A defined medium for a fastidious Spiroplasma.

A defined medium (H-1) was developed for cultivation of the suckling mouse cataract agent, Spiroplasma mirum, a fastidious member of the class Mollicutes that causes cataracts and chronic brain infection in inoculated neonate mice. The H-1 medium was used to show the importance of sphingomyelin as a growth factor for the culture of the spiroplasma in vitro. The growth of Spiroplasma mirum and the pathology it induces in sphingomyelin-rich tissues in vivo may be related to this dependency.

In vitro induction of tumor necrosis factor alpha, tumor cytolysis, and blast transformation by Spiroplasma membranes.

Membranes of Spiroplasma sp. strain MQ-1 (hereafter referred to as MQ-1) were potent inducers of tumor necrosis factor alpha (TNF alpha) secretion and of blast transformation. Specific anti-recombinant murine TNF alpha antibodies markedly inhibited macrophage-mediated tumor cytolysis of A9 fibrosarcoma target cells following activation by MQ-1 membranes. Thus, TNF alpha plays a major role in mediation of tumor cytolysis induced by MQ-1 membranes, which is similar to its role in lipopolysaccharide (LPS)-induced tumor cytolysis. Two findings, however, suggested that the mechanism of macrophage activation by MQ-1 membranes differs from that by LPS: (a) macrophages, taken from C3H/HeJ mice showing a low responsiveness to LPS, were activated by MQ-1 membranes to enhanced TNF alpha secretion, resulting in a high-level tumor cytolysis compared with the negligible tumor cytolysis induced by LPS; and (b) MQ-1 membranes and LPS synergized to highly augment TNF alpha secretion by macrophages of C57BL/6 mice. MQ-1 membranes were capable of inducing blast transformation of murine lymphocytes as well. In addition, they activated human monocytes to secrete high levels of TNF alpha. Further studies need to be carried out using in vivo models to evaluate the therapeutic potential of MQ-1 membranes in the treatment of malignant diseases.

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.

The cell membrane of Mycoplasma penetrans: lipid composition and phospholipase A1 activity.

Analysis of Mycoplasma penetrans membrane lipids revealed that, in addition to large amounts of unesterified cholesterol, M. penetrans incorporated exogenous phospholipids, preferentially sphingomyelin, from the growth medium. The major phospholipids synthesized de novo by M. penetrans were phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG). In vivo labeling of PG and DPG by growing the cells with radioactive palmitate or oleate, followed by snake venom phospholipase A2 treatment, enabled us to assess the positional distribution of fatty acids in these lipids. Saturated fatty acids were found preferentially in position 2 of the glycerol backbone, and not in position 1 as found elsewhere in nature, while unsaturated fatty acids prefer position 1. M. penetrans membranes contain phospholipase activity of the A1 type, removing a fatty acid from the sn-1 ester bond of phospholipids. The activity was neither stimulated by Ca2+ nor inhibited by EGTA and had a broad pH spectrum. The substrate specificity of the enzyme was investigated with various natural lipids and with a fluorescent analog of the phosphatidylcholine. The enzyme was equally active toward phosphatidylcholine and phosphatidylglycerol, but did not hydrolyze diphosphatidylglycerol. The enzyme did not act on triacylglycerol, diacylglycerol or cholesteryl ester, but low activity was detected toward monoacylglycerol. The enzyme was heat-sensitive and detergent-sensitive, and was almost completely inhibited by p-bromophenacylbromide (50 microM), but was not affected by SH reagents. This study is the first one reporting phospholipase A1 activity in Mollicutes. A possible role of this enzyme in forming lipid mediators upon the interaction of M. penetrans cells with eukaryotic cells is suggested.

Lipid distribution in Acholeplasma laidlawii membrane. A study using the lactoperoxidase-mediated iodination.

The lactoperoxidase-mediated radioiodination has been applied to study the transbilayer distribution of phospho- and glycolipids in Acholeplasma laidlawii membranes. After radioiodination, about 5% of the 125I-iodine was found in membrane lipids. A comparison of the labeling intensities of the various lipid species between iodinated intact cells and isolated membranes revealed that the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride are located almost exclusively in the outer half of the bilayer, whereas the phospholipids phosphatidylglycerol and diphosphatidylglycerol as well as the phosphoglycolipids glycerophosphoryl-diglucosyldiglyceride and glycerophosphoryl-monoglucosyldiglyceride are almost equally distributed in the outer and inner halves of A. laidlawii membranes.

Small unilamellar vesicles are able to fuse with Mycoplasma capricolum cells.

We have investigated the fusion characteristics of intact Mycoplasma capricolum cells and small unilamellar vesicles (SUV). The rate and extent of fusion was monitored continuously by octadecylrhodamine B (R18) fluorescence dequenching assay, as well as by intracellular contents mixing, and by sucrose density gradient analysis. The fusion of SUV with M. capricolum cells was found to be dependent on poly(ethylene glycol) (PEG 8000), divalent cations in the medium, and on the cholesterol content of the lipid vesicles. Maximal levels of fusion were obtained with SUV containing 40 mol% cholesterol in the presence of 5% PEG. The rate and extent of fusion were affected by temperature, pH, osmotic pressure, and SUV/mycoplasma ratio. Under optimal fusion conditions, PEG did not increase the rate of exchange of either cholesterol or phospholipids between M. capricolum cells and SUV. Throughout the fusion process, M. capricolum cells remained intact as measured by the retention of [3H]thymidine-labeled components, and viable. M. capricolum cells were rendered nonfusogenic by treatment with glutaraldehyde (greater than 0.01%) or chlorpromazine (greater than 10 microM). Fusion was partially inhibited by treating the cells with the uncoupler CCCP (5 microM) or proteolytic enzymes, suggesting that a proton gradient across the cell membrane is required for the fusion, and that the cells possess proteinase-sensitive receptors that are responsible for a tighter contact with the lipid vesicles.

Symmetrical distribution and rapid transbilayer movement of cholesterol in Mycoplasma gallisepticum membranes.

The exchange of cholesterol between [14C]cholesterol-labeled Mycoplasma gallisepticum cells and an excess of sonicated egg phosphatidylcholine/cholesterol vesicles (molar ratio of 0.9) was measured. More than 90% of the radioactive cholesterol underwent transfer from intact cells to the vesicles. The kinetics of the transfer was biphasic. About 50% of the radioactive cholesterol was exchanged with a half-time of about 4 h. The residual was exchanged at a slower rate with a half-time of about 9 h at 37 degrees C. Bovine serum albumin had a pronounced effect in enhancing both the fast and slow rates of cholesterol exchange, but did not affect the pool sizes significantly. The half-time for equilibration of the two pools in the presence of 2% albumin, calculated using a reversible two-pool method of analysis, was 6.2 h. The effect of albumin was also obtained with isolated membrane preparations and with cells treated with growth inhibitors, suggesting that this effect is independent of albumin preservation of cell viability. The rate enhancement of albumin was concentration dependent with maximal effects observed with greater than or equal to 2%, where the rates of exchange of both the rapidly and slowly exchanging pools were twice as fast. The mechanism by which albumin may affect the exchange rates is discussed.

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.

The outer membrane of Proteus mirabilis. I. Isolation and characterization of the outer and cytoplasmic membrane fractions.

1. The crude envelope preparation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was separated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a single trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped structures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. The outer membrane fraction consisted of protein (similar to 40%), lipopolysaccharide (similar to 36%) and lipid (similar to 18%) and had a density of about 1.22 g/cm3. The cytoplasmic membrane fraction consisted mostly of protein (similar to 56%) and lipid (similar to 38%), had a density of about 1.16 g/cm3, and contained almost all the NADH oxidase, succinate and D-lactate dehydrogenase activities of the crude envelope preparation. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6-7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.

Is the vertical disposition of Mycoplasma membrane proteins affected by membrane fluidity?

The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-to-protein ratio through treatment with chloramphenicol. The lactoperoxidase-mediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.

Lipid and protein membrane components associated with cholesterol uptake by Mycoplasmas.

Membranes of Mycoplasma species take up 2--4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A2. Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake. Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.

Phospholipid and cholesterol uptake by Mycoplasma cells and membranes.

The ability of growing mycoplasma cells and their isolated membranes to take up exogenous phospholipids was correlated with their ability to take up cholesterol. Horse serum or vesicles made of phosphatidylcholine and cholesterol served as lipid donors. Growing cells of five Mycoplasma species took up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three Acholeplasma species failed to take up any of the exogenous phospholipids, and only incorporated low amounts of free cholesterol and no esterified cholesterol. Hence, the ability of mycoplasmas to take up large quantities of cholesterol appears to be correlated with an ability to take up exogenous phospholipids. Isolated membranes of Mycoplasma capricolum and Acholeplasma laidlawii took up lower amounts of cholesterol than did membranes of growing cells and did not take up phospholipids. Inhibition of M. capricolum growth decreased the ability of the cells to take up exogenous phospholipids and cholesterol. The possibility that the contact between the lipid donors and the membrane involves specific receptors best exposed in actively growing cells is discussed.

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures.

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.

Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity.

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.

Interaction between tetanolysin and Mycoplasma cell membrane.

1. A partially purified tetanolysin preparation lysed the sterol-requiring Mycoplasma capricolum cells but had no effect on M. capricolum cells adapted to grow with no or very little cholesterol. The sterol-non-requiring Acholeplasma laidlawii cells grown either in a cholesterol-rich or a cholesterol-poor medium were unaffected by the tetanolysin preparation. 2. The lysis of M. capricolum cells by the tetanolysin preparation was temperature dependent, inhibited by cholesterol, sublytic concentrations of lucensomycin, and Mg2+. The sensitivity to lysis was greatly affected by the age of the culture, being highest in cells from the early logarithmic phase of growth and declining sharply thereafter. 3. Isolated M. capricolum membranes were capable of binding large amounts of the tetanolysin activity (up to 30 hemolytic units per mug membrane protein), 20 times as much as membranes of the adapted strain. The binding of tetanolysin activity to membranes was almost the same at 4,22, or 37 degrees C, and was very little affected by the age of the culture. The binding capacity of the membranes was not affected by the removal of 60-70% of membrane proteins by pronase digestion but markedly decreased with the removal of membrane lipids. 4. Of the five polypeptide bands detected in electrophorograms of the partially purified tetanolysin preparation, two bands (mol. wt. 44 000 and 42 000) were found to bind to the cholesterol-containing mycoplasma membrane preparation. EPR spectrometry revealed that the freedom of motion of fatty acid spin labels in the tetanolysin-treated membranes was markedly higher than that in untreated membranes. 5. The concept that tetanolysin interacts specifically with membrane cholesterol resulting in the shielding of cholesterol from its interaction with membrane phospholipids is discussed.

Beware of mycoplasmas.

Mycoplasma infection of cell cultures is widespread and has major detrimental effects on cellular physiology and metabolism. Since cell culture is used extensively, both in research and in industrial production processes, questions of primary concern arise, such as: how can mycoplasma contamination be detected; what are the effects of such contamination on cellular functions; what methods are available for eliminating contamination?

31P-NMR studies of Mycoplasma gallisepticum cells using a continuous perfusion technique.

31P-NMR studies of Mycoplasma gallisepticum cells have been carried out using a continuous perfusion technique; these are the first such studies with this organism. Using this technique, glucose metabolism was monitored in the intact organisms, and cell extracts were prepared to identify the intermediates. Under glycolytic conditions, high levels of fructose-1,6-diphosphate were observed, indicating that this sugar may play a key role in the regulation of metabolism. The level of phosphoenolpyruvate was low under normal glycolytic conditions, and did not increase during starvation. From the position of the internal inorganic phosphate peak, the intracellular pH was estimated. The cells were found to maintain an intracellular pH of approximately 7.1 over an investigated external pH range of 6.6-8.6.

Phosvitin kinase activity in Acholeplasma axanthum.

Incubating the soluble fraction derived from A. axanthum with phosvitin and [gamma-32P]ATP results in the phosphorylation of phosvitin. Casein, histone and kemptide were practically ineffective substrates, whereas a 55 kDa protein of M. gallisepticum was efficiently phosphorylated. The enzymatic activity has an optimal pH in the pH range 6.0-6.2 and requires divalent cations. The activity was inhibited by ammonium sulfate, heparin and sulphydryl blocking reagents, but was not affected by calmodulin with or without Ca2+ or by cyclic AMP.

Effect of trichothecenes on growth and intracellular pool size of Mycoplasma gallisepticum.

The mycotoxin T-2 inhibited the growth of Mycoplasma gallisepticum. The growth inhibition was most pronounced with the hydrophobic derivatives T-2 acetate and very little with the hydrophilic T-2 tetraol. The toxin had no effect on the biosynthesis of either protein, DNA, RNA or complex lipids but markedly reduced the intracellular pool size of soluble low molecular mass precursors. It seems that T-2 acetate, by virtue of its hydrophobic nature, may accumulate within the lipid backbone affecting the permeability properties of the cell membrane.

Effect of 7 beta-hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum.

7 beta-OH cholesterol in a cholesterol rich growth medium (5-10 micrograms/ml) extended the lag period and slowed down the growth rate of Mycoplasma capricolum cells. In a cholesterol poor medium (0.5 micrograms/ml) inadequate to support growth, 7 beta-OH cholesterol exerts a synergistic effect on growth. The 7 beta-OH cholesterol was incorporated unchanged from the growth medium and could be recovered exclusively in the membrane fraction. The incorporation of the 7 beta-OH cholesterol has no effect on the total phospholipid content but the DPG to PG ratio was markedly decreased. Exchange studies with lipid vesicles revealed that whereas most of the cholesterol underwent exchange, only about 20% of the 7 beta-OH cholesterol was exchanged.