The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose. The parasite is unable to take up galactose, suggesting that it may depend on the action of UDPglucose 4 -epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases. In this paper, we describe the cloning of T. brucei galE, encoding T. brucei UDP-Glc-4 -epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein. A tetracycline-inducible conditional galE null mutant of T. brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene. Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T. brucei proceeds via UDP-Glc-4 -epimerase and is essential for parasite growth. After several days without tetracycline, cultures spontaneously recovered. These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene. The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis.UDP-Gal ͉ galE ͉ epimerase T he tsetse fly-transmitted protozoan parasite Trypanosoma brucei causes human African sleeping sickness and the related cattle disease Nagana. There are 300,000-500,000 cases of the human disease per year in sub-Saharan Africa (1). The bloodstream form of the parasite divides by binary fission in the blood, lymph, and interstitial fluids of the mammalian host, and causes cachexia and anaemia in cattle and neurological disturbances in man. These conditions are fatal if not treated, and existing chemotherapies are toxic and difficult to administer. The need for new, less-toxic therapeutics is widely acknowledged (1).The bloodstream form of T. brucei is rich in galactosecontaining glycoproteins, most notably the variant surface glycoprotein (VSG) that, at 5 ϫ 10 6 homodimers per cell, forms a dense cell-surface coat. The VSG coat is a macromolecular diffusion barrier that protects the parasite from the innate immune system and also enables the parasite to undergo antigenic variation. Thus, although an individual parasite only expresses one VSG gene at a time, the parasite population can stay ahead of the host's specific immune response to expressed VSGs when a few parasites switch expression to one of several hundred genes encoding immunologically ...

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAc␣1-6-D-myo-inositol-1-HPO 4 -sn-1,2-diacylglycerol (GlcNAc-PI) to D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -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 brucei and Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12 and 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 TbGPI12 conditional 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 that TbGPI12 is an essential gene for the tsetsetransmitted 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-Nacetylase activity, but they retained their ability to make GlcNAc-PI and to process D-GlcN␣1-6-D-myo-inositol-1-HPO 4 -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.

African sleeping sickness is a debilitating and often fatal disease caused by tsetse fly transmitted African trypanosomes. These extracellular protozoan parasites survive in the human bloodstream by virtue of a dense cell surface coat made of variant surface glycoprotein. The parasites have a repertoire of several hundred immunologically distinct variant surface glycoproteins and they evade the host immune response by antigenic variation. All variant surface glycoproteins are anchored to the plasma membrane via glycosylphosphatidylinositol membrane anchors and compounds that inhibit the assembly or transfer of these anchors could have trypanocidal potential. This article compares glycosylphosphatidylinositol biosynthesis in African trypanosomes and mammalian cells and identifies several steps that could be targets for the development of parasite-specific therapeutic agents.