Inducible Expression of Suicide Genes in Leishmania donovani Amastigotes*

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. TheLeishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani3′-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitroand in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.

The Leishmania species are parasitic protozoa responsible for various clinical forms of leishmaniasis ranging from selfcuring skin ulcers to an often fatal visceral disease. The Leishmania parasite oscillates between two distinct host-specific developmental stages in the course of their life cycle. Flagellated promastigotes are found in the gut lumen of the sandfly vector, while nonmotile amastigotes exist intracellularly in the vertebrate host macrophages (1).
The Leishmania donovani A2 gene family of proteins was shown to be amastigote stage-specific (2) and essential for survival of this parasitic protozoan in its mammalian host (3). Previously, it was demonstrated that the A2 gene regulatory system could be used as a model to study the regulation of gene expression in Leishmania cells (4). The developmental expression of A2 gene transcripts and protein products can be induced in vitro in cultured promastigotes by a combination of pH and temperature shifts, conditions associated with the passage of the parasite from the insect vector to the phagolysosomal en-vironment within the mammalian macrophage (2,5). A2 gene expression is post-transcriptionally regulated, and selectable marker transcripts carrying the A2 mRNA 3Ј-untranslated region display amastigote-specific expression in transfected Leishmania cells (4).
In this study, we tested whether the A2 regulatory sequences could be used to developmentally express suicide genes toward developing an attenuated L. donovani strain. The suicide genes tested were the herpes simplex virus thymidine kinase gene (hsv-tk) previously used by LeBowitz et al. (6) as a negative selectable marker in Leishmania major and a truncated version of the L. donovani 3Ј-nucleotidase/nuclease (3ЈNT/Nu). The native L. donovani 3ЈNT/Nu is an externally oriented surface membrane enzyme capable of hydrolyzing 3Јnucleotides and nucleic acids (7)(8)(9). Developmental expression from both episomal and integrated A2-toxic gene chimeric constructs was tested in vitro in differentiated amastigotes. We demonstrate that the pattern of expression of these toxic genes under the control of A2 gene sequences was differential, although not completely amastigote-specific. The truncated 3ЈNT/Nu expression affected growth of amastigotes, and TK 1 expression induced sensitivity of amastigotes to the anti-herpes drug, ganciclovir.
Axenic amastigotes used in Western and Northern blot experiments were obtained by transferring late log-phase promastigotes to medium at 37°C, pH 5.5. Cells were used 12-18 h after the transfer.

Nucleic Acids Preparations and Analyses
Total RNA was extracted from promastigotes and axenic amastigotes by the phenol-chloroform-guanidium isothiocyanate method using TRIzol (Life Technologies, Inc.). DNA samples were prepared from promastigotes using the "mini-prep" procedure described by Medina-Acosta and Cross (13). Southern and Northern blot hybridizations were carried out in 1 M NaCl, 1% SDS, and 10% dextran sulfate for 18 h at 65°C. DNA probes were prepared from agarose gel-purified fragments * This work was supported in part by a grant from the Medical Research Council of Canada (MRC). Research at the Institute of Parasitology is partially funded by the Fonds pour la formation de chercheurs et l'aide à la recherche du Québec (FCAR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18

Plasmid Constructs
Schematic representations of plasmids used in this study are depicted in Fig. 1.
NEOPT and pKSneo-The NEOPT plasmid was designed to allow the differential expression of the neo gene in amastigotes (4). The construct carries the Pro (P) element, which represents the A2/A2rel intergenic region and the 5Ј end of the A2rel gene coding sequence, a synthetic 92-base pair trans-splicing element (pyt), the neomycin resistance gene, and the Tail (T) element, which represents the 3Ј-untranslated region of A2. The construction of the pKSneo plasmid was described in Zhang et al. (5). In distinction with the NEOPT plasmid, the neo gene in pKSneo is inserted upstream from the Pro element. The Pro element provides a polyadenylation site for the neo gene, whereas trans-splicing of neo at the 5Ј end is controlled by a pyt element. Thus, in pKSneo, the neo gene mRNA is constitutively expressed in both promastigotes and amastigotes.
pKSneo-3ЈNT-A truncated version of the 3Ј-nucleotidase/nuclease gene (3Јnt/nu) was amplified by a polymerase chain reaction (PCR) from plasmid Cl-2 (14), which contains a 4.5-kb fragment carrying the 3Јnt/nu coding sequence (1.4 kb). The signal peptide and the terminal anchor sequences were not included in the PCR product. Hence, the sense oligonucleotide (5Ј-gactccatgg atg acgctcctcagtactgtcgca-3Ј) created an initiation codon, whereas the antisense oligonucleotide (5Ј-gacttgatca cta cacggcgacaatcgccgtcac-3Ј) created a stop codon. The PCR product (1.1 kb) was first inserted into the pCRII cloning vector (T/A cloning system, Invitrogen). Subsequently, the SpeI and XbaI restriction sites from the pCRII vector restriction cassette were used to subclone it into the SpeI site of the pKSneo vector.

Constructs for Targeting tk into the A2 Locus
The neo gene was amplified from the pALTneo vector along with an ␣-tubulin intergenic region from L. enriettii, which provides a transsplicing acceptor site and a polyadenylation acceptor site (15). The following primers were used: sense, 5Ј-gg agatct acctttctctcttccgac-3Ј (bases in bold represent a BglII site); antisense, 5Ј-tacgactcactatagggc-3Ј. The neo-intergenic region (Neo-IG) product (1.8 kb) was subcloned BglII/BamHI in the BamHI site of vector pGEM-11zf (Promega). The Pro and Tail elements were inserted on either side of the neo-intergenic or neo-intergenic-tk sequences to permit targeting by homologous recombination (Fig. 1B). For the pGEMneo construct, the Tail was subcloned directionally at the XbaI and HindIII restriction sites. For the pGEMneo-TK plasmid, the tk gene was first inserted upstream from the Tail element on a KS pBluescript (Stratagene, LaJolla, CA) (KSTail plasmid). The 1.3-kb tk coding region was amplified from the pFG5 vector which contains the tk gene (16). The following primers were used: sense, 5Ј-gg tctaga cgcttaacagcgtcaaca-3Ј; antisense, 5Ј-gg tctaga ggtctcggtggggtatcg-3Ј (bases in bold represent a XbaI site). The PCR product (1.3 kb) was first inserted in the PCRII cloning vector and then subcloned with the XbaI restriction sites from the PCRII vector in front of the Tail element. The plasmid-created KS/TK-Tail was digested BglII/HindIII (the BglII site is located upstream from the tk initiation codon), and the TK-Tail element was subcloned BamHI/HindIII at the 3Ј end of the pGEM construct.

Transfections
Transfections and selection of clones were performed as described previously (4). Uncloned populations of recombinant promastigotes were used for experiments with episomal copies of the truncated 3Јnt/nu and neo genes. Recombinant promastigotes carrying neo and tk genes targeted into the chromosomal A2 gene cluster were selected with minimal doses of G418 (10 g/ml), and clonal populations were selected by limiting dilutions.
The 3Ј-nucleotidase activities of the native 3ЈNT/Nu and the truncated 3ЈNT/Nu were visualized in the SDS-PAGE gels by in situ staining using 3Ј-AMP as a substrate and malachite green as the staining agent (18). The 3ЈNT/Nu proteins were detected in Western blot analyses using a rabbit anti-3ЈNT/Nu peptide serum and a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad) (as described in Ref. 14). Bound secondary antibodies were detected using the ECL Western blotting detection system (Amersham Pharmacia Biotech). In Fig. 4B, the secondary antibody used was a biotinylated goat anti-rabbit IgG, and bound antibodies were detected with streptavidin-conjugated horseradish peroxidase (Amersham Pharmacia Biotech) and 3,3Ј-diaminobenzidine (Sigma) in a 0.03% H 2 O 2 solution.

Infection of Macrophages in Culture
Mouse bone marrow cell preparations and infections with L. donovani cells were performed as described previously (19). Late log-phase promastigotes were used to infect cultures of macrophages at a ratio of 20 parasites/macrophage for 24 h at 37°C, after which nonphagocytosed parasites were washed out by low speed centrifugation. Infected cells were incubated at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum, and intracellular infection levels were monitored daily by Giemsa staining.

Infections in Mice
Infections were performed as described in Zhang  burdens are expressed as Leishman-Donovan units (number of amastigotes/1000 cell nuclei ϫ liver weight (g)) (20).

RESULTS
Neomycin (G418) Resistance of neo Transfectants-As a first step toward assessing the A2 gene regulatory sequences for the developmental expression of suicide genes in L. donovani, the effect of gene copy number versus basal protein expression levels was examined in promastigotes. Promastigotes were transfected with the neo gene, used both as a selectable marker and as a reporter gene, present in single copy (targeted) or in a multicopy plasmid. The effect of the differential expression of NEO was monitored indirectly by determining the growth of promastigotes cultured in the presence of various concentrations of the drug G418. The resistance to G418 of L. donovani promastigotes transfected with the NEOPT plasmid (episomal) and of a targeted clone (R2) carrying the neo gene into the A2 genomic locus are shown in Fig. 2. R2 and NEOPT are described in Charest et al. (4). Wild-type 1S2D promastigotes were very sensitive to G418 (Fig. 2, lower panel); the EC 50 was estimated to be 4 g/ml.
R2 promastigotes multiplied slowly in medium containing up to 100 g/ml G418; however, growth was inhibited at Ն150 g/ml. In contrast, promastigotes carrying the neo gene in episomal vectors readily multiplied in culture medium containing up to 500 g/ml. Thus, episomally transfected promastigotes, which contain plasmid constructs in multiple concatenated copies (21), were more resistant than targeted promastigotes. Nevertheless, an increase in drug concentration in the medium correlated with a decrease in growth for both targeted and plasmid-carrying promastigotes.
Because drug pressure was shown to increase the plasmid copy number in transfected Leishmania cells (21,22), the concentration of G418 in the culture medium was used to modulate the plasmid copy number, and therefore the expression of episomal toxic genes, for the experiments described below.
Differential Expression of the Truncated 3ЈNT/Nu from an Episomal Construct-The A2 gene regulatory sequences were tested for the expression of toxic gene products from an episomal construct (refer to Fig. 1 for plasmid constructs). As a potential toxic gene, a truncated version of the L. donovani 3ЈNT/Nu was used in which the N-terminal signal and the C-terminal anchor sequences were deleted from the 3ЈNT/Nu sequence (pKSneo-3ЈNT). The assumption was that the lack of signal sequence would prevent the targeting of the protein to the endoplasmic reticulum, which is required for its expression to the surface of the cell. 3ЈNT/Nu activity would then be expressed in the cytoplasm and potentially be toxic to Leishmania.
Expression of the truncated 3ЈNT/Nu RNA in pKSneo-3ЈNT transfected cells grown in various G418 concentrations (ranging from 0 to 400 g/ml) is shown in Fig. 3A. The size of the 3ЈNT/Nu transcript (2.5 kb) derived from the pKSneo-3ЈNT plasmid was consistent with RNA processing at an artificial trans-splicing acceptor site located at the 3Ј end of the Tail element (pyt). As expected for episomal copies, the truncated 3ЈNT/Nu mRNA was differentially expressed in axenic amastigotes and was increased in proportion to the G418 concentration. Densitometric analyses revealed that truncated 3ЈNT/Nu transcripts accumulated to an 8-fold higher level in axenic amastigotes grown in 400 g/ml than in 25 g/ml G418. Further, when grown at identical concentrations of G418, axenic amastigote expression of truncated 3ЈNT/Nu mRNA was on average 5-fold higher than in promastigotes.
Protein accumulation paralleled transcript expression in the transfected cells (data not shown). The endogenous 3ЈNT/Nu has an apparent molecular mass of 43 kDa whereas that of the truncated 3ЈNT/Nu is 38 kDa. In both promastigotes and axenic amastigotes there was a dose-dependent expression of the truncated 38-kDa protein associated with concentrations of G418 in the culture medium. Also in agreement with the Northern blot results of above, more truncated 38-kDa 3ЈNT/Nu was detected in axenic amastigotes than in promastigotes.
Activity of the truncated 3ЈNT/Nu was determined in promastigotes and axenic amastigotes by staining renatured proteins for 3Ј-nucleotidase activity using 3Ј-AMP as substrate. The levels of endogenous and truncated 3ЈNT/Nu proteins expressed in promastigotes and axenic amastigotes are shown in the left panel of Fig. 3B, whereas the right panel shows the activity stain for 3Ј-nucleotidase activity. The Western blot clearly demonstrated the overexpression of the truncated 3ЈNT/Nu in the amastigote stage compared with the promastigote stage. Enzyme activity of the truncated 3ЈNT/Nu was detected in the transfected axenic amastigotes but was not detectable in promastigotes. Clearly, the specific activity of the truncated 3ЈNT/Nu was remarkably low compared with that of the endogenous enzyme, probably because it was not targeted to the endoplasmic reticulum where its normal processing occurs (i.e. glycosylation).
Induction of the Truncated 3ЈNT/Nu Leads to Reduced Amastigote Viability-To test whether differential expression of the truncated 3ЈNT/Nu was toxic to the Leishmania cells, pKSneo-3ЈNT plasmid transfected promastigotes and axenic amastigotes were grown in their media supplemented with various concentrations of G418. Axenic amastigote growth was inhibited in proportion to the increasing drug concentrations in the medium (Fig. 4A, lower panel). This corresponded to the up-regulation of expression of the truncated enzyme in these cells. At 400 g/ml, axenic amastigote growth was completely inhibited. In contrast, promastigotes transfected with the pK-Sneo-3ЈNT plasmid were not affected by the truncated 3ЈNT/Nu enzyme (Fig. 4A, upper panel). In preliminary experiments, pKSneo transfected cells showed no diminution of growth in G418 concentrations ranging from 25 to 400 g/ml. Further, there was no significant difference in cell growth between control pKSneo promastigotes cultured at 25 g/ml G418 and pKSneo-3ЈNT promastigotes grown at G418 concentrations ranging from 0 to 400 g/ml (Fig. 4A). These results strongly suggested that the accumulation of the truncated 3ЈNT/Nu, despite its apparent low specific activity, was toxic to Leishmania axenic amastigotes.
We examined in parallel the effect of the truncated 3ЈNT/Nu on amastigote survival in macrophages. Mouse bone marrow macrophages were infected for 24 h with transfected promastigotes previously cultured in G418 concentrations ranging from 0 to 400 g/ml. Proliferation of the resulting amastigote population within macrophages was monitored over a 5-day period. There was no significant difference in the ability of pKSneo-and pKSneo-3ЈNT transfected promastigotes to infect macrophages (72% of macrophages were infected versus 65-82%, respectively). However, over the 5-day incubation period, there was a significant difference in growth among these transfected amastigotes (Fig. 4B). The growth of truncated 3ЈNT/Nuexpressing amastigotes was significantly reduced when promastigotes were previously cultured at G418 concentrations Ն25 g/ml, compared with the drug-free control and pKSneo transfected amastigotes. These results are in agreement with the in vitro cell growth studies (Fig. 4A), which showed that cell growth was hindered by increasing levels of the truncated 3ЈNT/Nu expression in axenic amastigotes. Further, macrophage infection levels were also reduced for amastigotes expressing the truncated 3ЈNT/Nu. At day 5, 48% of macrophages were infected with pKSneo-3ЈNT amastigotes previously grown as promastigotes in 25 g/ml G418, whereas virtually all macrophages were infected with pKSneo amastigotes and pK-Sneo-3ЈNT amastigotes from promastigotes grown in G418-free medium.
The effect of pKSneo-3ЈNT transfection on the ability of Leishmania cells to establish an in vivo infection in mice was also determined. Promastigotes used to initiate the mouse infections were cultured in G418 concentrations ranging from 0 to 400 g/ml. Four weeks post-infection, mice were sacrificed, and parasite burdens were determined by counting the number of amastigotes in liver impressions. Values are expressed as Leishman-Donovan units in Fig. 4C. No significant difference in the parasite burden was observed among mice infected with wild-type promastigotes, promastigotes transfected with the pKSneo control plasmid, or promastigotes transfected with the pKSneo-3ЈNT plasmid and cultured without G418. However, significant differences in amastigote burdens were observed in mice infected with transfected promastigotes previously cultured in 100, 200, and 400 g/ml G418. These results are consistent with those obtained in the in vitro macrophage infection experiments above showing that expression of plasmidderived 3ЈNT/Nu decreased either the growth or viability of amastigotes.
Targeting of a Suicide Gene into the A2 Coding Locus-In subsequent experiments, the efficacy of the developmental expression of a gene with toxic potential was tested from a construct targeted into the A2 locus. Because the truncated 3ЈNT/Nu did not appear to affect the viability of transfected cells carrying low episomal gene copy number, presumably because of its low specific intracellular activity, the herpes FIG. 3. A, Northern blot analyses showing differential expression of the truncated 3ЈNT/Nu and increased accumulation through drug pressure. pKSneo-3ЈNT transfected promastigotes were cultured in media at different concentrations of G418. Total RNA was extracted from log-phase promastigotes. Amastigote RNA was obtained by switching log-phase promastigotes to amastigote culture conditions and extracting RNA 12 h later. Membranes were hybridized with a labeled 1.1-kb sequence from the 3Јnt/nu gene. Equal loading was verified by staining the denatured RNA with ethidium bromide in the agarose gel prior to Northern blot (shown on the bottom panel). B, truncated 3ЈNT/Nu protein expression and activity. 2.5 g of total protein from promastigotes (Pro) grown in 400 g/ml G418 and amastigotes (Ama), obtained as above, were subjected to SDS-PAGE, Western blot analysis (left panel), and an enzyme activity assay (right panel). 3ЈNT activities were visualized in these gels by in situ staining using 3Ј-AMP as a substrate. The endogenous 3ЈNT/Nu is present at 43 kDa while the truncated 3ЈNT/Nu migrates at 38 kDa. Note: truncated 3Ј-nucleotidase activity was only observed in amastigotes. simplex virus thymidine kinase gene (tk) was used for gene targeting experiments. The expression of tk per se does not affect Leishmania cell growth (6) but renders such transfectants sensitive to nucleoside analogues (e.g. anti-herpes drug ganciclovir).
The strategy used to target both the neo and tk genes into the A2 locus by homologous recombination is outlined in Fig. 5A. Results of the Southern blot analyses confirmed the correct insertion of the neo and tk genes into the A2 locus (Fig. 5B) and the localization of the neo, intergenic, and tk sequences upstream from the 3Ј-untranslated region in the genome.
Northern blots of the TK-targeted clone (R.tk) and of the control clone (R.ctl), which only contains the neo gene, demonstrated that induction of TK transcripts was up-regulated in the axenic amastigotes (Fig. 5C). The TK transcript length (2.8 kb) was consistent with trans-splicing occurring at the 3Ј-end of the A2 Tail sequence, and the ␣-tubulin intergenic region provided a polyadenylation site. As expected, the NEO transcript is not differentially regulated (cf. Fig. 1B). Its size (1.4 kb) corresponds to processing occurring in the Pro and the ␣-tubulin intergenic regions.
The cell growth of promastigotes and axenic amastigotes cultured in media containing different concentrations of ganciclovir were analyzed (Fig. 6A). L. donovani lines were inoculated into media containing the indicated amounts of ganciclovir and allowed to proliferate until control cultures lacking ganciclovir had reached late log phase (1 ϫ 10 7 cells/ml) at which time the EC 50 (defined as the concentration of ganciclovir that reduced the cell numbers by 50%) was calculated (23). R.tk promastigotes and axenic amastigotes were differentially sensitive to ganciclovir, with estimated EC 50 of 25 M and 4.5 M, respectively. In contrast, the growth of control (R.ctl) promastigotes and axenic amastigotes was unaffected by ganciclovir (Fig. 6A). In vitro infection studies using bone marrow macrophages were performed with the R.tk clones (Fig. 6B). Mouse bone marrow macrophages were infected for 24 h with FIG. 4. A, effect of the truncated 3ЈNT/Nu on cell growth is shown. pKSneo-3ЈNT transfectants were grown at different G418 concentrations. Optical densities at 600 nm were measured over time, as indicated. WT, wild-type cells; pKSneo, cells transfected with the control vector and grown in 25 g/ml G418. B, growth of pKSneo-3ЈNT transfected Leishmania amastigotes in macrophages in vitro. Promastigotes transfected with the pKSneo control vector were grown in 25 g/ml G418; pKSneo-3ЈNT were grown in concentrations of G418 ranging from 0 to 400 g/ml G418. Late log-phase promastigotes were used to infect macrophages. C, liver parasite burdens of infected mice are shown. Mice were infected with wild-type or transfected Leishmania grown at various G418 concentrations (0, 100, 200, and 400 g/ml). pKSneo control tranfectants were grown in 25 g/ml G418. Mice were injected with promastigotes via the tail vein (1.5 ϫ 10 8 promastigotes/mouse; n, 3/group). Four weeks post-infection, the parasite burden was determined by counting amastigotes from liver impressions. Liver parasite burdens are expressed as Leishman-Donovan units calculated as the number of amastigotes/1000-macrophage nuclei ϫ liver weight (g). The means per group are shown.
R.tk promastigotes. Infected macrophages were incubated in medium at seven different ganciclovir concentrations ranging from 0 to 1 mM. Infection of macrophages and growth of amastigotes within macrophages were monitored over a 5-day period. At day 0, there was on average for all ganciclovir concentrations a ratio of 7 amastigotes to 1 infected macrophage. At day 5 this ratio was 8:1 for R.tk in drug-free medium and in 1 M ganciclovir. At 10 M ganciclovir and higher concentrations, amastigote growth was dramatically affected with infection levels at a ratio of 2:1 (Fig. 6B).
These data show that promastigotes that carried the tk gene in the A2 genomic locus were relatively unaffected in culture by the presence of ganciclovir until high concentrations of the drug were reached. In contrast, axenically cultured amastigtk gene; Neo, 0.8-kb PCR fragment amplified from the pALTneo vector. The bottom panels show the total RNA present in each lane.  6. Drug sensitivity of TK transfectants. A, R.tk and R.ctl clones were inoculated as promastigotes (pro) and amastigotes (ama) into media at a density of 2 ϫ 10 5 cells/ml. Ganciclovir was added to the media at the indicated concentrations. Cells were allowed to grow until control cultures lacking ganciclovir had reached late log phase (approximately 1 ϫ 10 7 cells/ml). The EC 50 was defined as the concentration that reduced the cell numbers by 50%, in comparison to late log-phase drug-free control cultures. B, infection and growth of R.tk amastigotes in macrophages in vitro. Late log-phase promastigotes were used to infect macrophages, which were then incubated with the addition of ganciclovir at various concentrations (0, 1, 10, 100, 500, and 1000 M). Amastigote infection levels in macrophages were evaluated daily. At day 0, data were collected before the addition of ganciclovir. The control corresponds to clone R.ctl. otes or amastigotes within macrophages displayed a dose-dependent sensitivity to the drug. These results demonstrated the feasibility of targeting negative selectable markers into the A2 locus for their developmental up-regulation in amastigotes. DISCUSSION In this study, we have demonstrated the feasibility of using the A2 gene regulatory system to generate L. donovani recombinant lines in which attenuation is developmentally regulated during the promastigote to amastigote cytodifferentiation. The pattern of expression of toxic genes under the control of A2 gene sequences was differential, although not completely amastigote-specific. Whereas promastigotes were generally unaffected by low expression of the suicide genes, the growth of axenic amastigotes was hampered. Similarly, amastigote growth within host macrophages could be completely inhibited. We also showed by gene replacement that the A2 locus can control the expression of negative selection genes.
Two different suicide gene systems carried on episomal and targeting constructs were tested. Both suicide genes were placed under the control of the A2 untranslated regions in recombinant L. donovani cells. In a first series of experiments, a truncated version of the 3Ј-nucleotidase/nuclease, an endogenous L.donovani enzyme, was inserted between two A2 noncoding sequences to be developmentally expressed in amastigotes. The 3Ј-nucleotidase/nuclease is an externally oriented surface membrane glycoprotein responsible for the hydrolysis of 3Ј nucleotides and single strand nucleic acids (24). The truncated 3ЈNT/Nu used in our constructs, which lacked both the signal peptide and the membrane anchoring sequences, was expressed in the cytosol (data not shown), which led to reduced amastigote cell viability. The truncated 3ЈNT/Nu was not post-translationally modified by glycosylation because it was not targeted to the endoplasmic reticulum, where its normal processing occurs. Campbell et al. (25) have demonstrated that the glycosylation of the native enzyme plays an important role in regulating its specific activity. This is consistent with the observation that the 3ЈNT/Nu expressed in Escherichia coli had a 50-fold lower activity than the native enzyme of Leishmania. 2 To modulate the expression of the truncated 3Јnt/nu gene in amastigotes as well as in promastigotes, we took advantage of the increase in plasmid copy number resulting from the G418 selective pressure. Despite its low activity, high levels of truncated 3ЈNT/Nu expression resulted in a reduced amastigote viability. Decreased cell growth of axenic amastigotes correlated with a decreased replication of amastigotes in macrophages and mice. In the absence of G418, pressure-transfected DNA is maintained in Leishmania cells for a relatively long period of time (26). However, in vivo, transfectants would be expected to eventually begin to lose their plasmids. Thus, a generation of stable attenuated cell lines requires the chromosomal integration of the suicide gene. Clearly, the low specific activity of the truncated 3ЈNT/Nu that was observed, even at high copy number, was not suitable for the generation of attenuated lines.
To directly assess the feasibility of differentially inducing in amastigotes the expression of suicide genes when targeted into the chromosomal A2 locus, we used the tk/ganciclovir suicide gene system. LeBowitz et al. (6) and Muyombwe et al. (27,28) describe the expression of the hsv-tk gene on episomal vectors in L. major for the creation of ganciclovir-sensitive strains. We showed that the hsv-tk gene under the control of A2 regulatory sequences is differentially expressed in amastigotes. As expected, the expression of the herpes virus tk gene in transfected promastigotes results in sensitivity to nucleoside analogues, such as the anti-herpes drug ganciclovir. However, the EC 50 calculated for axenic amastigotes was 5 times lower than for promastigotes, correlating with an up-regulation of the tk gene expression in amastigotes.
The major advantage of the A2 gene regulatory system to establish attenuated cell lines is that attenuation can be developmentally regulated. Such attenuated L. donovani cell lines should prove useful in examining certain aspects of the immune response during infection, under conditions where the infection is not lethal. Further, it may hold potential for the development of an attenuated vaccine.