Michael Salman

Phosphatidylinositol synthesis in mycobacteria

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy.

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.

Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum

Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer. It is suggested that the role of cholesterol in mycoplasma-SUV fusion is not at the level of bulk bilayer viscosity but rather, affecting local lipid-lipid or lipid-protein interactions that are relevant to the fusion event.

Mycoplasma fermentans (incognitus strain) induces TNFα and IL-1 production by human monocytes and murine macrophages

We have demonstrated that Mycoplasma fermentans (incognitus strain), as well as M. fermentans KL4, PG 18 and IM 1 strains have the ability to activate human peripheral blood monocytes and murine macrophages of two inbred strains to secrete a high level of tumor necrosis factor alpha (TNF alpha) in a dose-dependent manner. Secretion of interleukin-1 (IL-1) was also stimulated following the incubation of human monocytes with the organism. We suggest that cytokine secretion following infection with M. fermentans (incognitus strain) that was detected in AIDS patients may contribute to the pathological manifestations, including cachexia, in this disease.

Mycoplasma penetrans infection of Molt-3 lymphocytes induces changes in the lipid composition of host cells

The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. The ability of M. penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of polyethylene glycol 8000 (PEG). The effect of PEG was more pronounced in the more alkaline pH range, where the binding kinetics were much faster and almost unaffected by temperature (4-37 degrees C). Incubation of [14C]oleic-acid-labelled Molt-3 cells with viable M. penetrans resulted in a substantial release of radioactive fatty acids, whereas treating the host cells with heat-inactivated mycoplasmas, isolated M. penetrans membrane preparations, or M. penetrans growth medium, had no effect. Total lipid analysis of Molt-3 lymphocytes infected by M. penetrans revealed an augmented level of the neutral lipid fraction that was associated with a decrease in the relative amounts of polar lipids, mainly a decrease in the amount of phosphatidylserine and diphosphatidylglycerol. Analysis of the neutral lipid fraction in the infected Molt-3 cells revealed a fivefold increase in the relative amount of diacylglycerol and a marked increase in the free fatty acid (FFA) fraction. The profile of the FFAs released was dominated by a relatively high concentration of the polyunsaturated fatty acid docosahexaenoic acid. The release of lipid intermediates suggests that the degradation of Molt-3 cell phospholipids induced by M. penetrans may initiate a signal transmission cascade in the host cell.

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.

D7 – PHOSPHOLIPASE ACTIVITY IN MYCOPLASMAS

This chapter discusses phospholipase activity in mycoplasmas. A membrane-bound lysophospholipase was described in Acholeplasma laidlawii and Mycoplasma gallisepticum. This enzyme hydrolyzes micellar dispersions of lysophosphatidylcholine and lysophosphatidylglycerol, as well as endogenous lysophospholipids generated by treating membranes with purified preparations of phospholipase A 2 . The high levels of free fatty acids in the cell membrane of Spiroplasma species were taken to suggest an endogenous phospholipase activity in spiroplasmas. A phospholipase activity, markedly stimulated by low concentrations of non-ionic detergents, was detected in M. gallisepticum membranes. Phospholipase A and C activities were detected in Ureaplasma urealyticum and a phospholipase A 1 activity was demonstrated in the cell membrane of M. penetrans. Indication for an endogenous phospholipase activity can be obtained by following the intramembrane hydrolysis of lipids. The mycoplasmal activity has a broad pH spectrum and is not affected by cyclic adenosine monophosphate (cAMP) but is inhibited by lyso components, suggesting that it may be regulated by intracellular substrate and/or product levels.

Uptake of a fluorescent-labeled fatty acid by spiroplasma floricola cells

Abstract12-(1-pyrene)dodecanoic fatty acid (P12) uptake by Spiroplasma floricola BNR-1 cells was characterized with regard to its kinetics, specificity, metabolism and susceptibility to protein and lipid inhibitors. The uptake process depended on temperature and pH, and exhibited biphasic saturation kinetics with a very low (2.7 μM) and a high (37 μM) apparent Km value. Lauric, myristic, palmitic, stearic and oleic fatty acids did not compete with P12 for transport. The fluorescence of P12 was exclusively recovered in the neutral lipid fraction, suggesting that this fatty acid is not further utilized for phospholipid biosynthesis. Valinomycin, carbonylcyanide m-chlorophenyldrazone (CCCP), dicyclohexylcarbodiimide (DCCD), and pronase strongly reduced P12 uptake by cells, but not by membrane vesicles, affecting the high affinity (low Km) component of the uptake system. Uptake of P12 by cells, as well as by membrane vesicles, was very sensitive to glutaraldehyde, chlorpromazine, phospholipase A21 and ascorbate with FeCl3, which affected the low affinity (high Km) component of a transport system. Digitonin stimulated P12 uptake. We suggest that the incorporation of P12 into spiroplasma cell membrane is a two-step process: a high specificity energy-dependent and protease-sensitive binding to the outer surface of membrane, and a low specificity and energy-independent diffusion and partition into the membrane lipid environment.