Modulation of myosin A expression by a newly
established tetracycline repressor-based inducible system in Toxoplasma gondii
Markus Meissner, Susan Brecht, Hermann Bujard and Dominique Soldati*
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69102 Heidelberg, Germany
Received June 27, 2001; Revised and Accepted October 3, 2001
ABSTRACT
We have developed a control system for regulating gene activation in Toxoplasma gondii. The elements of this system are derived from the Escherichia coli tetracycline resistance operon, which has been widely used to tightly control gene expression in eukaryotes. The tetracycline repressor (tetR) inter- feres with transcription initiation while the chimeric transactivator, composed of the tetR fused to the activating domain of VP16 transcriptional factor, allows tet-dependent transcription. Accordingly, tetracycline derivatives such as anhydrotetracycline, which we found to be well tolerated by T.gondii, can serve as effector molecules, allowing control of gene expression in a reversible manner. As a prerequisite to functionally express the tetR in T.gondii, we used a synthetic gene with change of codon frequency.
Whereas no activation of transcription was achieved using the synthetic tetracycline-controlled trans- activator, tTA2s, the TetRs modulates parasite tran- scription over a range of ~15-fold as measured for several reporter genes. We show here that the tetR-dependent induction of the T.gondii myosin A transgene expression drastically down-regulates the level of endogenous MyoA. This myosin is under the control of a tight feedback mechanism, which occurs at the protein level.
INTRODUCTION
An inducible control of individual gene expression is a prerequisite to study the function of essential genes, and is still lacking in the current repertoire of genetic tools available in apicomplexan parasites including Toxoplasma gondii and the Plasmodium species. In the last few years, several powerful strategies and tools associated with DNA transformation have been developed in these human and animal pathogens. The only method available to date to study essential genes is based on antisense RNA and ribozymes approaches (1,2). However, the development of a controlled gene expression system would not only permit the generation of conditional knockouts but
also allow the study of mutated forms of endogenous genes and the expression of toxic genes.
In the originally described tetracycline-controlled inducible expression system (3), the fusion of the tetracycline repressor (TetR) with the activating domain of the Herpes simplex virion protein 16 (VP16) has converted the repressor into an efficient tetracycline-controlled transactivator (tTA). In that case a minimal promoter fused to tetracycline operator (tetO) sequences is activated in cells expressing tTA and becomes silent in the presence of tetracycline. This system is highly efficient in regulating genes in diverse eukaryotic organisms but has not been established in any protozoan parasite so far. In contrast, the TetR system regulates gene expression in a number of protozoan parasites, including Trypanosoma brucei (4), Entamoeba histolytica (5) and Giardia lambia (6). As in bacteria, TetR interferes with initiation of transcription by binding to tetO sequences, placed properly in the vicinity of the promoter region of protozoan genes. In the presence of tetracycline the repressor ceases to bind to the tetO sequence and thus interference is abolished, rendering the promoter active.
Here we report the establishment of a repressor system that controls gene activities in T.gondii. Such an inducible system in apicomplexan parasites is highly suitable for genetic manipu- lation aiming at the study of essential mechanisms such as host cell invasion. We have applied this system to conditionally express a second copy of T.gondii myosin A gene. This myosin has been proposed to power gliding motility, an essential process for spreading parasites throughout the infected organism and for host cell invasion (7). In this study, we show that the intracellular concentration of MyoA is stringently controlled by a feedback mechanism. A dramatic down-regulation of endogenous MyoA occurs when the second copy of the TgMyoA gene is turned on by the Tet system.
MATERIALS AND METHODS
Parasite strains, transfection and selections
Toxoplasma gondii tachyzoites (RH strain wild type and RH hxgprt–) were grown in human foreskin fibroblasts (HFF) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and 25 µg/ml gentamicin. To generate stable trans- formants, 5 × 107 freshly released RH hxgprt– parasites were
*To whom correspondence should be addressed at present address: Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK. Tel: +44 20 7594 5342; Fax: +44 20 7584 2056; Email: d.soldati@ic.ac.uk
transfected and selected in the presence of mycophenolic acid and xanthine (MPA/X) as previously described (8). The selec- tion based on chloramphenicol resistance was achieved as described earlier (9).
Construction of plasmids
The plasmid p5RT70tetR was constructed by cloning the PCR product corresponding to the coding sequence of TetR between the EcoRI and PacI sites of p5RT70/HX (12). The PCR frag- ments were amplified with primers Rep-1 (5′-GGCGCT- GCAGTCTAGATTAGATAAAAGTA-3′) and Rep-2 (5′- GGCCCTGCAGTTAATTAAGACCCACTTTCAC-3′) using pUHD14-1 as a template (10). For expression of a p5RT70myc tTA, the PCR product corresponding tTA was cloned between the NsiI and PacI sites of p5RT70mycGFP as previously described (7). To express the synthetic tetR (TetRS) and synthetic tetR fused to three minimal VP16 transactivating domains (tTA2S), we used pUHD16-3 as a template (11) and primers Rep-4 (5′-CGGAATTCCTTTTTCGACAAAATGT- CGCGCCTGGACAAGAGCAAAGTCATCAACTCTGC-3′) and Rep-5 (5′-CCTTAATTAACCGCTTTCGCACTTCA- GCTG-3′) or Rep-4 and Rep-6 (5′-GCTTAATTAACCGGG- GAGCATGTCGAGGTC-3′) for amplification. Note that Rep-4 and Rep-5 show a slightly modified codon bias, which fits better to the one of T.gondii. The PCR products encoding either TetRS or tTA2S were cloned into p5RT70/HX (12) between the EcoRI and PacI sites. To express the transgenes stably in T.gondii, HXGPRT was used as a selection marker gene. Responsive plasmids were constructed for the repression and transactivation systems. To introduce one tetO at position +2 relative to the transcription start of the TUB-1 promoter, we undertook an inverse PCR strategy with primers TetO-1 (5′- CCGCTCGATCTCTATCACTGATAGGGATCTGACACA- TCCCCTTCGTGC-3′) and TetO-2 (5′-GGCCTCGAGAA- CATTTTGTTTGTTCTCTGTGAACTT-3′) using p5RT70GFP or plasmid pT230mycGFP as a template for the PCR amplifi- cation. After restriction enzyme digestion of the amplified plasmid with XhoI and ligation to generate p5RT70Tet and pT230Tet with one tetO placed at +2 relative to the transcrip- tion start. Additional tetO sequence motifs were introduced using oligonucleotides TetO-3 (5′-TCGACATCCCTAT- CAGTGATAGAC-3′) and TetO-4 (5′-TCGAGTCTATCAC- TGATAGGGATG-3′) yielding double-stranded oligonucleotides with XhoI and SalI compatible overhanging ends, which can be introduced into p5RT70Tet1 and pT230Tet1 in the XhoI site.
The orientation of all tetO sequences were checked by sequencing. To generate reporter plasmids that use lacZ as a reporter gene, we exchanged the promoter region of plasmid p5RT70lacZ with the tetO-containing promoter flanked between the KpnI and NsiI sites. A chloramphenicol acetyl transferase (CAT) gene under control of the constitutively active promoter (pT230) was introduced in all reporter plas- mids in the unique SalI.
The reporter plasmids for the tet-dependent transactivation system were based on the SAG1 minimal promoter, pS1-70LacZ and the bradyzoite-specific promoter of SAG4, pS4-70LacZ. Both constructs exhibit no detectable expression over background (12; M.Soete, C.Hettmann and D.Soldati, manuscript in preparation). Insertion of seven tetO sequences into the unique site XhoI at position –70 of SAG1 and SAG4 promotors was achieved by cloning 7tetO sequences flanked
by SalI and XhoI sites derived from ptet7-T81-luc (10). To generate pS4-48Tet7LacZ and pS4-33TetLacZ, the promoter region of SAG4 was amplified using the primer pairs SAG4-48 (5′-CCGCTCGAGCGCCGCTGAGACTAACTAG), binding at position –48, or SAG4-33 (5′-CCGCTCGAGCTAGAAA- GAAGTGTGCAACAG), binding at position –33, and LacZ- antisense (5′-GACGGCCAGTGCCAAGCTTG), binding in the lacZ coding region. The resulting PCR products were inserted into pS4-70Tet7LacZ between the XhoI and XbaI sites.
The cDNA coding for MyoA was placed under the tet- dependent promoter by exchanging the promoter regions in pS1mycMyoA with p5RT70TetO4GFP. A summary of the constructs used in this study is given in Table 1.
SDS–PAGE, immunoblot and indirect immunofluorescence assay
Total proteins were solubilized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 7.5) followed by centrifugation for 15 min at 13 000 r.p.m.
in a Beckmann TL-100 centrifuge at 4°C. The supernatant was mixed with SDS–PAGE loading buffer. Western blot analysis was done as described (7). The polyclonal antibodies against MIC4 were previously characterized (13). Indirect immuno- fluorescence assays (IFAs) were performed as previously described (14). The monoclonal antibodies 9E10 recognized the myc epitope. The anti-MyoA antibodies were characterized previously (7). Alexa secondary antibodies were purchased by Molecular Probes (The Netherlands). The anti-tetR antibodies were kindly provided by Wolfgang Hillen, Friedrich-Alexander Universitat Erlangen-Numberg, Germany. The micrographs were obtained with a Zeiss Axiophot camera (Photometrics Type CH-250). Adobe Photoshop (Adobe Systems, Mountain View, CA) was used for processing of images.
Electrophoretic mobility shift assay (EMSA)
Freshly released parasites (108) were harvested, extensively washed in phosphate-buffered saline (PBS), centrifuged, resuspended in 250 µl lysis buffer and incubated for 20 min on ice before they were quickly frozen and thawed three times.
(Lysis buffer: 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluo- ride.) NaCl was added to a final concentration of 250 mM and after incubation for 20 min on ice, the samples were centri- fuged in a Beckmann TL-100 ultracentrifuge at 435 000 g for 15 min on ice. Extracts were mixed with 10 µl binding buffer (20 mM MgCl2, 20 mM Tris pH 7.5, 10% glycerol, 2 mg/ml HS-DNA and 1 mg/ml BSA) with 2 fmol 32P-labeled tetO-DNA isolated from pUHC13-3 (15). The 42 bp TaqI frag- ment was labeled by filling the protruding ends with T4 DNA polymerase in the presence of [α-32P]dCTP. After 25 min incu- bation at room temperature the reaction mixture was loaded onto a 5% polyacrylamide/0.13% bisacrylamide gel containing 5% glycerol. Electrophoresis was carried out in 45 mM Tris, 45 mM boric acid and 1 mM EDTA at 7 V/cm.
Reporter gene assays
Enzymatic assays on fixed and live cells were carried out as previously described (16). The CAT assay was done as described previously (17). Time-lapse green flourescent protein (GFP) detection was achieved by fixation of the
cultures at indicated time points and analysis of each prepara- tion by fluorescence microscopy without changing the expo- sure parameters.
Semi-quantitative RT–PCR
Total RNAs were extracted from parasites grown in the presence or absence of anhydrotetracycline (ATc) using RNAClean from HYBAID, Germany, according to the manu- facturer. RT–PCR was done with the Titan one tube PCR system (Roche). The primers used to amplify endogenous MyoA correspond to a sense primer in the tail and an antisense in the 3′UTR of MyoA. The mycMyoA transcripts were selec- tively amplified using the same sense primer combined with an antisense primer corresponding to the 3′UTR of SAG1.
RESULTS
Toxicity of tetracycline and its derivatives
Depending on its concentration, tetracycline has limited pleiotropic effects on eukaryotic organisms; however, Apicomplexa are particularly sensitive to macrolides and the anti-toxoplasmodial activity of tetracycline derivatives ranges between 5 and 40 µg/ml (18). Since numerous tetracycline derivatives exist, we screened for compounds with negligible side effects in T.gondii, a prerequisite for the establishment of a tetracycline-responsive system. The LacZ-expressing parasites were incubated for 5 days in the presence of different concen- trations of tetracycline or derivatives. The number and size of plaques formed by the parasites were proportional to the extent of X-Gal staining. As illustrated in Figure 1A, ATc was less toxic than tetracycline or doxycycline, with no significant alteration of
growth up to 2µg/ml compared to untreated parasites (Ct). Intra- cellular parasites replicate every 6–12 h and, therefore, most experiments involving an inducible system are anticipated to be accomplished within 48 h. To quantify the toxicity effect of ATc over a shorter period of time, we used parasites constitutively expressing CAT (p5RT70CAT). No significant difference in intracellular parasite growth was detectable in the presence of 1 or 2 µg/ml ATc either by measurement of CAT activity (Fig. 1B) or by estimate counting of the numbers of parasites per vacuole after 48 h (data not shown). Tetracycline-inducible systems established in other organisms, specifically in protozoan parasites (4) allowed a tight regulation of gene expression at concentrations of tetracycline <1 µg/ml. Thus, ATc appears to be appropriate for controlling gene expression in T.gondii without causing deleterious effect.
Establishment of transgenic parasites expressing TetRS and tTA2S necessitated a remodeling of the codon usage Our initial attempts to express the bacterial TetR and the chimeric transactivator tTA in T.gondii failed to detect either TetR or tTA proteins by IFA or western analysis although the corresponding transcripts were detectable by northern blot analysis (data not shown). These latter data suggested that the respective mRNAs were very inefficiently translated in T.gondii, which is in accordance with previous findings concerning several other heterologous genes including GFP (19). This barrier was overcome in the case of GFP by N-terminal fusion with an efficiently expressed protein such as CAT or SAG1 (19) or by remodeling of the codon frequency of the gene (7). Similarly, in the case of TetR and tTA, the addition of a myc epitope tag at the N-terminus of TetR was sufficient to restore the expression of the protein at a detectable
Table 1.
Plasmid name Promoter Gene expressed Selectable marker
genes
Description and purpose
p5RT70tetR TUB1 + SAG1 repeats Tet repressor HXGPRT Expression of the Tet-Repressor
p5RT70myctTA TUB1 + SAG1 repeats Myc-tagged transactivator HXGPRT Expression of the myc tagged Tet repressor fused to VP16
p5RT70tetRs TUB1 + SAG1 repeats synthetic tet Repressor HXGPRT Expression of the synthetic Tet repressor p5RT70tTA2s TUB1 + SAG1 repeats Synthetic Transactivator HXGPRT Expression of the synthetic Tet repressor fused
three VP16 minimal activation domains pS1-70Tet7LacZ SAG1 minimal promoter
+7tetO
β-galactosidase CAT Tetracycline responsive promoter for analysis of transactivator system. 7tetO were placed upstream of SAG1 minimal promoter at position – 70.
pS4-70Tet7LacZ pS4-48Tet7LacZ pS4-33Tet7LacZ
SAG4 promoter + 7tetO β-galactosidase CAT Tet-responsive promoter for analysis of transactivator system The 7tetO were placed at –70, –48 and –33 of SAG4 transcription start site.
p5RT70Tet1LacZ p5RT70Tet2LacZ p5RT70Tet4LacZ
TUB1 + SAG1 repeats + tetO β-galactosidase or GFP
CAT Tet-responsive promoter with x tetO, used for analysis of the repression system.
pT230Tet4lacZ TUB1 + tetO β-galactosidase CAT Tet-responsive promoter for repression system.
LacZ was used for quantification, GFP was used in time course experiments
p5RT70Tet4mycMyoA TUB1 + SAG1 repeats + tetO myc-tagged myosin A DHFR-TS co-transfection
Expression of myosin A under control of Tet-responsive promoter.
level by IFA in transient transfection (Fig. 2A). However the DNA-binding activity of TetR is formed by a helix–turn–helix motif located at the N-terminus (20) and, as a consequence, the N-terminal fusion could potentially compromise the DNA-binding ability of the TetR. To circumvent this risk, we tested synthetic genes encoding TetRS and tTA2S, respectively.
These genes were generated synthetically and adapted to the human codon frequency (11). Note that the tTA2S contains three minimal VP16 activating domains. The human codon frequencies fitted better with the codon bias of the parasite genome, which is slightly GC rich (Fig. 2B). The vectors p5RT70TetRS-HX and p5RT70tTA2S-HX were stably integrated in the T.gondii genome using hypoxanthine-xanthine-guanine- phosphoribosyltransferase (HXGPRT) marker gene. A western blot analysis of stable parasite clones expressing TetRS or tTA2S revealed products of the expected size corresponding to 23 and 28 kDa, respectively (Fig. 2C). Individual clones
exhibited distinct levels of the transgene products suggesting that the respective genes were integrated in differently active loci or in multiple copies. The overall abundance of tTA2S or TetRS produced in the parasites appeared sufficient to sustain regulation of gene expression.
Functional analysis of TetRS and tTA2S expressed in T.gondii
In other systems, the extent of regulation was reported to correlate with the level of TetR expression (6). Based on western blot analysis, we chose the two parasite lines expressing the highest level of TetRS and tTA2S for further investigation. To assess whether TetRS and tTA2S produced in T.gondii were functional, we examined extracts from trans- genic parasite clones in EMSA using radiolabeled tetO DNA.
We found that all clones were capable of generating a strong and specific mobility shift in the absence of tetracycline (Fig. 3). The ability of the repressor to bind to tetO is lost upon conformational change in the presence of tetracycline. The binding to tetO was tetracycline dependent, as shown by adding ATc to the cell extracts prior to incubation with the tetO fragments or by growing parasites in the presence of ATc followed by thorough washing steps prior to preparation of cell extracts. In both cases DNA-binding activity was abolished by using 0.75 µg/ml ATc. These results indicated that TetRS and tTA2S were functional with respect to DNA-binding activity, and indicated that ATc was capable of entering the parasites during normal growth in host cells and of abolishing binding of TetRS and tTA2S to tetO.
Characterization of reporter plasmids for ATc-dependent repression
To investigate the ability of TetRS to repress transcription in T.gondii, we constructed several Tet-responsive promoters derived from the constitutive T.gondii tubulin 1 (TUB1) promoter. A unique transcription initiation site was previously mapped in TUB1 (21) and the promoter region roughly charac- terized (12). The constructs used in this study are depicted in Figure 4A. p5RT70LacZ is composed of TUB1 promoter region up to –70 TUB1 and preceded by five SAG1 27 bp repeat elements. One, two or four tetO sequences were inserted at position +2. In addition, we introduced four tetO elements at position +2 in pT230, which extended upstream to position –230 in the promoter region of TUB1. To evaluate the potential of using these constructs in the repression system, we analyzed their promoter strength using lacZ as a reporter gene. Wild- type parasites were transiently transfected with equimolar amounts of each plasmid and lacZ activity was monitored 16 h later. The presence of elements inserted at the transcriptional initiation site significantly diminished the promoter activity (Fig. 4B). Insertion of one tetO (p5RT70Tet1) decreases reporter activity to ~30% while p5RT70Tet2 and p5R70Tet4 only gave rise to 15 and 10% of the original promoter activity, respectively. It is conceivable that the integration of one or more tetO disrupted an essential cis-element or that the tetO sequences interfere with efficient translation initiation.
Comparison of promoter strengths between pT320 and p5RT70 was previously reported (12), and the insertion of four tetO sequences into pT230Tet4LacZ also caused a drastic reduction in promoter activity.
Figure 1. Toxicity assay of tetracycline and its derivatives on T.gondii tachyzoites in tissue cultures. (A) HFF cells were inoculated with 2 × 102 parasites constitutively expressing pT5RT70lacZ in 12-well plates. The cultures were treated with 0.1, 0.3, 1.0 or 3.0 µg/ml of tetracycline (Tc), anhydrotetracycline (ATc) or doxycycline (Dc). The non-treated parasites were included as control (Ct). Five days later, the cultures were fixed and stained with X-Gal. ATc showed the lowest toxicity effect on growth of parasites when compared to an untreated control culture. (B) Freshly released parasites (1 × 104) constitutively expressing CAT were inoculated on HFF cells in the absence or presence of 0.25, 0.5, 1.0, 1.5 or 2.0 µg/ml ATc. After incubation at 37°C for 48 h, the parasites underwent up to eight cycles of replication. The infected cells were lysed and assayed for CAT activity. Results of five independent experiments are pooled in this analysis.
To monitor transactivation in T.gondii, we used either the CMV promoter carrying seven tetO motifs (3), pS170, a minimal SAG1 promoter previously described (12), or pS4- 70LacZ, a construct inactive in tachyzoites and derived from the bradyzoite-specific promoter SAG4 (M.Soete, C.Hettmann and D.Soldati, manuscript in preparation). Removal of the 27 bp repeat elements upstream of the SAG1 promoter were shown to abrogate reporter gene expression to background
level (12). The seven tetO sequences were introduced at posi- tion –70 to generate pS1-70Tet7LacZ (Fig. 4C). In the case of SAG4 promoter, it was shown that a chimeric promoter p5RS4-70LacZ bearing the five 27 bp repeated elements of the SAG1 promoter is highly active, whereas pS4-70 itself is only inactive in tachyzoites (M.Soete, C.Hettmann and D.Soldati, manuscript in preparation). Introduction of seven tetO sequences into p5RS4-70Tet7LacZ showed no activity over
Figure 2. Establishment of stable parasite cell lines expressing functional TetRS or tTA2S genes after remodeling of their codon usage. (A) IFA on intracellular parasites expressing tTA, myctTA, tTA2S or TetRS, using a cocktail of monoclonals raised against TetR. The recombinant proteins are distributed homogeneously in the cytosol of the parasites. (B) The percentages of each codon present in tTA2 and synthetic tTA2S is compared to the frequency of codon usage in T.gondii, based on the EST database information. Only the modified codons are shown. Except for the triplet coding for Ile, all codons used for the humanized tTA2S sequence fitted better to the overall codon frequency of T.gondii. (C) The expression level of several independent clones for TetRS and tTA2S was examined by western blot analysis. Cell lysates from 1 × 107 wild-type parasites, RH (lane 1) and parasite clones expressing different TetRS (lanes 2–4) or tTA2S (lanes 5 and 6) were probed with monoclonal anti-TetR. Both proteins have the expected size of 23 kDa (TetRS) and 28 kDa (tTA2S), indicated by the arrows.
the background measured with p5RS4-70LacZ in wild-type parasites (data not shown).
ATc-dependent regulation of gene expression in T.gondii The reporter constructs designed to monitor repression and transactivation were introduced stably in transgenic parasites expressing high levels of TetRS and tTA2S, respectively, using CAT as a selectable marker (9). The β-galactosidase activity of the lacZ reporter was measured 24 h after incubation of the parasites in the absence or presence of 0.75 µg/ml ATc. The X-Gal staining on fixed parasites revealed a significant differ- ence in LacZ expression, depending on the reporter plasmid used. No visible difference in X-Gal staining was observed in parasites transformed with p5RT70LacZ or p5RT70Tet1LacZ (data not shown). In contrast, a clear discrepancy in staining was detectable between the non-induced and induced state (addition of ATc) in parasites expressing 5RT70Tet2LacZ and 5RT70Tet4LacZ. Measurement of the LacZ enzymatic activity allowed quantification of inducibility while CAT activity was used as internal reference. In agreement with our qualitative data, the TetRS-responsive promoter p5RT70Tet4 yielded the best regulation factor between ∼15- and 20-fold, whereas p5RT70Tet1lacZ and p5RT70Tet2lacZ had an induction ratio of only ~2- and 5-fold, respectively (Fig. 5A). Analysis of pT230Tet4LacZ did not reveal any tighter regulation.
Increasing the concentration of ATc did not improve the results (data not shown).
To test the transactivator system, we initially transfected the pCMVTet7LacZ, a promoter found to be highly inducible in the mammalian system (3), in transgenic parasites expressing tTA2S. This vector was completely inactive both in wild-type parasites and in tTA2S expressers, either in the presence or absence of Atc (data not shown). We then established double
cell lines expressing tTA2S and the promoter constructs described above and failed to monitor any transactivation as a result of incubation with ATc even though functional tTA2S was present in the cells (Fig. 5B). Since tTA2s proved to be fully functional in mammalian cells (3,11), this negative result suggests an absence of crosstalk between the VP16 transacti- vation domain and the transcription machinery of T.gondii.
Additionally we moved the seven tetO sequences with respect to the initiation of transcription at positions –48 and –33 to generate pS4-48Tet7LacZ and pS4-33Tet7LacZ, respectively.
Like the previous constructs, these reporter plasmids remained inactive when introduced into parasites expressing tTA2s (data not shown).
Kinetics of TetRS-dependent gene expression
The time course of reporter gene expression in response to ATc treatment was examined in cells expressing p5RT70Tet4GFP directly by monitoring the fluorescence produced by GFP.
Parasites were first cultivated for 3 days in the presence of ATc. The freshly released parasites were inoculated again on HFF cells in the absence of drug for different time periods. A significant decrease in fluorescence was visible after 24 h and no fluorescence over background level was detectable after 48 h (Fig. 6A). In a reverse experiment, parasites previously cultivated in the absence of drug showed appearance of GFP 4 h after the addition of ATc (Fig. 6B). A full level of GFP expression was achieved 24 h later, confirming that the inducible repression system functions in a reversible manner in T.gondii.
Modulation of endogenous MyoA level by ATc-induced mycMyoA transgene expression
To determine whether the system developed here can be applied to relevant endogenous genes, we chose to generate stable cell lines expressing MyoA under the control of the TetRS system. Like other apicomplexan parasites, T.gondii actively invades host cells by a mechanism depending on its intact actomyosin system. MyoA is likely to be responsible for powering the gliding locomotion and host cell penetration (22). Our attempts to disrupt this gene have failed so far. In a previous study, we noticed that the integration of a second myc-tagged copy of MyoA gene caused a drastic down-regulation of the endogenous MyoA level (7). Here, we generated stable transformants with p5RT70tetO4mycMyoA and selected posi- tive clones, which exhibited a non-detectable level of mycMyoA expression in the absence of ATc (Fig. 7A). A 10-fold increase in mycMyoA expression after removal of ATc was estimated on western blot analysis (Fig. 7B). The abun- dance of endogenous MyoA and mycMyoA was directly compared with anti-MyoAtail antibodies. The presence of a stretch of histidine residues and the myc tag caused a small mobility shift, distinguishing between the two proteins. Inter- estingly, we observed that turning on the transgene induced a drastic decrease in the level of endogenous MyoA confirming our earlier observations (Fig. 7B). The secretory protein MIC4 was used as reference for equal parasite proteins loading. A semi-quantitative RT–PCR analysis was then undertaken to assess at which level endogenous MyoA was down-regulated during expression of the transgene. The transcripts corre- sponding to the mycMyoA transgene fluctuate in function of ATc while the level of endogenous MyoA transcripts remains constant (Fig. 7C). This result indicates that the down-regulation
Figure 3. EMSA made with lysates of parasites expressing TetRS or tTA2S. Extracts of wild-type (wt) or transgenic parasites expressing TetRS or tTA2S were incubated in presence of radiolabeled DNA-probe bearing one copy of the tetO sequence. No band shift was observed with lysates prepared from wild-type RH parasites whereas a strong shift is generated by incubation with lysates from transgenic parasites in the absence of ATc (–). The DNA-binding activity illustrated by the mobility shift of tetO is abrogated when ATc was either added to the lysates, (+) or when lysates were prepared from parasites growing in media containing Atc, (+*). The parasites cultivated in presence of 1 µg/ml Atc were extensively washed in PBS before preparation of the cell extracts.
of endogenous MyoA most likely occurred at the post- transcriptional or post-translational level.
DISCUSSION
Since T.gondii and Plasmodium species have been success- fully transfected, several laboratories have made intense
efforts to establish a system controlling gene expression in these organisms. Towards this goal, we previously attempted unsuccessfully to hormonally control the site-specific Cre recombinase activity in T.gondii (17). We report here the development of an inducible expression system in T.gondii based on elements of the Escherichia coli tetracycline resist- ance operon. To reach this goal, we have generated transgenic
Figure 4. Construction of TetR- and tTA-responsible promoters. (A) Schematic representation of the reporter plasmids used to monitor tet-dependent repression of transcription. As reporter genes we used either lacZ or gfp placed under control of the promoters. The CAT gene, CAT, was used both as selection marker and as internal standard in quantitative reporter gene assays. To generate Tet-responsive promoter, one, two or four tetO sequences were placed at position +2, relative to the transcription start site of the TUB1 promoter pT230 or the chimeric SAG1–TUB1 promoter p5RT70. (B) Comparison of promoter strength upon insertion of tetO sequences. 2 × 108 freshly lysed wild-type RH parasites were transiently transfected with the same molar amount of reporter plasmids. The lacZ activity was determined 16 h post-transfection in cell lysates. Relative promoter activities obtained from three independent experiments are shown here. The modified promot- ers are significantly decreased in their activity upon insertion of tetO sequences. (C) Representation of the reporter plasmids designed to measure tTA-dependent transactivation. 7tetO sequences were inserted upstream of the minimal major surface antigen SAG1 promoter pS1-70 to generate pS1-70Tet7LacZ. The 7tetO were also introduced at position –70 of the bradyzoite-specific promoter SAG4, pS4-70 to generate pS4-70Tet7LacZ. The TUB1, SAG1 and SAG4 5′ flanking sequences are depicted in blue, green and yellow, respectively. The tetO sequences are depicted in red.
parasite lines expressing synthetic TetRS or tTA2S genes with a humanized codon usage (11). The problems encountered in this study in expressing the TetR underline the importance of an appropriate codon bias for heterologous gene expression in T.gondii. EMSAs revealed that both TetRS and tTA2S are indeed produced in a functional form in T.gondii, and that their binding to tetO sequences is ATc dependent. Despite these facts, tTA2S failed to transactivate genes in T.gondii. We cannot completely rule out that the minimal promoter/tetO combinations used here were topographically not appropriate.
However, several other promoter designs where the distance between the tetO and the transcription start site was varied, as well as constructs based on the bradyzoite-specific promoter SAG4 (23), were also tested without success. In eukaryotic
organisms, regulation of polymerase II transcription has been achieved efficiently by the transactivator system and such a system would be optimal for regulating the presumed pol II transcription in apicomplexans. Our data suggest that the acti- vating domain of VP16 is not able to stimulate the transcription machinery. Several viral promoters derived from CMV or SV40 have been tested previously and shown to be inactive in T.gondii (D.Soldati, unpublished results). A transcription acti- vation domain derived from T.gondii will most likely be required for overcoming this problem. No transcriptional factors have been identified in apicomplexans to date although some components of the machinery were characterized, for example the presence of a conserved histone acetyltransferase, T.gondii GCN5 (24). A better understanding of transcription and its regulation in apicomplexan parasites will be helpful in assisting in the development of a highly efficient transactivator-based system in these parasites.
Meanwhile, the TetRS is able to interfere significantly with parasite transcription, allowing 15–20-fold regulation. This is the first report of an ectopic reversible control of gene expres- sion achieved in apicomplexan parasites. Even though further refinements of this system aiming at a broader regulatory range will be very useful and appear feasible, the possibility of varying the activity of a gene 10–20-fold around its normal state will, in many cases, generate a phenotype and thus reveal insights into a gene’s function. For example, there is potential to improve the range of regulation further by testing very strong promoters such as that coding for the GRA genes that have been characterized (25) and NTP3 (26). Furthermore, an optimized reverse Repressor exists that shows the reverse binding properties to the tetO elements in the presence or absence of tetracycline (11). This novel reverse repressor exhibits vastly improved properties, which would deserve to be tested in T.gondii.
Thus, in the present form, the repressor system allows control of the activity of an individual gene such as MyoA and is sufficient for the creation of ‘on/off’ situations in a reversible way. The kinetics of induction and repression as determined using GFP demonstrated a rapid turn-on of expression observed within 4 h following addition of ATc.
The disappearance upon repression takes 24 h but obviously depends essentially on the half-life of the protein studied.
Applied to MyoA, it appears that this system is compatible with the generation of a conditional knockout of the gene.
Indeed, the mycMyoA transgene reaches a level of expression comparable with that of the endogenous gene and can be turned off to background level. This inducible system allows experimental mimicking and transiently a phenomenon of dosage compensation previously observed with this MyoA (7).
Upon induction of the mycMyoA transgene, the endogenous level of MyoA drops to <20%, with no apparent change at the level of its messenger RNA. This feedback loop mechanism acts at the protein level and potentially involves a titration of the myosin light chain(s). A limitation of light chains presum- ably hampers the proper folding of the myosin heavy chain rendering it very unstable.
In summary, the repressor system established here should be suitable for conditional expression of toxic genes and dominant negative mutants such as TgMyoA mutants that would be incapable of sustaining ATP hydrolysis. However, to generate conditional knockouts, it would be more appropriate
Figure 5. Measurement of tetracycline-dependent repression or transactivation of gene expression. (A) Tetracycline-dependent repression of transcription quantified by lacZ expression in double transgenic parasites expressing stably TetRS and the reporter plasmid indicated. Parasites (1 × 104) were inoculated on HFF cells in the presence or absence of ATc and, 48 h later, cells were lysed and assayed for lacZ and CAT activities. The lacZ activity of each clone was standardized against CAT activity. Relative lacZ activities reported here are based on three independent experiments. (B) Analysis of tTA2s activation of gene expression in two double transformants expressing tTA2s and pS1-70Tet7LacZ or pS1-70Tet7LacZ, respectively. Parasites (1 × 104) were inoculated on HFF cells in the presence or absence of ATc. Forty-eight hours later, cells were lysed and assayed for lacZ and CAT activities. LacZ activity of each clone was standardized against CAT activity. Relative lacZ activities reported here are based on three independent experiments. No tet-dependent activation of gene expression was detectable with these two reporter constructs. The double transformants expressing TetRS and modulating p5RT70Tet1LacZ were used as positive control.
to use a reverse repressor (11), which keeps the transgene turned on in the absence of ATc, during the selection proce- dure for homologous recombination clones. In combination with the elaborated tools associated with DNA transfection, we anticipate that such an inducible system will open new avenues of investigation and unravel the biological functions of a broad spectrum of relevant genes in Apicomplexa.
ACKNOWLEDGEMENTS
We are indebted to Drs Udo Baron and Maz Hasan for their precious assistance and we thank Dr Petra Gross for her contri- bution during the initial phase of the project. This work was mainly funded by the Deutsche Forschungsgemeinschaft (DFG grant number SO 366/1-2, SO366/1-3).
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Figure 7. TetRS-dependent expression of p5R70Tet4mycMyoA and the com- pensatory modulation of the endogenous TgMyoA level. (A) Detection of mycMyoA by IFA, on intracellular parasites incubated in the presence or absence of ATc, using mAb anti-myc. (B) Analysis of mycMyoA and endo- genous MyoA expression by western blot using anti-myc and anti-MyoA tail polyclonal antibodies. Detection of MIC4 with anti-MIC4 antibodies was included as reference. Before preparation of cell lysates, parasites were incubated for 48 h in the presence or absence of ATc. (C) Semi-quantitiative RT–PCR analysis using two dilutions of total RNAs isolated from wild type and parasites expressing myMyoA 48 h after incubation in the presence or absence of ATc. Specific sets of primers were chosen to amplify the endo- genous MyoA, the transgenic mycMyoA and as control MIC4 transcripts.
