Small unilamellar vesicles are able to fuse with Mycoplasma capricolum cells

Abstract

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 (> 0.01%) or chlorpromazine (> 10 μM). Fusion was partially inhibited by treating the cells with the uncoupler CCCP (5 μM) 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.