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A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.DNA topoisomerases are ubiquitous enzymes that catalyze changes in DNA topology by altering the linkage of DNA strands, solving topological problems caused by cellular processes such as DNA replication, transcription, or recombination (1, 2). These enzymes are classified on the basis of the number of DNA strands that they cleave and the covalent bond formed in the enzyme-DNA intermediate. Unlike type II DNA topoisomerases, type I enzymes are ATP-independent, which transiently break a single strand of DNA. Type I DNA topoisomerases are classified into two subfamilies: type IA and type IB. The enzymes of type IA subfamily, including bacterial DNA topoisomerase I and III, eukaryotic DNA topoisomerase III, and reverse gyrase (3, 4), form a tyrosyl linkage with a 5Ј-phosphate group of one of the DNA strands generated due to the enzyme action (2), whereas the enzymes of type IB subfamily, including eukaryotic and vaccinia virus DNA topoisomerases I (5) and DNA topoisomerase V, establish the tyrosyl bond with the 3Ј-phosphate group (2). Type 1A topoisomerases relax only negatively supercoiled DNA with Mg 2ϩ requirement, whereas type IB topoisomerases relax both negatively and positively supercoiled DNA even in the absence of a metallic cofactor, although Mg 2ϩ and Ca 2ϩ stimulate the relaxation activity (6, 7).Type IB DNA topoisomerases are monomeric enzymes, constituted by four domains (8, 9). The nonconserved amino-terminal domain contains putative signals for nuclear localization of the enzyme. The largest domain, the core, is essential for enzyme activity and shows high phylogenetic conservation, particularly in the residues closely interacting with DNA. The third domain is known as the linker, which is poorly conserved and highly variable in length and is not essential for the enzyme activity. Finally, the carboxyl-terminal domain is highly conserved and crucial for the catalytic activity. This domain contains a tyrosine residue (Tyr 723 in the human topoisomerase I), which interacts with one of the DNA strands, creating a transient covalent phosphodiester bond between the enzyme and the DNA.A type I DNA topoisomerase has been purified and characterized from Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis (10). Topoisomerases have...

BackgroundVisceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.Methodology/Principal findingsWe generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.Conclusions/SignificanceThe near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of s-adenosylmethionine (AdoMet), a metabolite that plays an important role in a variety of cellular functions, such as methylation, sulfuration, and polyamine synthesis. In this study, genomic DNA from the protozoan parasite Leishmania infantum was cloned and characterized. L. infantum MAT, unlike mammalian MAT, is codified by two identical genes in a tandem arrangement and is only weakly regulated by AdoMet. L. infantum MAT mRNA is expressed as a single transcript, with the enzyme forming a homodimer with tripolyphosphatase in addition to MAT activity. Expression of L. infantum MAT in Escherichia coli proves that the MAT and tripolyphosphatase activities are functional in vivo. MAT shows sigmoidal behavior and is weakly inhibited by AdoMet, whereas tripolyphosphatase activity has sigmoidal behavior and is strongly activated by AdoMet. Plasmids containing the regions flanking MAT2 were fused immediately upstream and downstream of the luciferase-coding region and transfected into L. infantum. Subsequent examination of luciferase activity showed that homologous expression in L. infantum promastigotes was dramatically dependent on the presence of polypyrimidine tracts and a spliced leader junction site upstream of the luciferase gene, whereas downstream sequences appeared to have no bearing on expression.

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