Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Genomic features and insights into the biology of Mycoplasma fermentans.

We present the complete genomic sequence of Mycoplasma fermentans, an organism suggested to be associated with the pathogenesis of rheumatoid arthritis in humans. The genome is composed of 977,524 bp and has a mean G+C content of 26.95 mol%. There are 835 predicted protein-coding sequences and a mean coding density of 87.6 %. Functions have been assigned to 58.8 % of the predicted protein-coding sequences, while 18.4 % of the proteins are conserved hypothetical proteins and 22.8 % are hypothetical proteins. In addition, there are two complete rRNA operons and 36 tRNA coding sequences. The largest gene families are the ABC transporter family (42 members), and the functionally heterogeneous group of lipoproteins (28 members), which encode the characteristic prokaryotic cysteine 'lipobox'. Protein secretion occurs through a pathway consisting of SecA, SecD, SecE, SecG, SecY and YidC. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The genes encoding DnaK-DnaJ-GrpE and Tig, forming the putative complex of chaperones, are intact, providing the only known control over protein folding. Eighteen nucleases and 17 proteases and peptidases were detected as well as three genes for the thioredoxin-thioreductase system. Overall, this study presents insights into the physiology of M. fermentans, and provides several examples of the genetic basis of systems that might function as virulence factors in this organism.

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.

The reducing antioxidant capacity of Mycoplasma fermentans.

Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.

MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly?

Although some activities of LPS are shared by other bacterial components, for half a century LPS has been regarded as unique in displaying many pathophysiological activities. Here we report on a synthetic lipopeptide, MALP-2 from Mycoplasma fermentans, which expresses potent endotoxin-like activity and whose lethal toxicity is comparable to that of LPS. With the exception of the Limulus lysate gelation test, in which MALP-2 was approximately 1000-fold less active than LPS, the synthetic lipopeptide induced all activities tested for, and in most cases to an extent comparable to that of LPS. Unlike LPS, the biological activities of MALP-2 were expressed both in LPS-responder and in LPS-non-responder mice (BALB/c/l, C57BL10/ScCr), indicating that MALP-2 signaling, unlike that of LPS, is not transduced via the Toll-like receptor (Tlr) 4 protein.MALP-2 expressed no toxicity in normal or sensitized Tlr2 knockout (Tlr2(-/-)) mice indicating that its toxic activity is induced via Tlr2 signaling. The phenomenology of the lethal shock induced by MALP-2 in normal or sensitized mice, i.e. the kinetics of its development and symptoms of illness exhibited by the treated animals, was very reminiscent of the lethal shock induced by LPS.

Choline-containing lipids in mycoplasmas.

Choline-containing lipids were identified and characterized in the cell membrane of Mycoplasma fermentans and were shown to participate in the adhesion to the surface of eukaryotic cells, to stimulate mycoplasma fusion with eukaryotic cells, and to induce cytokine secretion by cells of the immune system. These findings suggest that choline-containing lipids are important mediators of tissue pathology in the infectious process caused by M. fermentans.

Chronic fatigue syndrome: a risk factor for osteopenia?

No data documenting a possible depletion of bone mineral density in patients with chronic fatigue syndrome (CFS) are currently available. However, recent pathophysiological observations in CFS patients may have deleterious consequences on bone density. Firstly, the deregulation of the 2,5A synthetase RNase L antiviral pathway and its associated channelopathy, implicates increased demands for calcium and consequent increased calcium-re-absorption from the skeletal system. Secondly, Mycoplasma fermentans which has been frequently associated with CFS, produces a lipopeptide, named 2-kDa macrophage-activating lipopeptide (MALP-2), which stimulates macrophages. MALP-2 has been shown to enhance bone resorption in a dose-dependent manner, at least in part by stimulating the formation of prostaglandins. Thirdly, decreased levels of insulin-like growth factor I (IGF-I) have been reported in CFS-patients. IGF-I is critical to the proliferation of osteoblasts. Consequently, depleted levels of IGF-I may shift the balance between osteoclastic and osteoblastic activity towards bone resorption.

[Detection of Mycoplasma and its antibodies in the blood of HIV-positive patients and in healthy persons]. / Vyiavlenie mikoplazm i antitel k nim v krovi VICh-pozitivnykh i zdorovykh lits.

The comparative study of HIV-positive and clinically healthy persons has indicated that the antigens of M. pneumoniae, M. fermentans and U. urealyticum are encountered nearly twice more frequently in the blood of HIV-infected patients than in that of healthy individuals. Mycoplasma antibodies are detected in HIV-positive persons 12 times more frequently than in healthy ones. Among the HIV-infected persons there are those who have simultaneously antigens of some Mycoplasma species.

[Carbohydrate receptors for Mycoplasma fermentans adhesion on human epithelial tissues]. / Vuhlevodni retseptory dlia adheziï Mycoplasma fermentans na epitelial'nykh tkanynakh liudyny.

It has been stated that two terminal carbohydrates from polysaccharide complexes which are on the surface of human epithelial tissues, namely, alpha-D-glucose and N-acetylneuramino acid bound with subterminal galactose via alpha 2-->3-bond (NeuAc alpha 2-->3 Gal), may serve receptors for Mycoplasma fermentans adhesion on human epithelial cells. M. fermentans shows high selectivity to these receptors, though very low affinity. The latter, probably, explains why this mycoplasma is able to infect only the limited number of peoples. In the authors' opinion people with the lower content of glucose in urine, as well as those who suffer from diseases associated with hypothalamo-hypophyseal insufficiency are subjected to infection with M. fermentans. People with normal (3.33-5.55 mM) and elevated alpha-D-glucose content in blood and in urine are not susceptible to this mycoplasma. Results of the research carried have shown that alpha-D-glucose solutions of definite concentration may be used to eliminate M. fermentans from the urogenital tract of people who have it. The ability of M. fermentans to discriminate terminal structure of NeuAc alpha 2-->3 Gal provides it with the possibility to adhere human immunodeficiency virus virions on its cells as glycoprotein (gp120) of that virus has among its own oligosaccharides certain glycopolymers of the similar terminal structure. Then M. fermentans transports the virions directly to target cells for this virus. The target cells express receptor CD4 glycolized by oligosaccharides of the mentioned terminal structure. It provides adhesion of the mycoplasma on the receptor.

Fatal systemic infections of nonhuman primates by Mycoplasma fermentans (incognitus strain).

Four silvered leaf monkeys inoculated with Mycoplasma fermentans (incognitus strain) showed wasting syndromes and died in 7-9 months. Infected animals had a late and transient antibody response to mycoplasmal infection. Three monkeys revealed periodic mycoplasmal antigenemia. The one that had the most persistent antigenemia failed to mount a detectable antibody response and was the first to die of the infection. The control monkey was killed 8 months later, after the last of the infected animals had died, and revealed no evidence of seroconversion or antigenemia. Polymerase chain reaction, immunohistochemical, and electron microscopic studies identified systemic infections of M. fermentans in the infected animals. No other opportunistic infection or neoplastic disease was found. It is interesting to note the absence of an inflammatory reaction to the large number of mycoplasmas in the infected tissues. M. fermentans (incognitus strain) apparently suppressed normal inflammatory or immune responses, produced wasting syndromes, and caused a fatal systemic infection in these monkeys.

Mycoplasmas as cofactors in infection due to the human immunodeficiency virus.

Results obtained in vitro suggest that mycoplasmas act as cofactors with the human immunodeficiency virus (HIV) in the development of AIDS. Mycoplasmas, including Mycoplasma fermentans, Mycoplasma pirum, and Mycoplasma penetrans have since been isolated from HIV-infected individuals. In addition, M. fermentans has been detected by different investigators in numerous tissues and in the blood of HIV-infected patients. Higher titers of antibodies to M. penetrans have also been found in HIV-infected patients as compared with noninfected individuals. These mycoplasmas have been shown to have the capacity to invade cells and to be potent immunomodulators. Although there is no doubt that mycoplasmas are found in HIV-infected individuals and eventually produce systemic infections, their pathogenic role in association with HIV remains to be determined.

Differential protein expression and surface presentation generate high-frequency antigenic variation in Mycoplasma fermentans.

Mycoplasma fermentans, a wall-less prokaryote, is currently under investigation as a potential human pathogen. Recently, several surface lipoproteins have been shown to vary in expression between M. fermentans strains. Using specific antibodies to these lipoproteins, we investigated the extent and nature of antigenic variation within this species. Immunoscreening of type strain PG18 agar-grown colonies revealed marked heterogeneity in expression of distinct surface lipoproteins. Subsequent isolation and propagation of clonal isolates established isogenic lineages which displayed high-frequency (10(-2) to 10(-5) per generation) antigenic phase variation. [35S]cysteine-labeled protein profiles and Western immunoblots of phase-variant clones showed that several distinct integral membrane proteins undergo noncoordinate variation in expression. In addition to differential expression of epitope-bearing lipoproteins, differential accessibility of epitopes to antibodies was also documented as a mechanism generating surface phenotypic variation. Examination of one strain-variant antigen showed high-frequency phase variation to underlie previously observed antigenic differences between strains of this species. Thus, M. fermentans has a complex system capable of creating rapid changes in surface mosaics. This may profoundly affect mycoplasma-host interactions and may limit the methods by which populations of M. fermentans may be studied in vivo.

Mycoplasma fermentans binds to and invades HeLa cells: involvement of plasminogen and urokinase.

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

Cytopathogenicity of Mycoplasma fermentans (including strain incognitus).

Mycoplasma fermentans strain incognitus, an organism recently identified in tissues of patients with AIDS and in tissues of otherwise healthy adults with an acute fatal respiratory disease, was evaluated for cytopathogenicity for tracheal tissue in vivo and in vitro. In this study, the organism produced a chronic infection of the lower respiratory tract in LEW rats following intranasal inoculation and induced both ciliostasis and cytopathology in experimentally infected tracheal explants from rats. The time of onset of ciliostasis, type of cytopathogenicity, and localization of organism in strain incognitus were different from those in other strains of M. fermentans as well as other species of mycoplasmas isolated from humans. The results strongly support, but do not prove, that M. fermentans strain incognitus is an unusually invasive mycoplasma, as it was the only strain found within respiratory epithelial cells both in vivo and in vitro. Detection of the organism within the lamina propria also supported the organism's invasive potential. Further study of both the in vivo and in vitro models should provide insights into this potentially unique mycoplasma-host relationship.

Does HIV cause AIDS? A review.

It would require a detailed knowledge of virology, molecular biology, epidemiology, clinical medicine and politics, to appropriately compare and contrast the hypotheses on the causes of AIDS. The purpose of this review was not to do that, but to inform colleagues that alternative etiologies for AIDS have been considered. No doubt, this healthy questioning will continue until it has been demonstrated--via controlled studies of high-risk groups (both HIV positive and negative), matched for all other characteristics--that only those individuals with HIV positivity actually develop AIDS. It cannot be denied that a common theme to the hypotheses is the presence of high-risk activities. This has been used against the risk-AIDS hypothesis. How, for example, could it explain babies born with immunodeficiencies, K. Bergalis contacting AIDS from her dentist, the British nurse who died of AIDS after contracting HIV from her husband, or AIDS in the wives of hemophiliacs? It may be that these people died of specific diseases (leukemia, pneumonia, infections), which 20 years ago would have been diagnosed as such. Now, because these individuals are found to be HIV positive, they are viewed as AIDS patients. Alternatively, they may not have been asked about their nutritional status, use of psychoactive drugs, and immunosuppressive sexual practices. Additionally, it is possible that by the time AIDS was diagnosed they may have already received numerous antibiotic (immunosuppressive) drug treatments. In North America, for whatever reason, AIDS is associated with high-risk groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of serological evidence for Mycoplasma fermentans infection in army Gulf War veterans: a large scale case-control study.

Mycoplasma firmentans is suspected in the development of 'Gulf War illness' in veterans of Operation Desert Storm. We conducted a matched case-control study for the prevalence of M. firmentans-specific antibodies before and after the operation, as well as seroconversion rates in veterans with and without complaints of 'Gulf War illness'. Cases consisted of Gulf War veterans, who complained of various illnesses and were enrolled in the second phase of the health evaluation by the Army Comprehensive Clinical Examination Program (CCEP). Controls were selected from Gulf War veterans who did not participate in the registry and did not request a health evaluation by the CCEP. Before operation deployment, 34 out of 718 of the cases (48%) and 116 out of 2233 of the controls (5.2%) tested positive for M. fermentans-specific antibodies. There was no difference in rates of seroconversion between cases and controls (1.1 vs. 1.2%) to M. fermentans during Operation Desert Storm. Thus, there is no serological evidence that suggests infectionby M. fermentans is associated with development of 'Gulf War illness'.

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.

Phosphocholine-containing glycoglycerolipids (GGPL-I and GGPL-III) are species-specific major immunodeterminants of Mycoplasma fermentans.

Mycoplasma fermentans has unique glycoglycerophospholipids (GGPLs: GGPL-I and GGPL-III). Previously, the structure of these lipids was determined as phosphocholine-6'-alpha-glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-I) and 1"-phosphocholine-2"-aminodihydroxypropane-3"-phospho-6'-alph++ + a- glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-III). Thin-layer chromatography (TLC) immunostaining showed that the GGPLs were main lipid-antigens of the M. fermentans species. Anti-M. fermentans serum stained mainly the GGPLs, but the other anti-mycoplasma sera (anti-M. arginini, anti-M. hyorhinis, anti-M. pneumonia, anti-M. primatum, and anti-Acholeplasma laidlawii, anti-M. hominis, anti-M. orale, and M. salivarium) stained neither GGPL-I nor GGPL-III. The TLC analysis of glycolipids and phospholipids of various human related mycoplasmas showed clearly that GGPLs are specifically expressed in M. fermentans species. GGPL-I and GGPL-III ranged from 1.6 to 28% and from an undetectable level to 35% of total phospholipids, respectively. Although there was heterogeneity among the amounts of GGPL-I or GGPL-III of M. fermentans strains, all of the M. fermentans strains had GGPL-I and/or GGPL-III. These observations showed that GGPL structures are species-specific immunodeterminants of M. fermentans. The fact that the GGPLs are main phospholipid components of the M. fermentans species means the M. fermentans has a unique choline metabolic pathway. This observation may raise phylogenetic interest.

Identification and functional mapping of the Mycoplasma fermentans P29 adhesin.

Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).