The human parasite Leishmania uses adaptive gene rearrangements and amplification involving repeated sequences on a genome-wide scale as one strategy to adapt to a changing environment.

A variety of resistance mechanisms to SAG, most of them consistent with a model based on the study of resistance in vitro, were present in clinical isolates from the same geographical region.

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The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.

Transcription factor IIH (TFIIH) is a multisubunit complex required for transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNAdependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.RNA polymerase II (RNAPII) requires six auxiliary factors, called the general transcription factors (GTFs), to accurately initiate transcription from promoters of protein coding genes (1). These transcription factors (TF) include TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. Through a combination of specific protein-DNA and protein-protein interactions, RNA-PII is escorted to the promoter by the GTFs to form a transcription competent complex (2). Interestingly, the largest subunit of RNAPII contains an unusual carboxyl-terminal domain (CTD) consisting of 52 tandemly repeated copies of the heptapeptide YSPTSPS. This domain is essential for viability and is not present in any other RNAP (3,4). The CTD further distinguishes itself by the extensive phosphorylations on serine, threonine, and tyrosine residues. The presence of both hypo-and hyperphosphorylated forms of RNAPII in vivo (3) has been correlated with a function for this domain in transcription. The unphosphorylated form has been shown to form transcription preinitiation complexes, whereas the hyperphosphorylated form is present in actively elongating RNAPII complexes (5, 6). These observations suggested that transition from initiation to elongation by RNAPII is accompanied by phosphorylation of the CTD.Studies from a number of groups showed that the CTD is phosphorylated by TFIIH (for review, see ref. 7). TFIIH is a multisubunit complex consisting of approximately nine polypeptides ranging in size from 32 to 89 kDa. TFIIH also exhibits ATP-dependent helicase and DNA-dependent ATPase activities (7). Analysis of the TFIIH polypeptides revealed that many of them are proteins known to participate in DNA nucleotide excision repair (7). This intriguing connection between transcription and nucleotide excision repair proteins was extended by the demonstration that the TFIIH complex functions as an essential nucleotide excision repair factor, as well as a GTF (7,8).Recently, the CTD kinase component of TFIIH was identified as the cell cycle regulatory cdk (cyclin-dependent kinase)-activating kinase (CAK), composed of the catalytic subunit cdk7 and its regulatory subunit cyclin H (9-12). Studies in different species reveal...

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.Leishmania is a protozoan parasite affecting several million people throughout the world. The clinical manifestations of the infection depend on the species, the most life-threatening being visceral leishmaniasis caused by the Leishmania donovani complex. Treatment relies exclusively on chemotherapy, and pentavalent antimonials [Sb(V)] are still the mainstay against all forms of Leishmania infections (14, 18). While Sb(V) is used for treating patients, it is generally agreed that Sb(V) is reduced to trivalent antimony [Sb(III)], which constitutes the active form of the drug against the parasite. The exact site of drug reduction (inside the macrophages or inside the parasites) is not known, but activities were recently discovered in Leishmania that could be implicated in this reduction process (7,26,29,36). Resistance to Sb(V) is so widespread in part of India (33) that first-line treatment in this region is either based on miltefosine (31) or amphotericin B (32). Miltefosine is interesting because it can be taken orally, but single point mutations can lead to resistance (24), suggesting that resistance to this drug may occur rapidly.Leishmania has a relatively simple life cycle with two main stages, the flagellated promastigote in the insect stage and the intracellular amastigote living inside macrophages. Progress in culture techniques has allowed the growth of Leishmania amastigotes as part of axenic cultures. An increase in the temperature from 25°C to 37°C and a decrease in the pH of the culture medium to mimic the conditions encountered in the phagolysosome are the key parameters to transform promastigotes into amastigotes (reviewed in reference 37). It is nonetheless easier to grow promastigotes, and most of the work pertaining to resistance mechanisms to antimonials was performed in the insect stage of the parasit...

Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 M (0.12 g/ml), 55 g/ml, 95 g/ml, and 0.12 g/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferaseexpressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.Leishmaniasis is a significant cause of morbidity and mortality in several countries of the world (19). A vertebrate host is infected with flagellated extracellular promastigote forms via the bite of a sand fly. Promastigotes are rapidly transformed into nonflagellated amastigotes, which divide actively within the mononuclear phagocytes of the vertebrate host.The basic treatment for leishmaniasis consists of the administration of sodium stibogluconate (Pentostam), meglumine (Glucantime), or pentamidine. Treatment failure, especially in kala-azar, mucosal leishmaniasis, and diffuse cutaneous leishmaniasis, is becoming a common problem in many areas where the diseases are endemic. There are now strong indications that treatment failure may be partly due to the drug resistance of the parasite (15,18,21,25). In addition, numerous cases of relapse or unresponsiveness have been reported during the treatment of patients coinfected with human immunodeficiency virus and Leishmania spp. (1). Although rapid assays for drug screening of Leishmania promastigotes have been devised (5), promastigotes are usually less sensitive to various drugs than amastigotes (11,29). Although animal models are well established for drug testing, they are not suitable for largescale primary drug screens. The development of new drugs has been impeded by the lack of a simple, reliable, and rapid evaluation system allowing the simultaneous determination of drug activity at the mammalian stage under both axenic and intracellular conditions. The development of a system for the in vitro cultivation of amastigotes under axenic conditions enabled the development of models for in vitro drug sc...

The first line drug against leishmaniasis consists of pentavalent antimony [Sb(V)], but there is general belief that the active form of the metal is the trivalent form [Sb(III)]. In this study, we have quantified the accumulation of Sb(V) and Sb(III) in Leishmania by using inductively coupled plasma mass spectrometry. The accumulation was studied in three Leishmania species at various life stages, sensitive or resistant to antimony. Both Sb(III) and Sb(V) are accumulated in promastigote and amastigote parasites, but through competition experiments with arsenite, we found that the routes of entry of Sb(V) and Sb(III) are likely to differ in Leishmania. The level of accumulation of either Sb(III) or Sb(V), however, was not correlated with the susceptibility of wild-type Leishmania cells to antimony. This suggests that other factors may also be implicated in the mode of action of the drugs. In contrast to metal susceptibility, resistance to Sb(III) correlated well with decreased antimony accumulation. This phenotype was energy dependent and highlights the importance of transport systems in drug resistance of this protozoan parasite.