MYCOPLASMA-FERMENTANS (INCOGNITUS STRAIN) CELLS ARE ABLE TO FUSE WITHT-LYMPHOCYTES

Characteristics of the fusion of Mycoplasma fermentans (incognitus str ain) with cultured lymphocytes were investigated. The rate and extent of fusion were monitored continuously in an assay that measured lipid mixing on the basis of dequenching of a fluorescent probe, octadecylrh odamine (R18), incorporated into mycoplasmas. Fusion of M. fermentans was detected with CD4+ (Molt-3) cells, CD4- (12E1) cells, and primary peripheral-blood lymphocytes. The level of fusion was relatively low ( 8%-12%). Detection of a similarly low level of fusion by fluorescence microscopy suggested the involvement of a specific lymphocyte subpopul ation. After a short lag period, fusion at 37-degrees-C proceeded expo nentially for approximately 30 minutes and was virtually complete at 6 0 minutes. Throughout the process, lymphocytes remained intact. Fusion was stimulated by CaCl2 but not by MgCl2; its inhibition by antisera to M. fermentans and by pretreatment of M. fermentans with proteolytic enzymes implied that the mycoplasmas possessed a proteinase-sensitive receptor involved in fusion. Mycoplasmas were rendered nonfusogenic b y treatment with the uncoupler CCCP (carbonyl cyanide m-chlorophenylhy drazone; 5 muM) but were unaffected by treatment with chlorpromazine ( 10 muM) or DCCD (dicyclohexylcarbodiimide; 50 muM); these findings sug gested that a proton gradient across the cell membrane is required for fusion.