Shlomo Rottem

Cholesterol in mycoplasma membranes. Composition, ultrastructure and biological properties of membranes from Mycoplasma mycoides var. capri cells adapted to grow with low cholesterol concentrations.

Abstract 1. 1. Serial passages of the sterol-requiring Mycoplasma mycoides var. capri in a serum-free medium supplemented with decreasing concentrations of cholesterol resulted in the adaptation of the organisms to grow with no added cholesterol. The cells of the adapted strain were osmotically more fragile than cells of the native strain and were more permeable to erythritol. 2. 2. The cell membrane of the adapted strain contained low amounts of cholesterol (up to 3% of the total membrane lipid as against 22–25% in the native strain) but its polar lipids were more saturated than those of the native strain, as reflected by the preferential incorporation of [14C]palmitic acid when added to the growth medium together with [14C]oleic acid. 3. 3. The native strain was capable of growing at temperatures as low as 25°C, whereas the adapted strain could not. The lowering of the growth temperature of the native strain resulted in a decrease in the cholesterol content of the membrane (from 24–15% of the total lipid) but had no effect on the fatty acid composition of the membrane polar lipids. On the other hand, aging of the culture increased the ratio of saturated to unsaturated fatty acids in both adapted and native strains, and decreased the cholesterol content of the native strain. 4. 4. Freeze-etching of cells of the native and adapted strains, kept at 37°C prior to freezing, showed a random distribution of particles on the fracture faces of the cell membranes. Keeping the cells at 4°C prior to freezing caused the aggregation of particles on the fracture faces of the adapted strain but had no effect on the distribution of particles in the native strain. 5. 5. Our data support the thesis that cholesterol functions as a regulator of membrane fluidity and that changes in the fatty acid composition of membrane lipids may act to compensate for the lack of cholesterol.

Reassembly of Mycoplasma membranes disaggregated by detergents.

Abstract Plasma membranes of Mycoplasma laidlawii and Mycoplasma gallisepticum were solubilized by several ionic and non-ionic detergents. Solubilization of the membranes by sodium dodecyl sulfate (SDS) separated membrane lipid from protein as demonstrated by polyacrylamide gel electrophoresis of the solubilized material. The solubilized membrane material reaggregated spontaneously on removal of the detergent by dialysis or by Sephadex G-25, and formed vesicles limited by a triplelayered membrane of about the same thickness as the original Mycoplasma membrane. A divalent cation (e.g., Mg2+) was essential for membrane reassembly. The ratio of lipid to protein in membrane reaggregates varied considerably according to the Mg2+ concentration. At a low Mg2+ concentration reaggregates contained a higher percentage of lipid. The present results seem to bear out the suggestion of Engelman et al. [Biochim. Biophys. Acta135, 381 (1967)] that the SDS-solubilized membrane material does not consist of homogeneous lipoprotein subunits but of separate SDS-lipid and SDS-protein complexes. The reassembly of solubilized membrane lipid and protein on removal of the detergent indicates that these components contain sufficient structure-determining information to interact spontaneously in the presence of Mg2+ and produce membraneous structures.

Cholesterol in mycoplasm membranes. Correlation of enzymic and transport activities with physical state of lipids in membranes of Mycoplasma mycoides var. capri adapted to grow with low cholesterol concentrations

Abstract 1. 1. Membranes of a Mycoplasma mycoides var. capri strain adapted to grow with very low concentrations of cholesterol undergo a reversible phase transition detectable by differential-scanning calorimetry and fluorescence measurements. No phase transition could be detected in membranes of the cholesterol-containing native strain. 2. 2. EPR spectrometry, as well as fluorescence measurements, demonstrated that at temperatures above phase transition the membranes of the adapted strain were more fluid than membranes of the native strain, although the former contained a higher percentage of saturated fatty acids. 3. 3. Arrhenius plots of the ATPase activity of the adapted strain membranes showed breaks at temperatures corresponding to those of the phase transition of membrane lipids. The temperature of the break depended on the fatty acid composition of membrane lipids and on the age of the culture. No break could be detected in Arrhenius plots of the ATPase activity of the native strain. 4. 4. No break could be demonstrated in the Arrhenius plot of α-methylgucoside uptake by the adapted strain. Yet the activation energy of the uptake process by the adapted strain was much higher than that of the native strain. On the other hand, activation energies of α-methylglucoside phosphorylation were the same for membranes of both strains. However, Arrhenius plots of α-methylglucoside efflux from cells of the adapted strain showed breaks at temperatures corresponding to those of the lipid phase transition. 5. 5. It is concluded that cholesterol, by preventing the crystallization of membrane lipids maintains them in a state of fluidity essential for the optimal manifestation of several key activities of the membrane.

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?

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.

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.

Mycoplasmas in Cell Culture

This report will briefly review our current knowledge of: 1) the incidence, prevalence and sources of mycoplasma contamination in cell cultures; 2) the procedures for isolation, detection and identification of mycoplasmas, including procedures recommended for testing biological products produced for human use; 3) the effects of mycoplasma contamination and /or infection on the function and activities of various infected cell cultures; and 4) the recommended procedures for prevention and elimination of mycoplasma contamination. Extensive reviews on mycoplasma contamination in cell culture have been reported earlier (Barile, 1979; DelGiudice and Hopps, 1978; McGarrity and Kotani, 1985).

Mycoplasma fermentans Binds to and Invades HeLa Cells: Involvement of Plasminogen and Urokinase

ABSTRACT Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37°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 β-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.

Plasminogen Binding and Activation by Mycoplasma fermentans

ABSTRACT 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 ɛ-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 μM and the second with akd of 7.5 μM. 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 with125I-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 byM. 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.

Characterization of the mycoplasma membrane proteins IV. Disposition of proteins in the membrane

Abstract 1. 1. The disposition of proteins in membranes of Acholeplasma laidlawii and Mycoplasma hominis was studied by several techniques based on the use of proteolytic enzymes and labeling agents combined with electrophoretic analysis of membrane polypeptides in polyacrylamide gels containing sodium dodecylsulfate. 2. 2. Over 15 different polypeptide bands were detected in electrophorograms of A. laidlawii and M. hominis membranes, ranging in molecular weight from 30 000 to over 200 000. The electrophoretic pattern of M. hominis was dominated by a polypeptide band (mol. wt 110 000) estimated according to staining intensity to account for over 50% of the total membrane protein. 3. 3. The labeling intensity of isolated membranes by the lactoperoxidase-mediated iodination technique exceeded that of membranes of intact cells by a factor of 2.9–3.3 for A. laidlawii and 2.2–2.4 for M. hominis. Only 5–8% of the label was associated with cytoplasmic proteins. Electrophoretic analysis showed only a few of the membrane polypeptides to be labeled on treatment of intact cells as against the labeling of almost all the polypeptide bands on treatment of isolated membranes. 4. 4. The use of the diazonium salt of 35S]sulfanilic acid for the selective labeling of the proteins exposed on the outer membrane surface failed as the salt penetrated into the cells and labeled also the cytoplasmic proteins. 5. 5. The results of the simultaneous digestion of intact cells and isolated membranes by trypsin and papain corroborated the iodination data in that only a few polypeptide bands, mainly of high molecular weight, disappeared on treatment of intact cells as against the disappearance of most of the polypeptide bands on treatment of isolated membranes. Pronase was found to be inadequate for disposition studies since it damaged membrane permeability and digested most effectively membrane polypeptides on both membrane sides when intact cells were treated. The peptides retained in the membrane after prolonged pronase treatment were poorly labeled by the lactoperoxidase-mediated iodination, indicating their localization within the membrane lipid matrix. 6. 6. Our data point to an asymmetrical distribution of the proteins in mycoplasma membranes, with more proteins facing the cytoplasm than the exterior of the cell. The highest-molecular weight proteins in both membranes are apparently located on the exterior surface while some of the major proteins may be embedded in the membrane, projecting into cell interior, or even span the membrane, as may be the case with the major membrane protein of M. hominis.