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.     

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.     

Membrane lipids of Mycoplasma fermentans

Abstract Membranes of Mycoplasma fermentans , incognitus strain, were isolated by a combination of osmotic lysis and sonication. Analysis of membrane lipids revealed, in addition to free and esterified cholesterol, six major polar lipids dominated by a de novo synthesized compound (compound X), which accounts for 64% of the total lipid phosphorus. Compound X was labeled by palmitate, but not by oleate. Mass spectrometry and gas liquid chromatography analyses of compound X revealed two molecular species with molecular masses of 1048 and 1076 representing, a dipalmitoyl- and a stearoyl-palmitoyl-glycerodiphosphatidylcholine. Compound X has the ability to stimulate human monocytes to secret TNFα and to enhance the fusion of small unilamellar vesicles with MOLT-3 lymphocytes.     

Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity.

The interaction of hemoglobin (Hb) with endotoxins [i.e. with enterobacterial deep rough mutant lipopolysaccharide (LPS) Re and the "endotoxic principle" of LPS, lipid A] was investigated using a variety of physical techniques and with two biological assays, tumor necrosis factor (TNF)-alpha induction in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic binding to the hydrophobic moiety with a slight rigidification of the lipid A acyl chains, and an increase in the inclination of the lipid A backbone with respect to the membrane surface from 35 degrees to more than 40 degrees due to Hb binding, but no change of the predominantly alpha-helical secondary structures of Hb due to LPS binding. From isothermal titration calorimetry, the molar [Hb] : [endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a reorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q224. The LPS-induced TNF-alpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Although a detailed mechanism of action cannot be given, the enhancement of LPS bioactivity can be understood in the light of the previously presented conformational concept; Hb induces an increase in the conical shape of the lipid A moiety of LPS, higher cross-section of the hydrophobic than the hydrophilic part, and of the inclination angle of the diglucosamine backbone with respect to the direction of the acyl chains.     

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.     

Fusion of Mycoplasma fermentans strain incognitus with T-lymphocytes.

The ability of Mycoplasma fermentans (strain incognitus) to fuse with cultured lymphocytes was investigated and the fusion process was characterized. Fusion was measured using an assay to determine lipid mixing based on the dequenching of the fluorescent probe, octadecylrhodamine (R18), that was incorporated into the mycoplasma cells. Fusion of M. fermentans was detected with both CD4+ (Molt 3) and CD4- (12-E1) cells. The amount of fusion induced was relatively low and ranged from 5-10% with either cell culture. When primary peripheral blood lymphocytes were used the fusion yield was somewhat higher, reaching 12% of the cell population. Similar findings were obtained with fluorescent microscopy analysis suggesting that a predetermined, but unidentified subpopulation of cultured lymphocytes, were being fused. The rate of fusion was temperature dependent. Following a short lag period fusion at 37 degrees C was virtually completed in 60 min. The lymphocytes remained intact throughout the fusion process, as determined by the Trypan blue staining procedure. Fusion was almost completely inhibited by anti-M. fermentans antisera and by pretreatment of M. fermentans cells with proteolytic enzymes, suggesting that a surface-exposed proteinaceous component is involved in the fusion process.     

Biochemical diversity of Mycoplasma fermentans strains

In this study, the metabolism of a diverse range of Mycoplasma fermentans strains was investigated. It was shown that the ability to utilise glucose, fructose and N-acetylglucosamine differentiated strains, and that the patterns and kinetics of substrate utilisation were correlated with the site of isolation, i.e. joint fluid, respiratory tract, urinary tract or cell culture. Interestingly, isolates from the urogenital tract of AIDS patients used fructose in preference to glucose. There was also some correlation of fructose and N-acetylglucosamine utilisation of isolates with M. fermentans sub-groups, identified in an independent study, and based on the distribution of insertion sequence-like elements in the M. fermentans genome.     

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.     

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.     

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

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.     

Physicochemical and biological characterization of asialoerythropoietin. Suppressive effects of sialic acid in the expression of biological activity of human erythropoietin in vitro

Various partially or fully desialylated human erythropoietins were obtained by neuraminidase digestion of the hormone, without non-specific proteolysis and degradation of carbohydrates. Asialoerythropoietin showed a specific activity of 220-IU/mg protein in vivo, although that of the intact erythropoietin was 2.2 x 10(5) IU/mg. A linear relationship was found between the logarithm of the specific activity in vivo and the number of sialic acids. The asialoerythropoietin showed a four-times-higher specific activity in vitro compared with intact erythropoietin using mouse bone marrow cells. It also showed an approximately six-times-higher specific activity in a colony-forming assay for the erythroid colony-forming unit and the erythroid burst-forming unit. Partially or fully de-N-glycosylated erythropoietin derivatives also showed lower in vivo activity but higher in vitro activity than the intact erythropoietin, dependent on the number of sialic acids. To clarify the reason for the enhanced biological activity of asialoerythropoietin in vitro, the binding of intact 125I-erythropoietin or 125I-asialoerythropoietin to cells containing specific receptors for the hormone was analyzed. 125I-asialoerythropoietin bound to spleen cells from anemic mice approximately five times faster than did intact 125I-erythropoietin. The amount of 125I-asialoerythropoietin internalized by target cells, measured in the absence of NaN3, was four times higher than that of intact erythropoietin. These results demonstrate that asialoerythropoietin binds to its receptor faster than the intact form. This may be the main reason for the increased activity of asialoerythropoietin in vitro.     

An evaluation of PCR methods to detect strains of Mycoplasma fermentans.

A panel of 30 putative Mycoplasma fermentans strains, isolated from various sources including human, ovine and cell lines, were tested by a previously described polymerase chain reaction (PCR) to confirm their identity by amplification of a conserved 206 bp region of the insertion sequence IS1550. In addition, the application of another PCR based on the major part of the IS1550 element showed one or two products of different length (1144 and 1341 bp) enabling M. fermentans strains to be divided into two types designated as Type A and Type B. A PCR, which amplifies the macrophage activating lipopeptide gene (malp), supported the identification of all the strains as M. fermentans. Thirteen other species of Mycoplasma from human sources gave negative results in these tests, with the exception of Mycoplasma orale, which was detected by both IS1550-PCRs based on the major part and the conserved 206 bp region of the IS1550 element. This study suggests that all M. fermentans isolates possess both the IS1550 element and the malp gene. In contrast to the IS1550, the malp gene is shown to be species-specific and the use of a malp PCR described here could prove to be a useful adjunct to IS1550 detection as confirmation of the presence of M. fermentans in clinical material.     

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.     

Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles

Nucleic acid-based therapeutics have the potential to provide potent and highly specific treatments for a variety of human ailments. However, systemic delivery continues to be a significant hurdle to success. Multifunctional nanoparticles are being investigated as systemic, nonviral delivery systems, and here, we describe the physicochemical and biological characterization of cyclodextrin-containing polycations (CDP) and their nanoparticles formed with nucleic acids including plasmid DNA (pDNA) and small interfering RNA (siRNA). These polycation/nucleic acid complexes can be tuned by formulation conditions to yield particles with sizes ranging from 60 to 150 nm, zeta potentials from 10 to 30 mV, and molecular weights from approximately 7 x 107 to 1 x 109 g mol-1 as determined by light scattering techniques. Inclusion complexes formed between adamantane (AD)-containing molecules and the beta-cyclodextrin molecules enable the modular attachment of poly(ethylene glycol) (AD-PEG) conjugates for steric stabilization and targeting ligands (AD-PEG-transferrin) for cell-specific targeting. A 70 nm particle can contain approximately 10 000 CDP polymer chains, approximately 2000 siRNA molecules, approximately 4000 AD-PEG5000 molecules, and approximately 100 AD-PEG5000-Tf molecules; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The particles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. Uptake of the nucleic acid-containing particles by HeLa cells is measured by flow cytometry and visualized by confocal microscopy. Competitive uptake experiments show that the transferrin-targeted particles display enhanced affinity for the transferrin receptor through avidity effects (multiligand binding). Functional efficacy of the delivered pDNA and siRNA is demonstrated through luciferase reporter protein expression and knockdown, respectively. The analysis of the CDP delivery vehicle provides insights that can be applied to the design of targeted nucleic acid delivery vehicles in general.     

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 [Z?hringer 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.     

Purification and partial characterization of two extracellular endoglucanases from Cellulomonas fermentans

Avicelase assay of gel slices after non-denaturing polyacrylamide gel electrophoresis of concentrated supernatants from Cellulomonas fermentans revealed four active bands. One of them corresponded to the principal active band on CM-cellulose. Among the three others, at least one did not correspond to any active band on CM-cellulose and might reflect the presence of an exoglucanase (EC 3.2.1.91). The active band on CM-cellulose was composed of two endoglucanases (EC 3.2.1.4), called CFA and CFB, which we purified by the means of DEAE-Trisacryl chromatography and high performance liquid chromatography (anion exchange chromatography and gel chromatography). These two monomeric enzymes differ in their molecular weights (40,000 and 57,000 for CFA and CFB, respectively) and in their catalytic constants in the reaction with CM-cellulose (Km were 1.5 g/l and 59 g/l for CFA and CFB, respectively), but have similar modes of action on this substrate and similar substrate specificities.