Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol (GlcNAc-PI) tod-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity are PIG-L and GPI12 in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes from Trypanosoma bruceiand Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12and LmGPI12 were subsequently cloned, sequenced, and shown to complement a PIG-L-deficient Chinese hamster ovary cell line and restore surface expression of GPI-anchored proteins. A tetracycline-inducible bloodstream form T. brucei TbGPI12conditional null mutant cell line was created and analyzed under nonpermissive conditions. TbGPI12 mRNA levels were reduced to undetectable levels within 8 h of tetracycline removal, and the cells died after 3–4 days. This demonstrates thatTbGPI12 is an essential gene for the tsetse-transmitted parasite that causes Nagana in cattle and African sleeping sickness in humans. It also validates GlcNAc-PI de-N-acetylase as a potential drug target against these diseases. Washed parasite membranes were prepared from the conditional null mutant parasites after 48 h without tetracycline. These membranes were shown to be greatly reduced in GlcNAc-PI de-N-acetylase activity, but they retained their ability to make GlcNAc-PI and to processd-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol to later glycosylphosphatidylinositol intermediates. These results suggest that the stabilities of other glycosylphosphatidylinositol pathway enzymes are not dependent on GlcNAc-PI de-N-acetylase levels.