Transbilayer translocation of membrane phosphatidy lserine and its role in macrophage invasion in Leishmania promastigotes * Amit Tripathi, C.M. Gupta* Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India Received 5 November 2002; received in revised form 22 January 2003; accepted 30 January 2003 Abstract Infectivity of Leishmania promastigotes has been shown to be growth cycle-dependent and restricted to the stationary phase. By using annexin V-FITC binding and procoagulant activity measurement assays, we show here that the promastigotes in the stationary phase contain significantly higher amounts of phosphatidylserine (PS) on their surface as compared to the log phase promastigotes. We also demonstrate that the infectivity of the promastigotes is determined by the presence of PS on their surface. In addition, by using NBD-labelled phospholipids, we show that the promastigote plasma membrane contains ATP-dependent out-to-in and ATP-independent in-to-out PS translocases which regulate the PS localisation in two-halves of the membrane bilayer, and that the greater amounts of external PS observed in the stationary phase promastigotes is perhaps due to the slower ATP-dependent out-to-in PS movements in these cells, as compared to the log phase promastigotes. 1. !.,;U:: Keywords: Leishmania promastigotes; Infectivity; Phosphatidylserine; Plasma membrane; Transbilayer movements 1. Introduction Leishmania parasite is a protozoan organism, which causes the dreaded disease 'kala-azar'. The promastigote form of this parasite upon transfer by the infected vector to the vertebrate host invades its mononuclear macrophages wherein its flagellated, motile and spindle-shape body is transformed into a nonmotile and spherically-shaped form, called amastigote [1]. The invasion is a receptor-mediated process and may involve several macrophage receptors, namely, the mannosyl fucosyl receptor, CR3 receptor, re- Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanol- amine; PS, phosphatidylserine; NBD, N-(7-nitro-2,1,3-benzoxadiazol- 4-yl)amino; BSA, bovine serum albumin; C6-NBD-, l-palmitoyl-2-[6- N-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminol caproyl; Clz-NBD-, I-palmi- toyl-2-[ 12-N-(7 -nitro-2, I ,3-benzoxadiazol-4-yl)amino I dodecanoyl; RVV, Russel's viper venom; DMEM, Dulbecco's Modified Eagle Medium -It This is a communication no. 6085 from the Central Drug Research Institute, Lucknow (India). . Corresponding author. Tel.: +91-522-2223286/2210932; fax: +91-522-2223405/2223938. E-mail addresses:drcmg@rediffmail.com.drcmg@satyam.net.in (C.M. Gupta). ceptors for glycosylated molecules, and fibronectin receptor [2]. Two surface molecules of promastigotes, namely gp63, a glycoprotein, and LPG, a lipophosphoglycan, have been suggested as the ligands for attachment of these macrophage receptors [3]. However, recent studies [4-6] have revealed that phosphatidylinositol-anchored surface proteins, like gp63 and LPG, may not be essential for entry and survival of Leishmania promastigotes in the host macrophages. Phosphatidylserine (PS) is one of the phospholipid con- stituents of the promastigote plasma membrane [I]. Specific receptors for this phospholipid exist on the mononuclear macrophage surface [7]. Earlier studies have shown that the macrophages scavenge the aged cells from the circulation through these receptors [7]. Furthermore, several investiga- tors have reported that exposure of PS on the surface of the aged or apoptotic cells is the prerequisite for their uptake by the macrophages [8-15]. Since Leishmania amastigotes have recently been shown to invade the fresh macrophages by exposing their PS on the cell surface [16], we consid- ered it of interest to study the mechanisms that regulate the transbilayer movements of PS in the Leishmania promastig- otes and also the effect of PS exposure on the promastigote infectivity. Molecular and Biochemical Parasitology (2003),128.1.1-9