The oxidant scavenging capacity of the oral Mycoplasma salivarium.

OBJECTIVE: Mycoplasma salivarium is a human oral potential pathogen that preferentially resides in dental plaques and gingival sulci. It has been suggested that this organism may play an etiological role in inflammatory processes in the oral cavity. The aim of this work was to determine whether M. salivarium possesses a potent oxidant scavenging capacity (OSC). DESIGN: The OSC of M. salivarium was quantified by a highly sensitive luminal-dependent chemiluminescence assay in the presence of cocktails that induced a constant flux of luminescence resulting from the generation of peroxide, hydroxyl radical (cocktail A) and NO, superoxide and peroxynitrites (cocktail B). RESULTS: M. salivarium markedly reduced oxidative stress by scavenging both free reactive oxygen and nitrogen species. The OSC of M. salivarium was much higher than that of other Mycoplasma species. Most of M. salivarium OSC was confined to the cytosolic fraction and was markedly increased in the presence of tannic acid, red blood cells or mucin. The cytosolic OSC of M. salivarium was heat stable and not affected by sodium azide or prolonged proteolysis. However, it was markedly decreased upon dialysis, suggesting that the major reducing activity is not enzymatic but rather, a low molecular weight compound(s). CONCLUSIONS: The ability of M. salivarium to scavenge oxidants may play a role in the survival and pathogenicity of this microorganism. The enhanced OSC of M. salivarium in the presence of tannic acid, red blood cells or mucin might have a significant importance to assess complex interactions with polyphenols from nutrients, salivary proteins and red blood cells extravasated from injured capillaries during infection and inflammation in oral tissues.

Intracellular location and survival of Mycoplasma penetrans within HeLa cells.

Mycoplasma penetrans invades HeLa cells and survives within them for prolonged periods of time. The intracellular distribution of M. penetrans within HeLa cells was studied utilizing the acidotropic dye LysoTracker (green), which permeates cell membranes and upon protonation remains trapped in acidic compartments. The excitation and emission spectra of the green LysoTracker are suitable for colocalization studies with rabbit anti- M. penetrans antibodies and red Cy5 goat anti-rabbit IgG. The images collected by confocal laser scanning microscopy revealed that in the infected HeLa cells almost all Cy5 fluorescent foci (red) were located within the LysoTrack-labelled intracellular compartments, apparently endosomes. Viable mycoplasmas were detected within endosomes for prolonged periods of time, apparently due to a potent antioxidant activity detected in M. penetrans.

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells.

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.

Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes.

A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium.

The physico-chemical characteristics of the phosphocholine-containing glycoglycerolipid MfGL-II govern the permeability properties of Mycoplasma fermentans.

Mycoplasma fermentans seems to be involved in several pathogenic conditions in humans, and is among other things capable of fusing with T-cells and lymphocytes. The choline-containing phosphoglycolipid 6'-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-alpha-D-glucopyranosyl-(1'-->3)-1,2-diacylglycerol (MfGL-II) in the membrane of M. fermentans has been suggested to enhance the fusion process, and the characteristics of MfGL-II were therefore investigated. When a cell culture ages the fraction of MfGL-II increases, and the fraction of the other major membrane lipid, phosphatidylglycerol (PtdGro), decreases concomitantly. Swelling experiments showed that the permeability and osmotic fragility are markedly reduced in aged cells. MfGL-II is selectively released into the surrounding medium when aged M. fermentans cells are incubated in buffer containing EDTA. The physico-chemical properties of MfGL-II were studied by NMR spectroscopy and differential scanning calorimetry, and they can explain the biochemical results. The temperature for the transition between gel and lamellar liquid crystalline (Lalpha) phases is 35-45 degrees C higher for MfGL-II than for PtdGro, which most probably gives rise to the reduced permeability in aged cells. At high water contents MfGL-II forms an Lalpha phase and isotropic aggregates which were interpreted to be vesicles with a radius of approximately 450 A. It is proposed that MfGL-II forms vesicles in the surrounding medium when it is released from the cell membrane. Neither EDTA nor Ca2+ ions have a significant influence on the aggregate structures formed by MfGL-II. Our results indicate that MfGL-II has no fusogenic properties. It is more probable that a recently identified lysolipid in the M. fermentans membrane acts as a fusogen.

The phosphocholine motif in membranes of Mycoplasma fermentans strains.

Mycoplasma fermentans strains differ in the profile of choline-containing phosphoglycolipids (PGL) present in their cell membrane. MfGL-II [Zähringer et al. (1997) J. Biol. Chem. 272, 26262-26270] was found to be the major PGL in most strains tested. However, in the pulmonary isolates, M52 and M39 the major choline-containing PGLs were MfGL-I [Matsuda et al. (1994) J. Biol. Chem. 269, 33123-33129] and MfEL, a unique choline-containing ether lipid recently identified by us [Wagner et al. (2000) Eur. J. Biochem. 267, 6276-6286]. MfGL-I, MfGL-II and MfEL were metabolically labeled by growing the cells with radioactive choline but only MfGL-I and MfGL-II [corrected] reacted with antiphosphocholine antibodies. All tested strains fused with Molt-3 cells at almost the same rate and to about the same extent and in all the strains membrane proteins that reacted with anti-phosphocholine antibodies were detected, indicating that some membrane proteins are decorated with phosphocholine moieties.

Plasminogen binding and activation by Mycoplasma fermentans.

The binding of plasminogen to Mycoplasma fermentans was studied by an immunoblot analysis and by a binding assay using iodine-labeled plasminogen. The binding of 125I-labeled plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine analog epsilon-aminocaproic acid. Partial inhibition was obtained by a plasminogen fragment containing kringles 1 to 3 whereas almost no inhibition was observed with a fragment containing kringle 4. Scatchard analysis revealed a dual-phase interaction, one with a dissociation constant (kd) of 0.5 microM and the second with a kd of 7.5 microM. The estimated numbers of plasminogen molecules bound were calculated to be 110 and 790 per cell, respectively. Autoradiograms of ligand blots containing M. fermentans membrane proteins incubated with 125I-labeled plasminogen identified two plasminogen-binding proteins of about 32 and 55 kDa. The binding of plasminogen to M. fermentans enhances the activation of plasminogen to plasmin by the urokinase-type plasminogen activator (uPA), as monitored by measuring the breakdown of chromogenic substrate S-2251. Enhancement was more pronounced with the low-molecular-weight and the single-chain uPA variants, known to have low plasminogen activator activities. The binding of plasminogen also promotes the invasion of HeLa cells by M. fermentans. Invasion was more pronounced in the presence of uPA, suggesting that the ability of the organism to invade host cells stems not only from its potential to bind plasminogen but also from the activation of plasminogen to plasmin.

Ether lipids in the cell membrane of Mycoplasma fermentans.

Two new ether lipids, 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine and its lyso form, 1-O-alkyl/alkenyl-glycero-3-phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC-MS, MALDI-TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1' (plasmalogen-type lipid) or 9' in a ratio approximately 1 : 1. This is the first report of the presence of alkyl and vinyl (alk-1'-enyl) ether lipids in the cell membrane of aerobically grown mycoplasmas. Lipids of this type have been found in some Gram-positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2-O-acetylated lyso form lipid can mimic platelet-activating factor activity in isolated perfused and ventilated rat lungs.

Subversion and exploitation of host cells by mycoplasmas.

Mycoplasmas are minute wall-less bacterial parasites that exhibit strict host and tissue specificities. They enter, multiply and survive within the host for extended periods by circumventing host defenses. Their intimate interaction with eukaryotic cells, and in some cases the subsequent invasion into or fusion with these cells, mediates cell damage. Mycoplasmas also modulate the activity of host cells by a variety of direct mechanisms and/or indirectly by cytokine-mediated effects.

Protein kinase C activation and vacuolation in HeLa cells invaded by Mycoplasma penetrans.

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. This study investigated the invasion of HeLa cells and its consequences by confocal laser scanning microscopy. Invasion was dependent on the duration of infection and temperature, diminished by inhibiting microfilament assembly with cytochalasin D and almost completely abolished by disorganising microtubules with vinblastine or taxol. After a short infection period (< 20 min), pronounced activation of protein kinase C was detected in host cells, whereas prolonged infection resulted in intensive vacuolation of the host cells and a pronounced increment in intracellular organic peroxide levels. A marked decrease in the extent of vacuolation was observed when peroxide accumulation was partially prevented by alpha-tocopherol. The possibility that M. penetrans entry into HeLa cells involves the activation of protein kinases and the recruitment of cytoskeleton components is discussed.

Mycoplasma fermentans glycolipid triggers inflammatory response in rat astrocytes.

Mycoplasma fermentans glycolipid (MfGL-II) is a major lipid in the membranes of this AIDS-associated mycoplasma and constituting up to 20% of the total phospholipids of this organism. It was recently shown that MfGL-II, mainly through its phosphocholine moiety, is responsible for the attachment of M. fermentans to host cells. We now show that MfGL-II is also associated with the secretion of inflammatory mediators by cells of the central nervous system. Stimulation of primary rat astrocytes by MfGL-II caused activation of protein kinase C, secretion of nitric oxide (NO) and prostaglandin E2, and augmented glucose utilization and lactate formation in a dose-dependent manner. In an attempt to define the minimal structural requirements for MfGL-II activity, the two O-acylated fatty acids in the molecule were removed. Deacylation pronouncedly reduced the stimulatory activity of the glycolipid, suggesting that the fatty acyl residues are essential. Incubation of MfGL-II with polyclonal anti-MfGL-II antiserum or with monoclonal anti-phosphocholine antibody diminished NO release, whereas incubation of MfGL-II with normal rabbit serum had no effect. It is, therefore, likely that the terminal phosphocholine moiety plays an important role in MfGL-IIs stimulation of glial cells.

Antibody response to MfGL-II, a phosphocholine-containing major lipid of Mycoplasma fermentans membranes.

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.

Primary structure of a new phosphocholine-containing glycoglycerolipid of Mycoplasma fermentans.

The chemical structure of a novel phosphocholine-containing glycoglycerolipid, the major polar lipid in the cell membrane of Mycoplasma fermentans PG18, was investigated by chemical analyses, gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as one- and two-dimensional homo- and heteronuclear NMR spectroscopy and identified as 6'-O-(3"-phosphocholine-2"-amino-1"-phospho-1", 3"-propanediol)-alpha-D-glucopyranosyl-(1'-->3)-1,2-diacyl-glycerol (MfGL-II). Palmitate (16:0) and stearate (18:0), in a 3.6:1 molar ratio, constitute the major fatty acids present. MALDI-TOF mass spectrometry revealed two major pseudomolecular ions at m/z 1049.5 [MI + H]+ and 1077.3 [MII + H]+ representing a dipalmitoyl as the major component and a palmitoyl-stearoyl structure as a minor component. This is the first report of 2-amino-1,3-propanediol-1,3-bisphosphate present in a natural product. This glycoglycerolipid is the second phosphocholine-containing glycoglycerolipid found in M. fermentans.

The thioredoxin reductase system of mycoplasmas.

Representative species of the Mollicutes possess a thioredoxin reductase system (NTS) composed of a low-molecular-mass thioredoxin (TRX) and NADPH-binding thioredoxin reductase (NTR). The TRXs of Mycoplasma pneumoniae and M. capricolum have molecular masses of 11-2 and 12 kDa, respectively, and are stable at 90 degree C for 10 min. Both TRXs reacted with monospecific polyclonal antibodies generated against the Bacillus subtilis TRX, but not with anti-Escherichia coli TRX antisera. The M. capricolum and M. pneumoniae NTRs were partially purified and were found to be active with the homologous TRX, but not with the TRX of B. subtilis or E. coli. The NTS activity had an optimal pH of 6.5-7.5 and was dependent on NADPH as an election donor, a requirement which could not be fulfilled by NADH. The genes encoding the TRX and NTR (trxA and trxB) or M. pneumoniae were cloned and sequenced. The comparative analysis of the predicted amino acid sequence of trxA showed that the 11.2 kDa protein (102 aa) shared 26-68% sequence similarity with products of other known trxA genes and contained the conserved active site Cys-Gly-Pro-Cys. The predicted amino acid sequence of trxB contained 315 residues with a conserved NADPH binding domain and FAD binding domains I and II. The cysteine dithiol redox active region had isoleucine rather than threonine at the active site, as compared with other NTRs. The high activity of the NTS in mycoplasmas suggests that mycoplasmas may have evolved the NTS to protect themselves from the consequences of their self-generated oxidative challenge.

Mycoplasma in the spotlight of AIDS: partial biochemical characterization of mycoplasma-derived components inducing TNF alpha secretion and blast transformation.

We have previously demonstrated that the AIDS-associated Mycoplasma fermentans as well as Mycoplasma capricolum membranes activated bone marrow macrophages to secrete tumor necrosis factor alpha (TNF alpha) and induce blast transformation of splenic lymphocytes. Herein, we show that the membrane component of Mycoplasma capricolum capable of inducing TNF alpha secretion is a hydrophobic protein. This is supported by our findings that the TNF alpha inducing activity was eluted by a phenyl-Sepharose column in a peak distinct from bulk membrane lipids. The hydrophobic nature of the protein is indicated by the activity of the "hydrophobic protein" fraction of the membranes, and the pattern of elution obtained by the phenyl-Sepharose column. Fractionation of the M. capricolum membranes, solubilized by CHAPS (3-[(3-cholamidopropyl)-dimethylammoniol]1-propane sulfate) on a gel filtration column revealed a major peak of TNF alpha inducing activity of about 75,000 daltons, and a minor peak of about 55,000 daltons. The mitogenic activity, though spread throughout the column, peaked in the same fractions as the TNF alpha inducing activity. Both activities co-eluted by the phenyl-Sepharose column as well. However, the mitogenic activity of the membranes was much more resistant to elevated temperatures and extreme pH treatment.

The 57-Kilodalton Phosphoprotein of Spiroplasma melliferum Is Autophosphorylated

The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl2. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process.

Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein.

The ability of Mycoplasma penetrans to invade eukaryotic cells was studied using a HeLa cell line. The bactericidal antibiotic, gentamicin, in combination with low concentrations of Triton X-100, was utilized to kill mycoplasmas that had not entered the cells, allowing the quantitation of internalized organisms. The intracellular location of the mycoplasma was also documented by transmission electron microscopy. The actin polymerization inhibitor cytochalasin-D markedly inhibited the internalization process, whereas the tyrosine phosphorylation inhibitors, staurosporin and genistein had only a slight effect. As against the invasion of enteropathogenic Escherichia coli which depends on tyrosine phosphorylation of a 90-kDa (Hp90) HeLa cell protein, internalization of M. penetrans by HeLa cells was independent of the phosphorylation of Hp90. Nonetheless, tyrosine phosphorylation of a 145-kDa HeLa cell protein was found to be associated with the interaction of M. penetrans with HeLa cells.