purified by ion-exchange chromatography on DEAE-Sephacel and CM-Sepharose, followed by affinity chromatography on Con A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p-nitrophenyl phosphate was enhanced remarkably by Cu2+, Co2+ and Mg2+, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O-phospho-L-tyrosine as well as p-nitrophenyl phosphate, but not O-phospho-L-threonine, O-phospho-L-serine, glucose-1-phosphate, phosphoryl choline and adenosine triphosphate. The level of the O-phospho-L-tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas.

Acid phosphatase purified from Mycoplasma fermentans has protein tyrosine phosphatase-like activity.

Acid phosphatase purified from Mycoplasma fermentans dephosphorylated phosphotyrosine-containing lysozyme and Raytide, a peptide substrate for protein tyrosine phosphatases. The optimum pH for Raytide was about 5.5. Raytide phosphatase activity was inhibited by potassium fluoride, sodium molybdate, and sodium orthovanadate and was found to exist in some mycoplasmas.

[The physicochemical properties of the DNAse of Mycoplasma fermentans PG-18]. / Fiziko-khimicheskie svoistva DNK-azy Mycoplasma fermentans PG-18.

Physical and chemical properties, as well as polypeptide structure of DNAse from Mycoplasma fermentans PG-18, have been determined. The enzyme in a native form exists probably as a decamere (10X34 kD) and manifests maximal activity at weak alkaline pH range. The temperature optimum of the enzyme is --37 degrees C. DNAse appears to be Mg2+-dependent and has its maximal activity at 10 mM MgCl2. EDTA completely inhibits DNAse activity. The given DNAse has been determined to cleave a phosphodiether bond in 3'-position of deoxyribose and to have both exo- and endonuclease activity, since it has hydrolized both native linear doublestranded DNA and closed-circle plasmid DNA.

[The inhibitory action of 6-azacytidine on Mollicutes and its proposed mechanism]. / Ingibitornoe deistvie 6-azatsitidina na mollikuty i ego predpolagaemyi mekhanizm.

6-Azacytidine (6-AC) is shown to have an inhibitory effect on the Mollicutes of the different systematic position. The growth of type strains of Mollicutes (Acholeplasma laidlawii PG-8, Mycoplasma pneumoniae FH and M. fermentans PG-18) completely ceased in the nutrient medium at concentration of the above substance in it within the range of 125-250 micrograms/ml. 50% inhibiting concentration of 6-AC equaled: for M. fermentans PG-8: 23.43 micrograms/ml; M. pneumoniae FN: 46.8 micrograms/ml; Acholeplasma laidlawii PG-8: 62.5 micrograms/ml. 6-AC concentration 5 micrograms/ml decreased the process of DNA-dependent DNA synthesis in the in vitro system more than by 60%. 6-AC exerted less effect on the DNA-dependent RNA synthesis in the in vitro system: at different concentrations of 6-AC (up to 400 micrograms/ml) RNA synthesis decreased only by 20%. Translation on ribosomes of Mollicutes in the in vitro system completely ceased at 6-AC concentration 100 micrograms/ml. The results obtained indicate that for 6-AC in cells of Mollicutes and, possibly, for other microorganisms there are two targets: ribosomes and DNA-dependent DNA-polymerase. Total effect of blocking of the translation and replication processes by 6-azacytidine causes death of Mollicutes. Since 6-AC has no harmful effect on the human cells, it can be used as an efficient method for treatment of respiratory and urogenital diseases induced by Mollicutes.

[The isolation and purification of a nonspecific DNAse from Mycoplasma fermentans PG-18]. / Vydelenie i ochistka nespetsificheskoi DNK-azy Mycoplasma fermentans PG-18.

Nonspecific endogenic DNAase has been isolated from biomass of Mycoplasma fermentans PG-18 cells and purified to the homogeneous state. The scheme of isolation consists of purification stages on columns with phosphocellulose, DNA-cepharose CL-8B and phenylcepharose. DNAase was not bound to phosphocellulose, its volume was equal to zero. Then this DNAase was passed through column with DEA-cepharose CL-6B (elution by gradient KCl from 0.1 to 1.8M): enzyme was eluted at KCl concentration in the eluting buffer from 0.1 to 1.2 M. The enzyme was purified to the homogeneous state on column with phenylcepharose (elution by linear gradient of ethylene glycol from 30 to 80%): enzyme was eluted at the concentration of ethylene glycol in the eluting buffer from 43 to 80%. According to data obtained using gel-electrophoresis, under the denaturing conditions molecular mass of enzyme in acrylamide gel was 34 kDa.

[Mycoplasma restriction-modification system MunI and its possible role in pathogenesis processes]. / Mikoplazmennaia sistema restriktsii--modifikatsii MunI i ee vozmozhnaia rol' v protsessakh patogeneza.

The restriction-modification system, named RMMunI, has been purified and characterised from Friend murine erythroleukemia cells. The site-specific endonuclease recognizes and cleaves the 5'C1AATTG nucleotide sequence. RMunI is an isoschizomer of RMfeI from Mycoplasma fermentans. Site-specific methylase modifies the second adenine residue in the same sequence (5'Cam6ATTG). It was established that the discovered enzymatic system is from mycoplasma which contaminates cell lines. Mycoplasma's DNA hybridizes with species-specific DNA probed for Mycoplasma fermentans and Mycoplasma arginini. The possible role of mycoplasmic restriction-modification enzymes in the process of acquired immune deficiency syndrome are discussed.