Virulence of Trypanosoma brucei strain 427 is not affected by the absence of glycosylphosphatidylinositol phospholipase C

Abstract

The infectious stages of Trypanosoma brucei are covered by a homogenous coat consisting of 10 million molecules of a glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) (for a review, see [1]). The bloodstream forms express a phospholipase, the GPI-specific phospholipase C (GPIPLC), which has a predilection for cleaving the GPI anchor of cell-surface VSG when trypanosomes are disrupted (for a review, see [2]). During osmotic lysis, GPIPLC releases all of the cell-surface VSG within 5 min at 30°C, providing a convenient method for purifying soluble VSG (sVSG) [3]. GPIPLC also appears to be responsible for much of the degradation of the GPI precursor glycolipid A that occurs during lipid remodeling in vitro [4]. The normal cellular function of the GPIPLC is unknown, however, despite intensive study. The enzyme does not appear to be essential for normal differentiation to procyclic forms [5], when the surface VSG is released by proteolytic cleavage [6,7] and replaced by a small family of GPIPLC-resistant [8] GPIanchored surface proteins that are characterized by glutamate–proline-rich repeats [9–12]. On the other hand, it has been reported that GPIPLC is necessary for the accelerated differentiation of pleomorphic trypanosomes that is induced by mild acid stress [18]. In this brief report, we document the generation of a GPIPLC null-mutant in the virulent 427 strain of T. brucei. These cells, which retain the T7 RNA polymerase (T7RNAP) and Tet repressor (TETR) cassettes (the genotype designation is gpiplc::T7RNAP–NEO/ gpiplc::P10%T7TETR–HYG), will be useful for further biochemical and genetic studies of GPI synthesis and function, and for purification of GPI-linked proteins. The results also illustrate several issues concerning the use of genetically modified lines of T. brucei. There have been three reports of GPIPLC disruption. In the first, it was found that a GPIPLC null mutant was not impaired in its ability to complete the natural life cycle, but the moderate virulence of this T. brucei line was further reduced [5]. A GPIPLC null mutation in a more virulent monomorphic cell line of T. b. rhodesiense also reduced its virulence in mice [13]. In our previous work, for reasons that could only be speculated upon, we were unable to obtain a null mutant in the highly virulent 427 line of T. brucei, although a conditional null mutant, expressing exceedAbbre iations: BLE, phleomycin resistance gene; DAG, diacylglycerol; GPI, glycosylphosphatidylinositol; GPIPLC, GPI phospholipase C; GPIPLC, GPIPLC gene; HYG, hygromycin B phosphotransferase gene; NEO, neomycin phosphotransferase gene; T7RNAP, T7RNApolymerase gene; TETR, Tn10-encoded tetracycline-responsive repressor gene; VSG, variant surface glycoprotein. * Corresponding author. Tel.: +1-212-3277571; fax: +1-2123277845. E-mail address: george.cross@rockefeller.edu (G.A.M. Cross). 1 Present address: Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, UK. 2 Present address: Department of Biochemistry and Molecular Biology, University of Melbourne, Royal Parade, Parkville, Victoria 3052, Australia. 3 http://tryps.rockefeller.edu