Department of Bacteriology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan1
Department of Chemistry, Graduate School of Science, Tohoku University Aoba, Aramaki aza, Aoba-ku, Sendai 980-8578, Japan2
Author for correspondence: Sumio Arai. Tel: +81 942 31 7548. Fax: +81 942 31 0343. e-mail: sumiyuku{at}mx2.tiki.ne.jp
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ABSTRACT |
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Keywords: Acholeplasma laidlawii, adherence, glycoglycerolipids, lymphoid cells
Abbreviations: GAGDG, 3-O-[2'-O-(-D-glucopyranosyl)- 6'-O-acyl-
-D-glucopyranosyl]-1,2-di-O- acyl-sn-glycerol
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INTRODUCTION |
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The aim of the work described in this paper was to examine the capacity of glycoglycerolipids isolated from the membranes of Acholeplasma laidlawii, and of chemically synthesized glycoglycerolipids, to bind to human lymphoid cells.
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METHODS |
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Peripheral mononuclear cells from healthy donors were isolated using Lymphoprep (Nycomed Pharma AS Diagnostics) as described previously (Arai et al., 1983 ). Five million cells ml -1 were incubated in a Petri dish at 37 °C for 1 h to remove adherent cells. Nonadherent cells were collected and incubated with M-450 CD4 Dynabeads (Dynal) for 3 h at 37 °C, and CD4+ cells were then collected according to the manufacturers instructions. The purified CD4+ cells were cultured in RPMI1640 with 12% FCS, 100 units human recombinant interleukin-2 (Becton Dickinson Labware) ml-1 and 100 units of human recombinant interleukin-4 (Genzyme Diagnostics) ml-1 in a 24-well cell culture plate (Costar) coated with anti-human CD3 antibody (Serotec).
Cell binding assay.
Extracts from the membranes of A. laidlawii and synthetic 3-O-[2'-O-(-D-glucopyranosyl)- 6'-O-acyl-
-D-glucopyranosyl]-1,2-di-O-acyl-sn-glycerols (GAGDGs) were dissolved in chloroform and 50 µl samples were spread on individual 10 mm diameter coverglasses (Matsunami) to make thin films of GAGDGs. To avoid nonspecific adherence of cells, each coverglass was immersed in RPMI/FCS medium. After 1 h, the medium was removed by suction and replaced with serum-free tissue culture medium (Cosmo medium; Cosmo Bio). Cells were suspended in Cosmo medium at a concentration of 4x105 cells ml -1 and 0·5 ml of the suspension was added to each well of a 24-well cell culture plate with a GAGDG-coated coverglass. The plate was then incubated at 37 °C in a CO2 incubator for 4 h. The coverglass was then removed with forceps and gently dipped twice in warmed PBS (137 mM NaCl, 2·68 mM KCl, 1·47 mM KH2PO4 , 8·1 mM Na2HPO4). The adherent cells on the coverglass were scraped off with a rubber policeman. The cell suspensions from five coverglasses were centrifuged at 170 g for 5 min and the cells were resuspended in 0·2 ml RPMI/FCS. The cells were counted with a haemocytometer. For detection of adherence to A. laidlawii, 0·1 ml of a 1x107 c.f.u. ml -1 suspension of A. laidlawii was inoculated into each well of a cell culture plate and incubated in 1 ml RPMI/FCS medium for 3 d at 37 °C in a CO2 incubator. The medium was then removed and suspensions of eukaryotic cells in Cosmo medium were added to different wells. The adherent cells were counted as described above. An alternative method for estimation of cell numbers was also employed. Briefly, after removing nonadherent cells, residual cells on the coverglasses were incubated for 4 h at 37 °C in a CO2 incubator in the presence of 1 µCi (37 kBq) [6-3H]thymidine [specific activity 16·3 Ci (603 GBq) mmol-1 ; Dupont NEN]. The cells were lysed, DNA was precipitated onto glass filters and the amount of [6-3H]thymidine incorporation was determined. Both methods for estimation of numbers of binding cells were used in five independent experiments.
Fractionation of A. laidlawii membranes.
Membranes of A. laidlawii PG8 ATCC 23206 were isolated by osmotic lysis as described previously (Iyama et al., 1996 ). The membranes of A. laidlawii were extracted twice using the BlighDyer method with 40 vols water/chloroform/methanol (0·8:2:1, by vol.). Extracted lipids were dried on a rotary evaporator. Fifty milligrams of the lipid was resuspended in chloroform for application to a column of 10 g activated silica gel (Silica gel 60; Merck) prepared in chloroform. Sequential elution of the lipids from the column was performed with a discontinuous gradient starting with 400 ml of chloroform and followed by 400 ml each of the following mixtures of chloroform/methanol (v/v): 95:5, 90:10, 80:20, 70:30, 60:40, and finally 400 ml methanol. The fraction showing binding activity was further purified by TLC (Silica gel 60, 1·2 mm in thickness) using chloroform/methanol/acetic acid (85:15:0·5, by vol.).
Structure determination by NMR and GC-MS.
The structure of a component of the membranes of A. laidlawii with binding activity was determined by 13C-NMR, 1 H-NMR and GC-MS analysis. Detailed one- and two-dimensional NMR studies of +39 °C (c 0·1, CHCl3)} by 1H-1H DQFCOSY, HOHAHA (
m=60 ms) and HMQC experiments with a JEOL
-500 spectrometer in CHCl3-CH3OD (1:1, v/v) allowed sequential assignments of proton and carbon resonances. Molecular ion peaks of the fatty acid methyl esters from lipid X by GC- MS indicated that the fatty acyl groups of the lipid were a mixture of straight- and branched-chain fatty acids. The lipids (0·7 mg in 0·5 ml methanol) were treated with 15 µl 140 mM sodium methoxide in methanol and the resulting fatty acid methyl esters were analysed by GC-MS equipped with a 25 m PEGM capillary column and electron ionization-MS spectrometry (Huang & Anderson, 1995
).
Synthesis of GAGDGs.
Synthesis of glycoglycerolipids essentially followed a method established by van Boeckel & van Boom (1985a , b
, c
).
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RESULTS |
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DISCUSSION |
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Glycoglycerolipids are not only found in the cell membranes of mycoplasmas, but also in those of Gram-negative and Gram-positive bacteria, and their L-forms (Kates, 1990 ). However, their function in the membranes of prokaryotic cells is not clear. Several investigators have reported that glycolipids in the membranes of A. laidlawii are mono- and di-glycosyldiacylglycerol (Bhakoo et al., 1987
; Dahlqvist et al., 1995
; Gross & Rottem, 1979
; Shaw 1968
) and that they might play an important role in physiological functions such as membrane viscosity (De Kruyft et al., 1973
; McElhaney et al., 1970
; McElhaney, 1975
; Rottem et al., 1973
). Several reports suggest that the fusion of mycoplasmas with eukaryotic cells may result in the delivery of mycoplasma components into the host cells (Toole & Lowdell, 1990
; Franzoso et al., 1992
). Furthermore, Rottem (1980)
reported that the presence of cholesterol in small unilamellar vesicles is required to allow their fusion with mycoplasmas. These findings suggest that GAGDGs, a major component of A. laidlawii membranes (Shaw, 1970
; Silvius et al., 1980
), might play an important role not only in the membrane fluidity of prokaryotic cells, but also in interactions between micro-organisms lacking cell walls and eukaryotic cells.
Our results strongly suggest that glycoglycerolipids in A. laidlawii membranes might participate in the adhesion of bacterial cells to eukaryotic cells. The role of glycoglycerolipids in the pathogenesis of A. laidlawii infections is unknown. The induction of conformational changes in the membranes of eukaryotic cells as well as in those of prokaryotic cells by glycoglycerolipids may be responsible for these biological activities. In this study, we have clearly demonstrated that glycoglycerolipids possess the ability to bind to certain kinds of eukaryotic cells. However, we need to clarify the initial binding sites and the significance of this phenomenon in infections.
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REFERENCES |
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Received 7 January 2000;
revised 10 May 2000;
accepted 23 May 2000.
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