We report the results of an in vitro screening assay targeting the intraerythrocytic form of the malaria parasite Plasmodium falciparum using a library of 560 prenyl-synthase inhibitors. Based on "growth-rescue" and enzyme-inhibition experiments, geranylgeranyl diphosphate synthase (GGPPS) is shown to be a major target for the most potent leads, BPH-703 and BPH-811, lipophilic analogs of the bone-resorption drugs zoledronate and risedronate. We determined the crystal structures of these inhibitors bound to a Plasmodium GGPPS finding that their head groups bind to the ½Mg 2þ 3 cluster in the active site in a similar manner to that found with their more hydrophilic parents, whereas their hydrophobic tails occupy a long-hydrophobic tunnel spanning both molecules in the dimer. The results of isothermal-titration-calorimetric experiments show that both lipophilic bisphosphonates bind to GGPPS with, on average, a ΔG of −9 kcal mol −1 , only 0.5 kcal mol −1 worse than the parent bisphosphonates, consistent with the observation that conversion to the lipophilic species has only a minor effect on enzyme activity. However, only the lipophilic species are active in cells. We also tested both compounds in mice, finding major decreases in parasitemia and 100% survival. These results are of broad general interest because they indicate that it may be possible to overcome barriers to cell penetration of existing bisphosphonate drugs in this and other systems by simple covalent modification to form lipophilic analogs that retain their enzyme-inhibition activity and are also effective in vitro and in vivo.M alaria, caused by Plasmodium spp., causes approximately 1 million deaths each year (1), and there are ever-present problems due to drug resistance (2). There is, therefore, a need for new drugs and drug leads. In earlier work, we and others found that the bisphosphonate class of drugs (3) used to treat bonerelated diseases-osteoporosis, Paget disease, and hypercalcemia due to malignancy-also inhibited the growth of a range of parasitic protozoa, including Trypanosoma cruzi (4, 5), Trypanosoma brucei (4, 6), Leishmania spp. (4,7,8), Toxoplasma gondii (4, 9), Cryptosporidium parvum (10, 11), Entamoeba histolytica (4, 12, 13), and Plasmodium spp. (4, 13-15). In the case of Plasmodium spp., the most potent inhibitors were not, however, the nitrogencontaining bisphosphonates such as zoledronate or risedronate (Scheme 1) used to treat bone diseases, but more lipophilic n-alkyl bisphosphonates (13). Their target in Plasmodium falciparum was not determined. However, more recently, a Plasmodium vivax geranylgeranyl diphosphate synthase (PvGGPPS) has been cloned, expressed, purified, and crystallized, and its three-dimensional structure determined (16). The enzyme is inhibited by bisphosphonates (16), so it seemed possible that it might be a target for the inhibitors discovered earlier. To investigate this possibility, we recently determined the IC 50 values for 25 bisphosphonates against PvGGPPS and compared the results for enzyme inhi...

RNA polymerase II of trypanosomes, early diverging eukaryotes, transcribes long polycistronic messages, which are not capped but are processed by trans-splicing and polyadenylation to form mature mRNAs. The same RNA polymerase II also transcribes the genes coding for the spliced leader RNA, which are capped, exported to the cytoplasm, processed, and reimported into the nucleus before they are used as splicing donors to form mRNAs from pre-mRNA polycistronic transcripts. As pre-mRNA and spliced leader transcription events appear to be uncoupled, we studied how the RNA polymerase II is distributed in the nucleus of Trypanosoma cruzi. Using specific antibodies to the T. cruzi RNA polymerase II unique carboxy-terminal domain, we demonstrated that large amounts of the enzyme are found concentrated in a domain close to the parasite nucleolus and containing the spliced leader genes. The remaining RNA polymerase II is diffusely distributed in the nucleoplasm. The spliced leader-associated RNA polymerase II localization is dependent on the cell transcriptional state. It disperses when transcription is blocked by ␣-amanitin and actinomycin D. Tubulin genes are excluded from this domain, suggesting that it may exclusively be the transcriptional site of spliced leader genes. Trypomastigote forms of the parasite, which have reduced spliced leader transcription, show less RNA polymerase II labeling, and the spliced leader genes are more dispersed in the nucleoplasm. These results provide strong evidences that transcription of spliced leader RNAs occurs in a particular domain in the T. cruzi nucleus.Trypanosomes belong to a group of early diverging flagellated protozoa that causes several parasitic diseases, including Chagas' disease, leishmaniosis, and African trypanosomiasis. As with most eukaryotes, they also have three RNA polymerases characterized by a variable sensitivity to ␣-amanitin (15, 48). In trypanosomatids, the ␣-amanitin-sensitive RNA polymerase II (RNA Pol II), which transcribes most of the protein coding genes, catalyzes the transcription of long polycistronic messages (25, 47). These long polycistronic messages are then processed in a trans-splicing reaction by addition of a 30-to 40-nucleotide sequence derived from the spliced leader (SL) RNA at the 5Ј end of each cistron, followed by addition of a poly(A) tail at the 3Ј end (3, 42). The cap is added at the 5Ј end of the SL RNA during its transcription, and then it is transferred with the 30 to 40 nucleotides to the mature mRNA in the trans-splicing reaction (21, 29, 37). Transcription is constitutive for almost all genes characterized to date and varies in overall rates according to the parasite developmental stages (18). Thus, most regulation of gene expression in these organisms seems to occur posttranscriptionally either by the stability of the processed mRNAs or by translational controls as discussed in several reviews (8,23,44,49). Recently, a bidirectional promoter was identified in strand-switch regions of chromosomes 1 (31) and 3 (30) of Leishmania ...

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast -an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.

Parasite virulence genes are usually associated with telomeres. The clustering of the telomeres, together with their particular spatial distribution in the nucleus of human parasites such as Plasmodium falciparum and Trypanosoma brucei, has been suggested to play a role in facilitating ectopic recombination and in the emergence of new antigenic variants. Leishmania parasites, as well as other trypanosomes, have unusual gene expression characteristics, such as polycistronic and constitutive transcription of protein-coding genes. Leishmania subtelomeric regions are even more unique because unlike these regions in other trypanosomes they are devoid of virulence genes. Given these peculiarities of Leishmania, we sought to investigate how telomeres are organized in the nucleus of Leishmania major parasites at both the human and insect stages of their life cycle. We developed a new automated and precise method for identifying telomere position in the three-dimensional space of the nucleus, and we found that the telomeres are organized in clusters present in similar numbers in both the human and insect stages. While the number of clusters remained the same, their distribution differed between the two stages. The telomeric clusters were found more concentrated near the center of the nucleus in the human stage than in the insect stage suggesting reorganization during the parasite's differentiation process between the two hosts. These data provide the first 3D analysis of Leishmania telomere organization. The possible biological implications of these findings are discussed.