Imipramine Is an Orally Active Drug against Both Antimony Sensitive and Resistant Leishmania donovani Clinical Isolates in Experimental Infection

Background

In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported.

Methodology/Principal Findings

Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug was found to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF α, IFN γ and iNOS activity increased with the concomitant decrease in IL 10 and TGF β level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity.

Conclusions

This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar.     

Leishmania donovani: characterization and expression of ORFF, a gene amplified from the LDI locus

The LD1 locus is a 27.5-kb region of chromosome 35 that is conserved among all species of Leishmania and is amplified in several different isolates. Here, we report the genomic distribution of ORFF, a gene from the LD1 region, and its expression at the RNA and protein levels in two Indian isolates of Leishmania donovani. In both of these isolates, ORFF was present as a single copy on chromosome 35. Densitometric analysis of ORFF mRNA abundance revealed relative abundance of 0.2 and 1.0 in AG83 and S-Lal, respectively. Antiserum against recombinant ORFF protein detected a protein of the predicted size ( approximately 34 kDa) in both strains. The protein is most abundant in mid-log-phase promastigotes and has a nuclear localization. The ORFF protein is preferentially expressed in L. donovani amastigotes but, in contrast, is expressed at higher levels in L. major promastigotes.     

Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification

The methotrexate (MTX) and folate transport properties of five MTX-resistant lines of Leishmania major have been examined. These resistant lines all show a decreased Vmax for MTX influx, with no change in apparent affinity (Kt). The Vmax of folate influx is also proportionately decreased without alteration in Kt, supporting our proposal that there is a single carrier mediating influx of both ligands. Amplifications of two regions of DNA, the R region (encoding dihydrofolate reductase-thymidylate synthase) and the H region (Beverley, S.M., Coderre, J.A., Santi, D.V., and Schimke, R.T. (1984) Cell 38, 431-439), were also observed. In a given line, the amplifications occurred singly, in combination, or not at all. No other regions of amplification were detected. The phenotype of reduced MTX transport was moderately stable in the highly resistant R1000 line, being retained for more than 200 generations in the absence of MTX in vitro and during one passage through an infected mouse; in contrast, R- and H-amplified DNA were less stable. The lack of correlation of R and H amplification with reduced MTX transport suggests that alterations in transport are not causally mediated by gene amplification in Leishmania, but are a separate mode of MTX resistance. The onset of decreased MTX transport was also examined; wild-type Leishmania developed a reduced Vmax of MTX influx within 24 h following exposure to 1 microM MTX, which is extremely unstable in the absence of drug pressure. A comparable decrease in the Vmax of influx is seen in cells exposed to MTX in media in which cytotoxicity is eliminated. As the folate/MTX transporter is regulated by exogenous folate, these data suggest that the initial rapid decrease in MTX transport may be a cellular regulatory response, in contrast to that found within the R1000 line which resembles a more stable genetic mutation.     

Linear amplicons as precursors of amplified circles in methotrexate-resistant Leishmania tarentolae.

Gene amplification is frequently observed in Leishmania cells selected for drug resistance. By gene targeting we have tagged both alleles of the H locus of Leishmania tarentolae with the neomycin and hygromycin phosphotransferase genes ( neo and hyg ). Selection of these recombinant parasites for low level methotrexate resistance led to amplification of the H locus as part of linear amplicons. The availability of tags has permitted us to determine that both alleles can be amplified in the same cell and that chromosomal deletions are frequent. When methotrexate concentration was increased in subsequent selection steps, circles were observed in several mutants. We have introduced a hyg marker into linear amplicons to test whether the circles originated from linear amplicons. After selection with a high methotrexate concentration, circles with the hyg marker were observed, showing that circles can indeed be formed from linear amplicons. The tagging of H locus alleles permits appreciation of the extent of genetic rearrangements leading to amplicon formation in Leishmania cells selected for drug resistance.     

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.     

Formation of extrachromosomal circular amplicons with direct or inverted duplications in drug-resistant Leishmania tarentolae.

Selection for methotrexate resistance in Leishmania spp. is often associated with amplification of the H locus short-chain dehydrogenase-reductase gene ptr1 as part of extrachromosomal elements. Extensive sequences are always coamplified and often contain inverted duplications, most likely formed by the annealing of inverted repeats present at the H locus. By gene targeting mediated by homologous recombination, several repeated sequences were introduced in the vicinity of ptr1. Selection for methotrexate resistance in these transfectants led to ptr1 amplification as part of small circles with direct or inverted duplications whether the integrated sequences consisted of direct or inverted repeats. Hence, for a region to he amplified in L. tarentolae during drug selection, a drug resistance gene is required and must be flanked by (any) homologous repeated sequences. The distance between these repeats and their orientation will determine the length of the amplicon and whether it contains direct or inverted duplications.     

Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania

This paper demonstrates how the polymerase chain reaction can be used to increase the sensitivity of detection of Leishmania parasites by DNA hybridization methods through the amplification of the minicircle target sequence. The oligonucleotide primers used are able to direct the amplification of all Leishmania strains tested. In addition, the PCR products from L. mexicana and L. braziliensis strains can be distinguished by hybridization with kDNA probes. The method is sensitive enough to detect the kDNA from a single organism and this sensitivity allows the use of nonradioactive hybridization methods. This method can be used to detect Leishmania from human biopsy material.     

Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.     

Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine

Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene.     

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.     

Co-existence of circular and multiple linear amplicons in methotrexate-resistant Leishmania.

Circular and linear amplicons were analyzed in detail in Leishmania tropica cells resistant to methotrexate (MTX). Both types of elements presented sequences related to the H locus and coexisted in resistant cells. The linear amplicons appeared first during the selection process (at 10 microM MTX) and varied with regard to size and structure in cells exposed to increasing concentrations of drug. The circular element was evident at higher concentrations (50 microMs) but was the major amplified DNA in cells resistant to 1000 microM MTX while the level of amplification of the linear elements remained low. The extrachromosomal DNAs were unstable in the absence of drug and their disappearance coincided with an increase in sensitivity to MTX. Mapping of the minichromosomes and the circular element showed that they were all constituted by inverted duplications. The circular amplicon contained an inverted repeat derived from the H locus that encompassed the pteridine reductase gene (PTR1) responsible for MTX resistance. The amplified segment in the linear amplicons was longer and included the pgpB and pgpC genes that encode P-glycoproteins of unknown function previously characterized in different Leishmania species.     

A member of the aldoketo reductase family confers methotrexate resistance in Leishmania.

Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

Role of the locus and of the resistance gene on gene amplification frequency in methotrexate resistant Leishmania tarentolae.

The protozoan parasite Leishmania resists the antifolate methotrexate (MTX) by amplifying the R locus dihydrofolate reductase-thymidylate synthase ( dhfr-ts ) gene, the H locus ptr1 pterin reductase gene, and finally by mutation in a common folate/MTX transporter. Amplification of dhfr-ts has never been observed in Leishmania tarentolae MTX resistant mutants while ptr1 amplification is common. We have selected a L.tarentolae ptr1 null mutant for MTX resistance and observed dhfr-ts amplification in this mutant demonstrating that once a preferred resistance mechanism is unavailable, a second one will take over. By introducing the ptr1 gene at the R locus and the dhfr-ts gene at the H locus by gene targeting, we investigated the role of the resistance gene and the locus on the rate of gene amplification. Transfection studies indicated that ptr1 gave higher levels of MTX resistance than dhfr-ts. Consistent with this, when ptr1 was present as part of either the H locus or the R locus it was invariably amplified, while dhfr-ts was only amplified when ptr1 was inactivated. When dhfr-ts was present in a ptr1 null background on both the H locus and the R locus, amplification from the H locus was more frequent suggesting that both the gene and the locus are determining the frequency of gene amplification in Leishmania.     

Gene amplification in amphotericin B-resistant Leishmania tarentolae

Two Leishmania tarentolae cells were selected step by step for resistance to the polyene antibiotic amphotericin B, a second-line drug against the parasite Leishmania. One of the mutants was cross-resistant to ketoconazole. DNA amplification was observed in both mutants. The amplicons were extrachromosomal circles and were derived from different chromosomes. In one mutant the circle was unusually stable as it remained within the cell despite numerous passages in the absence of the drug. A circumstantial link between the copy number of amplicons and the resistance levels was established. Gene transfection experiments indicated that the link between the locus amplified and the resistance levels was not straightforward and possibly several mutations act together to lead to amphotericin B resistance.     

Fine needle aspiration of lymphadenopathy in visceral leishmaniasis

OBJECTIVE: To illustrate the cytomorphologic features of Leishmania lymphadenitis associated with visceral leishmaniasis (V/L) and post-kala-azar dermal leishmaniasis (PKDL) and to highlight the fact that Leishmania lymphadenitis must he included in the differential diagnosis of patients presenting with lymphadenopathy, particularly in areas endemic for the disease. STUDY DESIGN: Fine needle aspiration (FNA) was routinely done in 21 cases of lymphadenopathy in VL (18 cases) and PKDL (3 cases), and the detailed cytomorphologic features were correlated with the respective histopathologic findings. RESULTS: Amastigote forms of Leishman-Donovan (LD) bodies were seen in 19 cases both intracellularly, in histiocytes and multinucleate giant cells, and extracellularly. The FNA smears revealed a polymorphous population of cells composed of lymphocytes, histiocytes, plasma cells, giant cells and tingible body macrophages. In a few cases, epithelioid cell granulomas were also seen. The cytomorphologic features were confirmed and correlated on histopathology. CONCLUSION: Not all lymphadenopathy in VL and PKDL is due to Leishmania lymphadenitis. Demonstration of LD bodies on FNA smears helps with the early diagnosis of VL and PKDL with lymphadenopathy where the diseases are endemic.     

A novel antifolate resistance gene on the amplified H circle of Leishmania.

In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short-chain dehydrogenases, enzymes that are involved in several oxido-reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.     

Cloning,characterization and preliminary crystallographic analysis of Leishmania hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae hgprt gene contains a 633-nucleotide open reading frame that encodes a 23.4-kDa protein. A pairwise alignment of the different HGPRT's sequences revealed a 26%-53% sequence identity with the Leishmania sequences and 87% identity to the HGPRT of Leishmania donovani. A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The steady-state kinetic parameters were determined for the recombinant enzyme and the enzyme is active as a homodimer in solution. Single crystals were obtained for the L. tarentolae HGPRT representing the first Leishmania HGPRT crystallized and initial crystallographic data were collected. The crystals obtained belong to the orthorhombic space group (P2(1)2(1)2(1)) with unit cell parameters a=58.104 A, b=85.443 A and c=87.598 A and diffract to a resolution of 2.3 A. The availability of the HGPRT enzyme from Leishmania and its crystallization suitable for X-ray diffraction data collection should provide the basis for a functional and structural analysis of this enzyme, which has been proposed as a potential target for rational drug design, in a Leishmania model system.