Buddy Ullman

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium.

A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar. Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum. Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum. Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency. Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%.

Two high affinity nucleoside transporters in Leishmania donovani

A rapid sampling kinetic technique has been used to evaluate the nucleoside transport functions of Leishmania donovani. The results indicated that L. donovani promastigotes possessed two independent purine nucleoside transporters with nonoverlapping substrate specificity. The first transported inosine, guanosine, and their analogs, while the second carried adenosine, analogs of adenosine, and the pyrimidine nucleosides, uridine, cytidine, and thymidine. The apparent Km values of the two nucleoside permeases for their purine nucleoside substrates were extraordinarily low, in the micromolar range. The organisms were capable of concentrating purine nucleosides from the medium and converting them to the nucleotide level with great efficiency and rapidity. Inosine and adenosine transport could be distinguished by different sensitivities to sulfhydryl reagents, suggesting structural differences between the two transporters. Finally, the two nucleoside transport systems of L. donovani were virtually refractory to inhibition by 4-nitrobenzylthioinosine and dipyridamole, two potent inhibitors of nucleoside entry into mammalian cells.

Nucleoside and Nucleobase Transporters in Parasitic Protozoa

One distinctive feature of the biochemistry of parasitic protozoa is their absolute reliance upon the salvage of preformed purines from their vertebrate and invertebrate hosts. While many mammalian cells possess the innate ability to synthesize purines de novo, all protozoa so far examined that

Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

The proteins that mediate polyamine translocation into eukaryotic cells have not been identified at the molecular level. To define the polyamine transport pathways in eukaryotic cells we have cloned a gene, LmPOT1, that encodes a polyamine transporter from the protozoan pathogen, Leishmania major. Sequence analysis of LmPOT1 predicted an unusual 803-residue polytopic protein with 9-12 transmembrane domains. Expression of LmPOT1 cRNA in Xenopus laevis oocytes revealed LmPOT1 to be a high affinity transporter for both putrescine and spermidine, whereas expression of LmPOT1 in Trypanosoma brucei stimulated putrescine uptake that was sensitive to inhibition by pentamidine and proton ionophores. Immunoblot analysis established that LmPOT1 was expressed predominantly in the insect vector form of L. major, and immunofluorescence demonstrated that LmPOT1 was localized predominantly to the parasite plasma membrane. To our knowledge this is the first molecular identification and characterization of a cell surface polyamine transporter in eukaryotic cells.

An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Δarg), as well as wild-type and complemented Δarg controls (Δarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p < 0.05); 2) poorer survival of Δarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Δarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Δarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Δarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Δarg parasites produced more IFN-γ and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of Δodccells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The Δodc L. donovaniexhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the Δodc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L. donovani ldmdr1 gene. The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

Cloning of a novel inosine-guanosine transporter gene from Leishmania donovani by functional rescue of a transport-deficient mutant.

Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovanideficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2re-established their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K m = 0.3 μm) and guanosine (K m = 1.7 μm) and does not recognize other naturally occurring nucleosides. Expression ofLdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.

Genetic analysis of spermidine synthase from Leishmania donovani.

The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Deltaspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Deltaspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.