A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi

Fil: Ferella, Marcela. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Montalvetti, Andrea. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Rohloff, Peter. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Miranda, Kildare. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Fang, Jianmin. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Reina, Silvia. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Kawamukai, Makoto. University Matsue. Faculty of Life and Environmental Science. Department of Applied Bioscience and Biotechnology; Japón.Fil: Bua, Jacqueline. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Nilsson, Daniel. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Pravia, Carlos. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Katzin, Alejandro. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Casera, María B. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Áslund, Lena. Uppsala University. Department of Genetics and Pathology; Suecia.Fil: Andersson, Björn. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Docampo, Roberto. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Bontempi, Esteban. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén"; Argentina.We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ∼ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target

Mechanism of Action of 4-Phenoxyphenoxyethyl Thiocyanate (WC-9) against Trypanosoma cruzi, the Causative Agent of Chagas’ Disease

We investigated the molecular basis of the activity of 4-phenoxyphenoxyethyl thiocyanate (WC-9) against Trypanosoma cruzi, the etiological agent of Chagas’ disease. We found that growth inhibition of T. cruzi epimastigotes induced by this compound was associated with a reduction in the content of the parasite's endogenous sterols due to a specific blockade of their de novo synthesis at the level of squalene synthase

Mitochondrial Localization of the Mevalonate Pathway Enzyme 3-Hydroxy-3-methyl-glutaryl-CoA Reductase in the Trypanosomatidae

3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms