Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.     

The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance

We have induced drug resistance against methotrexate, an inhibitor of dihydrofolate reductase, and CB3717, an inhibitor of thymidylate synthetase in a strain of Leishmania tarentolae. The drug-resistant strains contain extrachromosomal DNA circles of 68 kilobases with a 30-kilobase inverted duplication flanked by 4- and 5 kilobase unique segments. We show that these circles are highly homologous to the drug-induced H circles of L. tropica (1). All three L. tarentolae strains analyzed contain a chromosomal copy of the H region without duplication, but two of the three strains contain extrachromosomal H circles as well, predominantly present as H circle dimers in one strain and as tetramers in the other. After induction of methotrexate resistance, monomeric circles, presumably derived from the oligomers, become the major type of circle. Our results indicate that the H region represents a genomic region that can be copied at very low frequency to yield circles by a precise, but unusual mechanism under natural conditions in wild-type cells. Although superficially analogous to the episomes of bacteria, the system is without precedent in nature.     

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.     

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.     

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.     

Multiple drug resistance and conservative amplification of the H region in Leishmania major

Amplification of the H region has been previously observed in methotrexate (MTX)-resistant strains of Leishmania major and in unselected laboratory stocks of L. tarentolae. We now show that selection of L. major with the structurally unrelated drugs primaquine or terbinafine generated resistant lines exhibiting H region amplification and 23- and 12-fold cross-resistance to MTX, respectively. These and other drug-resistant lines bearing H region amplification also exhibited weak cross-resistance to primaquine and terbinafine, associating the amplified H region with pleiotropic resistance to MTX and other drugs. In contrast, lines selected for chloroquine or pentamidine resistance did not show H region amplification or this pattern of drug cross-resistance. The primaquine- and terbinafine-selected lines exhibited wild-type levels of dihydrofolate reductase-thymidylate synthase and normal uptake and accumulation of MTX, and the MTX resistance of these lines was not reversed by verapamil. These data suggest that the mechanism of MTX cross-resistance associated with H region amplification is novel and distinct from that mediated by overexpression of MDR genes in multidrug-resistant mammalian cells. Structural studies indicated that the amplified H region DNA in these L. major lines was largely (possibly exclusively) extra-chromosomal and consisted of circular inverted repeats joined at two DNA rearrangement junctions. Southern blot analyses showed that these rearrangement junctions were identical in four independent cell lines, suggesting that these sites are "hotspots" for DNA rearrangement. H region amplification in all of these lines was conservative, defined as retention of the chromosomal H region locus without structural alteration or reduction in copy number. This finding is consistent with an over-replication/recombination model for amplification of the H region.     

The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei

The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1–3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1–3 substantially reduced folate transport into trypanosomes and reduced the parasite''s susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1–3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1–3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1–3 loss-of-function is a mechanism of antifolate drug resistance.     

Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate

We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic antifolates, respectively, were established. Increased antifolate resistance was associated with a prominent overexpression of dihydrofolate reductase as determined from the elevated folate reductase activity, cellular labeling with fluorescein-methotrexate, and steady-state mRNA levels as a result of a consistent dihydrofolate reductase gene amplification. However, upon subsequent incremental increases in trimetrexate, further resistance was also associated with amplification of the multidrug resistance gene. This resulted in overexpression of P-glycoprotein and a subsequent 20-50-fold collateral resistance to pleiotropic drugs such as adriamycin, actinomycin D, vinca alkaloids, etoposide, and colchicine. In contrast, initial resistance following selection with low piritrexim concentrations resulted from an unknown mechanism(s) not involving overproduction of either dihydrofolate reductase or P-glycoprotein. This piritrexim resistance was shared with trimetrexate but not with methotrexate. Upon further selection with piritrexim, resistant variants emerge with amplified dihydrofolate reductase but not with multidrug resistance genes. These variants were subsequently resistant to both hydrophilic and lipophilic folate antagonists but retained sensitivity to pleiotropic drugs. The pattern of resistance with methotrexate, trimetrexate, and piritrexim shared a common mechanism, dihydrofolate reductase gene amplification, but differed regarding the additional amplification of the multidrug resistance gene in trimetrexate-resistant cells as well as the emergence of an additional unknown mechanism(s) of resistance to lipid-soluble antifolates upon initial selection in piritrexim.     

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.     

Heavy metal resistance: a new role for P-glycoproteins in Leishmania.

P-glycoproteins are responsible for multidrug resistance in tumor cell lines and are thought to have a physiologic role in exporting cellular metabolites. We now report that a P-glycoprotein gene in the H region of the trypanosomatid protozoan Leishmania confers resistance to heavy metals when present in multiple copies. The Leishmania H region is frequently amplified in drug-resistant lines and is associated with metal resistance. Leishmania expression vectors were used to introduce multiple copies of segments of the Leishmania major H region into wild-type L. major promastigotes. Only constructs bearing a segment of L. major DNA containing the P-glycoprotein lmpgpA conferred arsenite resistance. Deletional analysis of the arsenite-resistant construct mapped resistance to the lmpgpA protein coding region. Lines expressing lmpgpA showed resistance to arsenite and trivalent antimonials, but not to pentavalent antimonials, zinc, cadmium, or the typical multidrug-resistant P-glycoprotein substrates vinblastine and puromycin. Transfection of the Leishmania tarentolae P-glycoprotein homologue ltpgpA resulted in a similar resistance profile. Thus, these pgpAs represent a functionally distinct group of P-glycoproteins which exhibit a substrate specificity similar to prokaryotic heavy metal pumps. Additionally, several arguments suggest that pgpAs may play a role in the susceptibility of Leishmania to clinically utilized antimonials.     

A new type of high affinity folic acid transporter in the protozoan parasite Leishmania and deletion of its gene in methotrexate-resistant cells

The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here that Leishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistant Leishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (K(m) 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions.     

Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.

We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.     

Amplification of a new region of DNA in an unselected laboratory stock of L. tarentolae: the T region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae. This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania, or the maxicircle of L. tarentolae, nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae.     

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.     

Pterin transport and metabolism in Leishmania and related trypanosomatid parasites

The folate metabolic pathway has been exploited successfully for the development of antimicrobial and antineoplasic agents. Inhibitors of this pathway, however, are not useful against Leishmania and other trypanosomatids. Work on the mechanism of methotrexate resistance in Leishmania has dramatically increased our understanding of folate and pterin metabolism in this organism. The metabolic and cellular functions of the reduced form of folates and pterins are beginning to be established and this work has led to several unexpected findings. Moreover, the currently ongoing sequencing efforts on trypanosomatid genomes are suggesting the presence of several gene products that are likely to require folates and pterins. A number of the properties of folate and pterin metabolism are unique suggesting that these pathways are valid and worthwhile targets for drug development.     

Amplification of a New Region of DNA in an Unselected Laboratory Stock of L. tarentolae: The T Region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae . This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania , or the maxicircle of L. tarentolae , nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae .     

Isolation of genes mediating resistance to inhibitors of nucleoside and ergosterol metabolism in Leishmania by overexpression/selection

We tested a general method for the identification of drug resistance loci in the trypanosomatid protozoan parasite Leishmania major. Genomic libraries in a multicopy episomal cosmid vector were transfected into susceptible parasites, and drug selections of these transfectant libraries yielded parasites bearing cosmids mediating resistance. Tests with two antifolates led to the recovery of cosmids encoding DHFR-TS or PTR1, two known resistance genes. Overexpression/selection using the toxic nucleoside tubercidin similarly yielded the TOR (toxic nucleoside resistance) locus, as well as a new locus (TUB2) conferring collateral hypersensitivity to allopurinol. Leishmania synthesize ergosterol rather than cholesterol, making this pathway attractive as a chemotherapeutic target. Overexpression/selection using the sterol synthesis inhibitors terbinafine (TBF, targeting squalene epoxidase) and itraconazole (ITZ, targeting lanosterol C(14)-demethylase) yielded nine new resistance loci. Several conferred resistance to both drugs; several were drug-specific, and two TBF-resistant cosmids induced hypersensitivity to ITZ. One TBF-resistant cosmid encoded squalene synthase (SQS1), which is located upstream of the sites of TBF and ITZ action in the ergosterol biosynthetic pathway. This suggests that resistance to "downstream" inhibitors can be mediated by increased expression of ergosterol biosynthetic intermediates. Our studies establish the feasibility of overexpression/selection in parasites and suggest that many Leishmania drug resistance loci are amenable to identification in this manner.     

Isolation and characterisation of an antifolate insensitive (afi1) mutant of Arabidopsis thaliana

Antifolates can impair the synthesis and/or function of folates in living organisms. Mechanisms of resistance or tolerance to antifolates have been mainly described in plants using the drug methotrexate. In this work, the antifolate trimethoprim (TMP) was used with the aim of revealing a novel mechanism of resistance. EMS mutagenised seeds from Arabidopsis were screened to isolate individuals insensitive to TMP. Genetic analysis revealed a homozygous recessive mutation that segregates with the phenotype of tolerance to 50 μm TMP. Mapping analysis localised the mutation at the end of the short arm of chromosome 3. Preliminary characterisation demonstrated up‐regulation of several genes from the folate biosynthetic pathway in the TMP insensitive mutant, and a slight increase in total folate content in the mutant as compared with the Col‐0 control. Moreover, sequence analysis of the DHFR (dihydrofolate reductase) genes, which encode a known target for resistance to antifolates, did not reveal any changes. This study is the first report of a stable mutant insensitive (afi1) to the antifolate trimethoprim in plants, and suggests the existence of a novel mechanism of resistance to antifolates.     

Expression of two soybean resistance gene candidates shows divergence of paralogous single-copy genes

The cloning of several plant genes directly involved in triggering a disease resistance response has shown that numerous resistance genes in the nucleotide binding site (NBS)/leucine-rich repeat (LRR) class have similar conserved amino acid sequences. In this study, we used a short soybean DNA sequence, previously cloned based on its conserved NBS, as a probe to identify full-length resistance gene candidates. Two homologous, but genetically independent genes were identified. One gene maps to the soybean molecular linkage group (MLG) F and a second is coded on MLG E. The first gene contains a 3,279 nucleotide open reading frame (ORF) sequence and possesses all the functional motifs characteristic of previously cloned NBS/LRR resistance genes. The N-terminal sequence of the deduced gene product is highly characteristic of other resistance genes in the subgroup of NBS/LRR genes which show homology to the Toll/Interleukin-1 receptor genes. The C-terminal region is somewhat more divergent as seen in other cloned disease resistance genes. This region of the F-linked gene contains an LRR region that is characterized by two alternatively spliced products which produce gene products with either a four-repeat or a ten-repeat LRR. The second cloned gene that maps to soybean MLG E contains 1,565 nucleotides of ORF in the N-terminal domain. Despite strong homology, however, the 3′ region of this gene contains several in-frame stop codons and apparent frame shifts compared to the F-linked gene, suggesting that its functionality as a disease resistance gene is questionable. These two disease resistance gene candidates are shown to be closely related to one another and to the members of the NBS/LRR class of disease resistance genes. Received: 29 November 1999 / Accepted: 22 December 1999