Trypanosoma cruzi:Use of Herpes Simplex Virus–Thymidine Kinase as a Negative Selectable Marker

EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR974163

86, 171–180 (1997)

Trypanosoma cruzi: Use of Herpes Simplex Virus–Thymidine Kinase as a Negative Selectable Marker Frederick S. Buckner,*,1 Aaron J. Wilson,* and Wesley C. Van Voorhis*,† *Department of Medicine, Division of Allergy and Infectious Diseases, and †Department of Pathobiology, University of Washington, Seattle, Washington 98195, U.S.A. BUCKNER, F. S., WILSON, A. J., AND VAN VOORHIS, W. C. 1997. Trypanosoma cruzi: Use of herpes simplex virus–thymidine kinase as a negative selectable marker. Experimental Parasitology 86, 171–180. Trypanosoma cruzi, the protozoan that causes Chagas’ disease, was transfected with a fusion gene of hygromycin phosphotransferase and herpes simplex virus–thymidine kinase, HyTK. Transfectants selected in hygromycin had thymidine kinase activity, whereas controls did not. In vitro growth of the mammalian life-stage forms, amastigotes and trypomastigotes, was inhibited 98% by the nucleoside analogue ganciclovir (5 mg/ml). Growth of the insect-stage form, epimastigotes, was not inhibited by ganciclovir (up to 250 mg/ml) or other nucleoside analogues. Intracellular uptake of ganciclovir by epimastigotes was found to be 10-fold less than that by amastigotes. Mice infected with the HyTK-expressing parasites and treated with ganciclovir had a statistically significant reduction of parasitemia by 57%; however, complete eradication of parasites was not achieved. The parasites recovered from the treated mice continued to be susceptible to ganciclovir in vitro. Parasite clones with higher expression of thymidine kinase were more sensitive to ganciclovir, suggesting that greater expression of the thymidine kinase gene may lead to parasites that can be fully eradicated from infected experimental animals. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Chagas’ disease; negative selection; herpes simplex virus thymidine kinase (HSV-TK); ganciclovir (GCV); hygromycin.

INTRODUCTION Genetic manipulations of Trypanosoma cruzi have been facilitated by the use of positive selectable markers such as neomycin phosphotransferase and hygromycin phosphotransferase (Hariharan et al. 1993; Buckner et al. 1996; La Flamme et al. 1996; Cooper et al. 1993; Otsu et al. 1995). Transfection with a negative selectable marker, a gene whose expression is lethal to cells, has not yet been performed on this protozoan parasite. The herpes simplex virus thymidine kinase gene (HSV-TK) is widely used as a negative selectable marker in mammalian systems. It is employed to enrich for correctly targeted recombinants in cultured cells (Mansour et al. 1988) and to delete specific cell lineages in transgenic mice after fusion of the negative marker to cell-type-specific gene control elements (Plautz et al. 1991; Palmiter et al. 1987). HSV-TK is used in gene therapy for various types of cancer (Moolten and Wells 1

To whom correspondence should be addressed.

1990; Ezzeddine et al. 1991; Ram et al. 1993; Takamiya et al. 1992) and infections (Caruso and Klatzmann 1992). In kinetoplastids, HSVTK has been expressed in Leishmania major and rendered procyclics susceptible to ganciclovir (Lebowitz et al. 1992). The gene has been introduced into Trypanosoma brucei procyclics, where it was useful to define rates of mutagenesis (Valdes et al. 1996) and for the study of the biogenesis of glycosomes (Lye and Wang 1996). A fusion gene of hygromycin phosphotransferase and herpes simplex virus–thymidine kinase, HyTK (Lupton et al. 1991), was used in the experiments described below. The expression of this fusion protein allows for positive selection with hygromycin and negative selection with drugs such as GCV. HSV-TK phophorylates a variety of nucleoside analogues which remain unphosphorylated in cells lacking this enzyme. The phosphorylated nucleoside analogues inhibit host cell replication by competing for DNA polymerase or acting as DNA

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chain terminators. A growing list of nucleoside analogues for use in negative selection has been developed primarily as antiviral drugs, including GCV, acyclovir, pencyclovir, and sorivudine. Transfection of a negative selection marker into T. cruzi would facilitate numerous avenues of investigation. Use of a negative selection marker could allow eradication of parasites in animal models of Chagas’ disease, enabling the study of pathologic progression of tissue damage following the parasitological cure. It is currently not known whether the parasitological cure will diminish the progression of chronic pathology which is believed to be mediated to a significant degree by autoimmune mechanisms (Cunha-Neto et al. 1996; Kalil and Cunha-Neto 1996). Studies of protective immunity could be conducted in mice whose infection was eradicated by drugs such as GCV. Treatment of antigen presenting cells with a drug in vitro to inhibit the outgrowth of parasites may be advantageous in efforts to derive lymphocyte lines directed against T. cruzi. Negative markers may be used to select for transcriptional regulatory elements and to study stage-specific gene regulation. In addition, a parasite transfected with a suicide gene might have potential applications as a vaccine candidate (particularly in animals) and would provide a safer laboratory strain, as accidental infection is always a serious concern.

In the experiments described below, T. cruzi expressing the HyTK gene were susceptible to GCV in the mammalian life-cycle stages. The insect-stage epimastigotes were resistant to GCV apparently due to exclusion of the drug from the intracellular space. Treatment of infected mice with GCV showed reduced parasitemia, but failure to completely eradicate the parasites. Data suggest that parasite clones with higher expression of TK were more sensitive to GCV. MATERIALS AND METHODS Parasites and culture procedures. The Tulahuen strain of T. cruzi was provided by S. Reed (Infectious Diseases Research Institute, Seattle, WA) and the CL strain was provided by H. Eisen (Fred Hutchinson Cancer Research Center, Seattle, WA). Trypomastigotes and amastigotes were grown on monolayers of mouse 3T3 fibroblasts as previously described (Van Voorhis and Eisen 1989). Epimastigotes were grown in liver infusion tryptone broth with 10% fetal calf serum, penicillin, and streptomycin as previously described (Van Voorhis and Eisen 1989). Plasmid constructs. Expression plasmids (pBS:BCHyTK-10 and pBS:BC-Hygro-10) for T. cruzi were constructed, incorporating the intergenic regions of the calmodulin–ubiquitin 2.65 locus of the CL strain (Fig. 1a). The starting plasmid (pBS:IL-2-CnFc) contained the BC and 10 intergenic regions which flanked the murine IL-2 cDNA and is described by La Flamme et al. (1996). This plasmid was cut with MluI, which released a 1.9-kb fragment spanning the BC region, the IL-2 cDNA, and the 10 region. This fragment was blunted with T4 polymerase and ligated into

FIG. 1. (a) Partial map of the calmodulin–ubiquitin 2.65 locus of Trypanosoma cruzi. Short boxes represent untranslated regions (designated CL, 01, BC, and 10); tall boxes represent translated genes (Ajioka and Swindle 1992). (b) Expression plasmid pBS:BC-HyTK-10. Intergenic regions containing a trans-splice acceptor site (in BC) and polyadenylation signal (in 10) flank the hygromycin phosphotransferase–herpes simplex virus– thymidine kinase (HyTK) gene. Restriction sites for PvuII within pBluescribe were used to prepare the linear DNA fragment (3050 bp) used in electroporation of epimastigotes.

NEGATIVE SELECTION OF the polylinker of pBluescribe [Vector Cloning Systems (renamed Stratagene), San Diego, CA]. The IL-2 gene was excised with XbaI and BamHI, giving pBS:BC × 10. The HyTK insert, a fusion product of the hygromycin phosphotransferase gene and the herpes simplex virus thymidine kinase gene, was obtained from the plasmid tg CMV/HyTK (Lupton et al. 1991) generously provided by S. D. Lupton (Targeted Genetics Corp., Seattle, WA). The HyTK gene was cut out with NheI and BglII and ligated into pBS:BC × 10 to give pBS:BC-HyTK-10 (Fig. 1b). The Hygro insert was obtained by PCR of the hygromycin phosphotransferase gene (plasmid template provided by J. Swindle, Seattle Biomedical Research Institute) using the following oligonucleotides: 59 sense primer (59-GCTGGCCAATGAAAAAGCCTGAACTCACC) and 39 antisense primer (59-GCGATATCCTATTCCTTTGCCCTCGGACG). The PCR product was blunted with T4 polymerase and ligated into the expression site of pBS:BC × 10, giving pBS:BCHygro-10 (not shown). For electroporation, the DNAs from pBS:BC-HyTK-10 and pBS:BC-Hygro-10 were linearized using PvuII, which cuts within pBluescribe 59 to the BC region and 39 to the 10 region. The resulting fragments were gel purified and resuspended in 1 mM Tris, 0.1 mM ethylenedinitrilotetraacetic acid at 2 mg/ml. Electroporation and cloning of parasites. Epimastigotes were electroporated with 10 mg of linearized DNA as previously described (Chung et al. 1994). Transfectants were selected by growth in hygromycin (Boeringer-Mannheim, Mannheim, Germany) at 250 mg/ml for Tulahuen strain parasites and at 500 mg/ml for CL strain parasites. Epimastigotes were cloned by limiting dilution. Clones were transformed to the mammalian forms (trypomastigotes/amastogotes) by inoculating the parasites onto mouse 3T3 monolayers and incubating for approximately 10 days at 37°C in a CO2 incubator. Southern analysis of transfected clones. Genomic DNA was cut with the restriction enzyme BglII and probed with a 32 P-labeled PCR product of the calmodulin A2 gene (Ajioka and Swindle 1992). In all experiments (except those using the CL clone A2), parasite clones were used that were shown by Southern analysis to have integrated the HyTK or Hygro gene into the Cal A2 locus (see Fig. 1). Sensitivity testing of transfected parasites using ganciclovir and other nucleoside analogues. Logarithmic growth-phase epimastigotes were placed in 24-well plates at 5 × 105/ml in the presence of hygromycin with dilutions of GCV (Syntex Laboratories, Inc., Palo Alto, CA) from 250 mg/ml (903 mM) down to 1 mg/ml (3.6 mM). Growth inhibition was determined by quantitating parasites at 3 and 5 days using a counting chamber with comparison to parasites grown in the absence of GCV. Growth inhibition by GCV in the mammalian forms of the parasite was determined after seeding monolayers of 3T3 fibroblasts with trypomastigotes (1 × 105/well on 24-well plates) in the presence of dilutions of GCV (from 10 to 0.1 mg/ml) and counting the parasites released into the media from the lysed mammalian cells on Days 7, 8, and 9 postinfection. The latter experiments were

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done in the absence of hygromycin due to its inhibitory effect on 3T3 cells. T. cruzi were also tested for sensitivity to acyclovir (Burroughs Welcome Co., Research Triangle Park, NC), pencyclovir (SmithKline Beecham Pharmaceuticals, King of Prussia, PA), and sorivudine (BV-araU, Bristol–Meyers Squibb Pharmaceutical Research, Princeton, NJ). Thymidine kinase assay. Epimastigotes were grown to a concentration of 107/ml in 10 ml of culture media, washed in phosphate-buffered saline (pH 7.4), and pelleted. The mammalian forms of T. cruzi grown on 3T3 cells (consisting of approximately 50% trypomastigotes and 50% amastigotes) were also grown to a density of 107/ml in 10 ml, washed, and pelleted. The procedure was performed on the samples as described by Brinster et al. (1981). Briefly, the cell pellets were suspended in 100 ml of buffer containing 10 mM KCl, 2 mM MgCl2, 10 mM Tris–Cl, 1 mM ATP, 1 mM NaF, and 50 mM e-aminocaproic acid (pH 7.4), freeze/ thawed on dry ice twice, and then centrifuged for 10 min at 15,000g. The supernatants were stored at −70°C until assayed. An aliquot of thawed lysate equivalent to 50 mg protein was mixed in a final volume of 25 ml of reaction mixture containing 150 mM Tris–Cl (pH 7.5), 10 mM ATP, 10 mM MgCl2, 25 mM NaF, and 10 mM b-mercaptoethanol and 5 mCi of [3H]thymidine (46 Ci/mmol; Amersham Corp., Arlington Heights, IL). Samples were tested in duplicate. The mixture was incubated for 2 hr at 37°C and absorbed to DE-81 paper (Whatman, Inc., Clifton, NJ); the filters were washed twice with 1 mM ammonium formate, then with H2O, and then with 95% ethanol (Gronowitz et al. 1984). The bound [3H]thymidine monophosphate was quantitated by scintillation counting. Background activity was determined by applying to the filter the radioactive reaction mixture alone; the counts per minute from nonspecific binding were substracted from the counts per minute for each sample. Identical assays were also conducted utilizing [3H]ganciclovir (13.1 Ci/mmol; Moravek Biochemicals, Inc., Brea, CA) as substrate. Uptake experiments with radiolabeled GCV or thymidine. Epimastigotes, trypomastigotes, and amastigotes were separately tested for uptake of [3H]GCV. Cultures with greater than 90% trypomastigotes were obtained from an early burst of parasites from 3T3 fibroblasts. Cultures with greater than 90% amastigotes were derived by allowing trypomastigotes to incubate 24 hr in liver infusion tryptone broth (no serum) at 37°C. Cells were adjusted to 5 × 106 in 250 ml of Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum and incubated with 5 mCi of [3H]GCV or 5 mCi of [3H]thymidine for 4 hr. The cells were then centrifuged at 1500g and washed four times with phosphate-buffered saline; the pellet was directly added to scintillation fluid and counted for b-emission. Mouse experiments. Six- to eight-week-old female C57BL/6 mice (B&K Universal, Kent, WA) were injected intraperitoneally on Day 1 with 5 × 104 Tulahuen strain trypomastigotes/amastigotes expressing HyTK or Hygro. Tulahuen strain parasites were selected as they produce eas-

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ily detectable parasitemia and tissue pathology with relatively low inocula compared to our lab strain CL T. cruzi. The mice received 25 mg/kg GCV twice daily intraperitoneally at approximately 8 AM and 5 PM on Days 1 through 14. The first GCV injection was administered 6 hr before injection of the parasites. Parasitemia was determined by examining two drops of tail blood under a coverslip at 400× magnification. Fifty fields were scanned and motile parasites counted (parasites/ml 4 no. parasites/field × 600,000). Statistical analysis. Two tailed T tests were performed on the data in Fig. 5 using the statistics software package in Prism, version 2.00 (Graphpad Software, Inc., San Diego, CA).

RESULTS Southern analysis of drug-resistant T. cruzi clones. In the Tulahuen strain, all clones transfected with BC-HyTK-10 (n 4 4) or BCHygro-10 (n 4 3) showed integration by gene replacement into the calmodulin–ubiquitin locus at the homologous site of the Cal A2 gene (see Fig. 1a). In the CL strain, this same integration event was confirmed for four BCHygro-10 clones and three BC-HyTK-10 clones. One additional BC-HyTK-10 clone (named A2) had an aberrant integration event outside of the Cal A2 locus but with linkage to a calmodulin gene (data not shown). Sensitivity of transfected parasites to ganciclovir. Epimastigotes transfected with the HyTK gene and grown in hygromycin were not inhibited with GCV in doses as high as 250 mg/ml. However, when the HyTK parasites were transformed into the mammalian forms (trypomastogotes/amastigotes) by culturing on mouse 3T3 cells, the parasite growth was inhibited 98% by GCV at 5 mg/ml and 96% by GCV at 1 mg/ml on Day 9 of culture (Fig. 2a). Hygro-transfected parasites were not affected by GCV in doses up to 5 mg/ml (Fig. 2b). GCV doses of 10 mg/ml or higher were inhibitory to mouse 3T3 cell growth. The HyTK trypomastigotes cultured in the presence of GCV eventually grew to a density of approximately 1 × 106/ml after 25 days (as opposed to 5 days for the HyTK parasites grown in the absence of GCV). These parasites were transferred to fresh 3T3 monolayers and tested again for sensitivity to GCV to see if a resistant subpopulation had emerged. Even after

a second exposure to 5 mg/ml GCV these parasites were equally sensitive (data not shown). Sensitivity of transfected parasites to other nucleoside analogues. Parasites expressing HyTK were compared to Hygro-transfected parasites for sensitivity to acyclovir, pencyclovir, and sorivudine, which are nucleoside analogues known to be phosphorylated by HSVTK. At doses determined to be just below toxic to control parasites, HyTK-expressing epimastigotes were only inhibited by approximately 25%; these concentrations were acyclovir, 250 mg/ml; pencyclovir, 100 mg/ml; and sorivudine, 500 mg/ml. Growth of trypomastigotes/amastigotes expressing HyTK was inhibited less than 25% by the drugs at the following concentrations: acyclovir, 5 mg/ml; pencyclovir, 10 mg/ ml; and sorivudine, 25 mg/ml. Thymidine kinase activity in transfected T. cruzi. Since the HyTK-transfected epimastigotes were not sensitive to GCV, these cells were tested for the presence of TK enzyme activity using [3H]thymidine as substrate. Lysates of epimastigotes expressing HyTK had somewhat greater TK activity than trypomastigotes in three clones that were tested (Fig. 3). Lysates of parasites that were untransfected or transfected with Hygro had TK activity at about background levels. One HyTK clone (A2), which had greater sensitivity to GCV (IC95, 0.2 mg/ml) than other clones (A4 and A5; IC95, 1.0 mg/ml), was found to have higher TK activity than the less sensitive clones. This suggested that greater expression of the TK gene resulted in greater sensitivity to GCV. A TK assay using [3H]GCV as substrate in place of [3H]thymidine demonstrated findings similar to those shown in Fig. 3 (data not shown). Uptake of ganciclovir or thymidine by parasites. To investigate why HyTk-transfected epimastigotes were resistant to GCV, the parasites (CL strain) were tested for the ability to take up radiolabeled GCV into the intracellular space. The amastigote form of the parasite had about 8–10 times as much intracellular [3H]GCV as was found in epimastigotes or the trypomastigotes in a 4-hr incubation (Fig. 4a). This suggested that the resistance of epimastigotes was

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FIG. 2. Growth of HyTK (a)- or Hygro (b)-transfected T. cruzi (CL strain) in the presence of GCV. Trypomastigotes were inoculated onto 3T3 fibroblasts on Day 0 and cultured with varying concentrations of GCV. Parasites released into the media following intracellular replications were quantitated on Days 7, 8, and 9. GCV had no effect on 3T3 cell growth at the doses of 5 mg/ml and less. Experiments were repeated with both strains of parasites, showing similar results.

due to low uptake of the drug. In addition, intracellular [3H]GCV was about 3 times greater in amastigotes expressing HyTK than in amastigotes expressing Hygro (Fig. 4a), probably due to the fact that phosphorylated nucleotides are trapped in the intracellular space (Plunkett and Cohen 1977). The uptake of [3H]thymidine by parasites in different life-cycle stages was tested in a similar manner. Over three times as much [3H]thymidine as [3H]GCV was taken up by amastigotes. In addition, epimastigotes had relatively high

uptake of [3H]thymidine (60% of that seen by amastigotes), unlike the uptake of [3H]GCV by epimastigotes (10% of that seen by amastigotes) (Fig. 4b). GCV treatment of mice infected with HyTKtransfected T. cruzi. Parasitemia with HyTKexpressing Tulahuen parasites was significantly lower on Days 7 and 9 postinfection in mice treated with GCV (Fig. 5). The differences were not statistically significant at Days 11 and 14. All five mice in the saline-treated group died on Day 14 or 15 postinfection. Deaths in the GCV-

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FIG. 3. Thymidine kinase assay on T. cruzi clones (CL strain) transfected with HyTK, Hygro, or no DNA. Lysates of epimastigotes (‘‘Epis,’’ the insect stage) and of trypomastigotes/amastigotes (‘‘T/As,’’ the mammalian stages) were normalized to protein concentration and tested for the ability to convert thymidine to thymidine monophosphate. Nonspecific binding of the reaction buffer containing [3H]thymidine to the filters (4406 counts per minute, CPM) was subtracted from each sample. Similar results were observed in two repetitions of the experiment.

treated group occurred on Days 20, 21, and 29; one mouse survived beyond 30 days. After 2 weeks of injections, mice treated with GCV had elevated leukocyte counts on peripheral blood smear, similar to the mice treated with saline, indicating that the GCV did not have a significant myelosuppressive effect. In a control experiment using Hygro-expressing Tulahuen parasites, parasitemia was not significantly different between GCV-treated (n 4 5) and salinetreated mice (n 4 5) at any time points (data not shown). Deaths occurred between Days 16 and 21 in both groups, except for one survivor beyond 30 days in the saline-treated group. Bloodstream parasites were recovered from mice infected with the HyTK transfectants after the completion of treatment with GCV and tested in vitro for sensitivity to GCV. The recovered parasites were found to have the same level of sensitivity to the drug as parasites not passaged through mice, indicating that the failure to eradicate parasites was not a result of mutation or deletion of the TK gene. Mice infected with the CL HyTK clone A2 (shown to be highly sensitive to GCV in vitro) developed low parasitemia and minimal tissue pathology even with very large inocula of parasites (108 trypomastigotes) without administration of GCV to the mice. Therefore, this clone was not employed for more extensive in vivo studies.

DISCUSSION Two objectives were desired in the expression of the suicide gene HSV-TK in T. cruzi. The first was to optimize stability of the transfected gene so that negative selection would not result in rapid development of resistant revertants. To this end, three strategies were employed. The HSV-TK gene was transfected into the parasite as a fusion gene with hygromycin phosphotransferase. This assured integration of both the positive and the negative selection markers. In constructs with separate genes arranged in tandem, some of the transfectants integrated only the positive selection marker (La Flamme et al. 1996). Next, the parasites were electroporated with linearized DNA, resulting in integration into the genome. In previous work we have shown that integrated DNA can be stably expressed in T. cruzi for months out of positive drug pressure (La Flamme et al. 1996). It was not possible to apply positive drug selection in tissue culture or in vivo since hygromycin levels of >250 mg/ml were required, which are toxic to cells and mice. Lastly, the parasites were cloned and the clones were characterized by Southern analysis to assure homogeneity. The in vitro experiments demonstrated that GCV suppressed outgrowth of parasites (98%) and that the cells surviving negative selection maintained the same level of susceptibility to GCV even after

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FIG. 4. Intracellular uptake of [3H]GCV (a) or [3H]thymidine (b) by different life-cycle stages of T. cruzi (CL strain). Parasites containing the HyTK gene or the Hygro gene were incubated with radiolabeled drug for 4 hr, washed, and directly pipetted into scintillation vials for quantitation of counts per minute (CPM). Similar results were observed in two repetitions of the experiments.

two further rounds (3-week in vitro cultures) of negative selection. Although the mutation rate cannot be precisely calculated from these data, it is probably less than 10−7 occurrences per cell generation. A single revertant developing early in the first passage (from an initial inoculum of 2 × 106 cells) would dominate the culture during the second 3-week passage, assuming that it established a normal growth rate of one generation per 24 hr, and this was not observed to occur. Parasites recovered from GCV-treated mice were also observed to retain their sensitivity to GCV in vitro. The stability of the TK gene in T. cruzi contrasts with the instability of the gene in TK-expressing T. brucei. Valdes et al. (1996) observed mutation rates of 10−6 per

cell generation in T. brucei. They speculated that T. brucei has a high overall mutation rate due to evolutionary pressure for diversity among the variable surface glycoprotein genes. The overall mutation rate of T. cruzi is not known, but from a practical standpoint, the development of GCV-resistant mutants was not problematic in our experiments. The second objective was to generate parasites that were fully sensitive to GCV for complete eradication either in vitro or in vivo. This goal was attained in part by demonstrating 98% growth inhibition of the mammalian form of T. cruzi with GCV in vitro. The same degree of inhibition, unfortunately, was not attained in vivo, where inhibition was maximum at 57% on

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FIG. 5. Parasitemia in mice infected with T. cruzi (Tulahuen strain) expressing HyTK and treated with twice-daily intraperitoneal injections of GCV (50 mg/kg/day) or saline. The data are not plotted at the point when deaths have occurred in a group. *Indicates statistically significant differences between the two groups (p < 0.02).

Day 11 of GCV treatment (Fig. 5). The most likely explanation for the incomplete inhibition in mice is difficulty delivering and maintaining sufficiently high drug concentrations in the animals. Dosages higher than the 50 mg/kg/day used in these experiments are toxic to mice (Buhles 1991). In addition, GCV has a relatively short plasma half-life (2.9 hr) and is rapidly cleared from tissues; thus, inhibitory levels of drug may be only transiently attained (Capparelli et al. 1991). Another possible explanation of why HyTK-transfected parasites are not eliminated with GCV in mice is that the TK gene may not expressed to the same level in vivo as it is in vitro. However, against this possibility is the finding that the b-galactosidase gene targeted to the same genomic site in T. cruzi is efficiently expressed in vivo (unpublished data). A parasite producing higher levels of TK might be expected to be more sensitive to GCV. This, in fact, was seen with the highly GCVsensitive HyTK clone A2 that was found to have the highest level of thymidine kinase activity in cell lysates (Fig. 3). This particular clone had an aberrant integration event with the HyTK gene recombining outside of the Cal A2 locus, perhaps in a region with greater transcriptional activity of the gene. The clone was not useful in mouse experiments, however, because it was

impaired in its ability to produce parasitemia or tissue pathology. The implication of this finding that higher gene expression resulted in greater GCV sensitivity is that a vector yielding more expression of HyTK may lead to a parasite with sufficient sensitivity to GCV to allow for eradication in vivo. Although the HyTK-bearing parasites were sensitive to GCV in vitro in the mammalian life-cycle stages, they were not sensitive to GCV in the insect stage, epimastigotes. The resistance in epimastigotes was not due to insufficient production of thymidine kinase, as TK levels were at least as high in epimastigotes as they were in trypomastigotes/amastigotes (Fig. 3). Rather, the resistance appeared to be due to the low intracellular levels of GCV obtained in epimastigotes (Fig. 4). The low intracellular levels of nucleoside detected in the trypomastigote form (Fig. 4) were probably because this is the nonreplicative form of the parasite, which may not take up nucleosides since it is not synthesizing new DNA. The intracellular accumulation of nucleosides is a function of surface transporters (Finley et al. 1988) although accumulated levels could theoretically be affected by efflux pumps. T. cruzi has at least two nucleoside transport processes to provide substrate for the salvage pathways of nucleotide synthesis (Finley et al.

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1988). These transporters have broad specificities compared to vertebrate nucleoside transporters and do not sharply discriminate between purines and pyrimidines (Finley et al. 1988). Despite these nonspecificities, the epimastigotes took up little of the purine analogue, GCV. The uptake experiments were conducted in the same growth medium (Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum) for the different life-cycle forms, so the differences observed were not due to variations in competing substrates in the medium. It is possible that new nucleoside transport processes are expressed in amastigotes when the parasite is introduced into the environment of the mammalian host from the invertebrate host. The transporter processes present in amastigotes appear capable of efficiently transporting GCV, whereas the processes present in epimastigotes are not. The marked difference between epimastigotes and amastigotes in the transport of GCV was not observed in the transport of the natural nucleoside, thymidine. This indicates the presence of a functional nucleoside transporter system in epimastigotes, as has been previously shown (Berens et al. 1981; Hammond and Gutteridge 1984). ACKNOWLEDGMENTS The authors thank Steven Lupton for providing the HyTK gene, John Swindle and Janet Ajioka for providing unpublished sequence data and advice on constructing the expression plasmid, Anne LaFlamme for advice, Lynn Barrett for technical assistance, and William Scott for providing us with sorivudine. This work was supported by American Heart Association Grant-in-Aid 95006700 and National Institutes of Health Grant AI01258.

REFERENCES Ajioka, J., and Swindle, J. 1992. The calmodulin–ubiquitin associated genes of Trypanosoma cruzi: Their identification and transcription. Molecular and Biochemical Parasitology 57, 127–136. Berens, R. L., Marr, J. J., LaFon, S. W., and Nelson, D. J. 1981. Purine metabolism in Trypanosoma cruzi. Molecular and Biochemical Parasitology 3, 187–196. Brinster, R. L., Chen, H. Y., Trumbauer, M., Senear, A. W., Warren, R., and Palmiter, R. D. 1981. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223–231. Buckner, F. S., Verlinde, C. L. M. J., La Flamme, A. C.,

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