Development of a Stable Episomal Shuttle Vector for Toxoplasma gondii*

Michael W. Black and John C. BoothroydDagger

From the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The rapid developments in the molecular genetics of Toxoplasma gondii have far reaching implications in treatment and vaccination strategies for this as well as closely related pathogens such as Plasmodium. Although stable transformation of this parasite through homologous and illegitimate genomic integration has provided many of the tools necessary for genetic analysis, subsequent manipulations of the DNA have proven laborious. This report describes the selection and subsequent characterization of a Toxoplasma sequence that permits the episomal maintenance of bacterial plasmids in this parasite. This sequence was isolated from the Toxoplasma genome through selection for episomal stability of a pUC19-based library in the absence of a selectable marker. A 500-base pair fragment was determined to possess the stabilization activity. Transformations of Toxoplasma using vectors possessing this fragment, referred to as EMS (episomal maintenance sequence), demonstrated an elevated stable transformation frequency compared with the vector alone. Mutants deficient in hypoxanthine-xanthine-guanine phosphoribosyltransferase activity were used as a test to see if this gene could be selected from a genomic library using a vector containing the EMS. The success of this test demonstrates the utility of EMS-containing vectors in complementation strategies and the ability of such constructs bearing large fragments of the Toxoplasma genome to be maintained episomally.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Toxoplasma gondii is an obligate intracellular protozoan of the phylum Apicomplexa which includes Plasmodium, Eimeria, and other medically and agriculturally important pathogens. Although many of the structural and biochemical attributes of these pathogens are conserved, Toxoplasma is unusual within the phylum in several characteristics that are amenable to molecular genetic manipulations (1, 2). For example, the lack of host cell specificity and rapid replication cycle allows one to quickly and inexpensively propagate this organism to high numbers in vitro. Since the parasite has a haploid genome in the asexual part of its life cycle, molecular genetic manipulations are not complicated by allelic copies, and generation of loss-of-function mutations can be done easily by a variety of methods (3-5). In addition, homologous and illegitimate recombination for the stable transformation of constructs into the genome of this parasite have been widely used in the analysis and complementation of different genetic loci (6-9).

Although genomic integration is conducive for a variety of genetic manipulations (e.g. increased stability of the transforming DNA, definitive replacement of a gene, etc.), some procedures are restricted by the nature of this transformation event. Using an episomal vector to stably transform a strain would bypass many of these obstacles and would allow easy recovery and/or removal of a given construct. The intrinsic instability associated with episomal DNAs allows definitive evidence for the association of a given phenotype to the DNA under examination using a molecular form of Koch's postulates. Since the vector would be independent of the parasite's genome, analysis of the activity attributed to the transformed DNA would simply require either isolating the episome to re-transform the parental strain or selecting against the episome using a negative selectable marker. In addition, as the episome does not directly interact with the genome, the probability of inducing a mutation as a result of integration is diminished.

To obtain sequences that would allow independent replication and stabilization of episomes in Toxoplasma, we searched through the parasite's genome to recover sequences that demonstrate these attributes. Although the majority of the procedures previously used to isolate autonomous replicating sequences (ARS)1 from eukaryotic organisms have utilized selectable markers (10-12), we have chosen to select for sequences that stabilize episomal copies in the absence of drug pressure to achieve a strong selection for sequences providing a highly efficient mode of replication and/or faithful segregation. The selection for these sequences and characterization of a 500-bp fragment that possesses this activity are described in this report as is a test of an engineered shuttle vector in a complementation experiment.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Parasite Strains and Growth Conditions-- The RH strain (13) and mutants generated from this strain were grown in the tachyzoite stage for all experiments presented. The parasites were propagated in vitro by serial passage in monolayers of human foreskin fibroblasts as described (14). The HXGPRT mutants in this strain consist of the RHDelta HXGPRT knock-out strain (1, 15) and the TXR-2 point mutant. TXR-2 was isolated from a population of RH mutagenized with 150 µg/ml N-nitroso-N-ethylurea (Sigma) for 60 min, syringe-released, and re-plated on a fresh monolayer for selection using 6-thioxanthine at 400 µg/ml (Sigma). After 2 weeks of continuous selection, clones were isolated that were incapable of growth under the selective pressure of mycophenolic acid confirming their status as deficient in HXGPRT activity (see below).

The selection of parasites transformed with the chloramphenicol acetyltransferase was performed using 20 µM chloramphenicol (Sigma) as described previously (8). Strains deficient in HXGPRT activity were selected for the transformation of a construct bearing a form of the HXGPRT gene using 50 µg/ml mycophenolic acid (MPA) and 50 µg/ml xanthine (Sigma). Individual plaques of MPA-resistant clones were picked from monolayers overlaid with selective medium containing 0.9% Bacto-agar as described previously (1).

Constructs-- All constructs used in the transformation of Toxoplasma in this report use the pANA vector backbone. This vector was generated by inserting a 90-bp sequence containing a central NotI site flanked by two AscI sites in pUC19 at the AflIII site. Modifications of this vector include the following: 1) pANA-0.5, adding the 500-bp EcoRI/KpnI fragment of pEM1 (constituting the episomal maintenance sequence (EMS)) in the respective sites of the multicloning site (MCS); 2) pANAE, adding a blunted version of the 500-bp EMS in a blunted NotI site (outside the MCS); 3) pCANA, adding a blunted BamHI/HindIII tubulin-driven chloramphenicol acetyltransferase cassette of pT/230 (16) in a SspI site; 4) pCANA-1.9, the pCANA vector with the 1.9-kb KpnI fragment of pEM1 in the MCS; 5) pHANA, adding a DHFR-driven HXGPRT cassette of pmini-HXGPRT (17) in a SspI site; 6) pHANA-0.5, the pHANA vector with the 500-bp EMS in the unique EcoRI and KpnI sites of the MCS. The blunted restriction sites were generated using the Klenow fragment of Escherichia coli DNA polymerase I, and all ligations were performed using T4 DNA ligase by standard techniques (18). The pACYC184 vector (New England Biolabs) was used as a control for DNA extractions and subsequent transformations of E. coli.

Library Construction-- A genomic library of the PDS strain of Toxoplasma gondii (a clonal isolate of ME49 (19)) was used to select sequences bearing episome-stabilizing activity in the RH strain. This library was generated in the BamHI site of pANA using a Sau3AI partial digest of genomic DNA from which fragments 4-8 kb in size were gel-purified (Geneclean) using standard procedures (18). Two additional libraries of RH genomic DNA were constructed similarly in pANA and pANAE for the isolation of the HXGPRT gene. The complexity of each of the three libraries was found to be 1-5 × 105 independent recombinants with >90% containing Toxoplasma genomic DNA as determined by the lack of beta -galactosidase activity and size analysis of random clones.

Selection of Episome Maintenance Sequences-- T. gondii (RH strain) were transformed with 50 µg of the PDS genomic library as described previously (20). In the first two rounds of selection for episomal maintenance, the transformed parasites were passaged twice in human foreskin fibroblasts monolayers and recovered to isolate genomic and episomal DNAs using the TELT procedure described below. The DH12 strain of E. coli was electroporated with the isolated DNA and plated on ampicillin plates to select for bacteria transformed with recovered episomal copies. Plasmid was extracted from the population of ampicillin-resistant bacteria for subsequent transformation into RH for additional rounds of selection. The population of plasmids carried through four passages of RH in the third round were re-electroporated into RH for an additional five passages in the fourth and final round of selection. Following recovery of episomal DNA from this population and transformation of E. coli, plasmids from 20 ampicillin-resistant bacterial clones were digested with RsaI and examined for the enrichment of certain library fragments as determined by their restriction pattern. Five distinct constructs were chosen based on the criterion of being represented at least twice among the bacterial clones examined.

DNA Extraction, Normalization, and Southern Blot Analysis-- T. gondii DNA used in the preparation of genomic libraries was isolated from a freshly lysed culture in a single T-175 flask. Recovered parasites were syringe-released using a 27-gauge needle and separated from host cell debris using a 3-µm nucleopore membrane (Costar). Parasites were pelleted, washed, and resuspended in a lysis solution containing 120 mM NaCl, 10 mM EDTA, 25 mM Tris (pH 8.0), 1% Sarkosyl, and 0.1 mg/ml of RNase A. After a 30-min incubation at 37 °C, proteinase K (1 mg/ml) was added for an additional incubation overnight at 55 °C. The genomic DNA was twice extracted with phenol:chloroform, ethanol-precipitated, and resuspended in 10 mM Tris (pH 8.0), 1 mM EDTA.

DNA of Toxoplasma used for the quantitation and analysis of episomal forms was extracted from parasites freshly lysed from T-25 monolayers using a modified version of the TELT extraction procedure (21). Parasites were syringe-released using a 27-gauge needle, pelleted, and lysed using a solution of 50 mM Tris (pH 8.0), 62.5 mM EDTA, 2.5 M LiCl, and 4% Triton X-100. Genomic and episomal DNA was extracted twice using phenol:chloroform, ethanol-precipitated, and resuspended in 10 mM Tris (pH 8.0), 1 mM EDTA.

To take into account the differences in recovery of episomal DNA and subsequent transformation of E. coli, equal amounts (~1 ng) of the tetracycline-resistant plasmid pACYC184 were added to each pellet of cells prior to DNA extraction (pACYC184 possesses a plasmid p15 origin of replication that is compatible with the ColE1-type origin on the pANA-based vectors). Using this, the number of ampicillin-resistant colonies could be normalized to the number of tetracycline-resistant colonies obtained from the same electroporation. The pACYC184 "spike" also allowed the transformation efficiency of the extracted plasmids to be determined which in turn enabled the actual number of plasmid molecules per parasite to be estimated. To determine the actual number of transformation-competent episomes per parasite, this normalized figure was then multiplied by the difference between the number of TetR obtained from a known amount of the pACYC184 plasmid and the number recovered from the cell pellet after the DNA extraction procedure.

For Southern blot analysis, genomic and episomal DNAs were digested with either EcoRI or PvuII, subjected to electrophoresis, and transferred to a nylon membrane for hybridization to random-primed radiolabeled probes corresponding to either the pANA vector or a 700-bp BamHI/NdeI fragment of the HXGPRT gene. Phosphorimaging analysis of hybridized membranes was done using the Storm 860 PhosphorImager (Molecular Dynamics) and quantitated using ImageQuant software (Molecular Dynamics).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Selection of Toxoplasma Episomal Maintenance Sequences-- A Toxoplasma genomic DNA library was generated from a Sau3AI partial digest of the PDS strain with an average insert size of 6 kb using the pUC19-based vector pANA. The PDS genomic DNA was used to generate the library as this strain has only recently been derived from an oocyst, whereas the RH strain has been in continuous lab passage for over 50 years. Thus, PDS is expected to have undergone fewer genetic alterations (i.e. deletions) under the selection for in vitro growth. The RH strain of Toxoplasma was electroporated with 50 µg of this library and passaged two times through confluent monolayers of human foreskin fibroblasts. After the second passage, genomic and episomal DNAs were extracted and electroporated into the DH12 strain of E. coli for selection of the episomes bearing the ampicillin resistance marker of pANA. Plasmids isolated from populations of ampicillin-resistant bacteria were re-transformed into the RH strain for an additional three rounds of selection. From the resulting population, 20 ampicillin-resistant E. coli colonies were picked and their plasmids analyzed by restriction endonuclease digestion using PvuII. Of these 20, 5 clones were chosen that showed distinct digestion patterns and appeared to be represented at least twice among those analyzed (data not shown).

As our ultimate goal was to generate a shuttle vector for Toxoplasma that could be efficiently retained in episomal form even in the absence of selection, the five isolated plasmids were analyzed for this property. After three passages of RH transformed with the isolated constructs and a pANA control, all five clones were at least 100-fold more efficient in maintaining the constructs as episomes in RH in the absence of selection when compared with the parental vector alone (see Fig. 1). Since the constructs of the library had an average insert size of 6 kb, a control construct in the same vector carrying a ~5.8-kb XmaI fragment from a cosmid clone of SAG1 was similarly tested and demonstrated no stabilizing activity (data not shown). One of the five clones, named pEM1 (episomal maintenance), was arbitrarily chosen for further analysis.


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Fig. 1.   Comparison of episomal stability in Toxoplasma of five constructs selected from a genomic library. Constructs were selected for the ability to be maintained episomally for extended periods in Toxoplasma without selective drug pressure. DNA from an approximately equal number of parasites of each passage was isolated and used to transform E. coli to select out the pUC19-based constructs that were still in an episomal form. The total number of colony-forming units correlates to the number of episomes recovered from DNA extracted from parasites of each passage. The construct corresponding to pEM1 was arbitrarily chosen for further analysis.

Isolation of EM Activity from of pEM1-- As the ~6.8-kb size of the pEM1 genomic insert is excessively large for use as a stability element in episomal vectors, we sought to isolate a smaller sequence from this clone that possessed the same EM activity. As a first step in identifying the critical region in this insert, a restriction map of pEM1 was generated, and overlapping fragments of the insert ranging from 0.75 to 4.2 kb were isolated and cloned into pHANA (the modified form of pANA carrying hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) driven by upstream and downstream non-coding sequences of dihydrofolate reductase (DHFR) (17)). A constant molar amount of each construct (5-10 µg each) was electroporated into a strain of RH deleted in HXGPRT open reading frame (RHDelta HXGPRT (1, 15)) and grown in the presence of 50 µg/ml mycophenolic acid (MPA) and xanthine to select for HXGPRT expression. Only parasites transformed with the construct carrying a 1.9-kb KpnI fragment (pHANA-1.9) were able to survive after the second passage under drug pressure. DNA isolated from the third passage of these parasites revealed that at least part of the population carried the marker episomally (data not shown, but see below).

It has been previously suggested that sequences in the dihydrofolate reductase gene of Toxoplasma are able to elevate the stable transformation frequency (6, 7). Although there are only short regions of the DHFR locus in the pHANA vector and the aforementioned activity was not observed using other fragments of the pEM1 genomic insert, we chose to use a construct bearing a tubulin-driven chloramphenicol acetyltransferase gene (pCANA) to examine the relative transformation frequency in a different context. Plasmids carrying the 1.9-kb fragment (pCANA-1.9) were compared with a control construct with a 2.0-kb PstI fragment of the parental EM1 clone that did not possess episomal maintenance activity (pCANA-2.0). Transformed parasites were passage three times in chloramphenicol and plated for plaque formation to compare the number of surviving parasites between the control and EMS-bearing constructs. Since chloramphenicol kinetics are such that parasite death is only detected during the third passage, approximately 16-20 generations would have occurred before the chloramphenicol acetyltransferase marker exerts its positive effects. The pCANA-1.9 fragment was determined to elevate the transformation frequency by ~53-fold when compared with the pCANA-2.0 size control (data not shown). Although this indicates that the 1.9-kb fragment elevates the transformation frequency, more definitive evidence will be presented below demonstrating that the augmentation of transformation is a result of stabilizing the constructs in an episomal form, thus eliminating the requirement for the less frequent genomic integration event.

As the 2.0-kb PstI fragment, which does not possess stabilizing activity, overlaps 1.4 kb of the 1.9-kb KpnI fragment, the remaining 500 bp of this DNA were examined for episomal maintenance activity. This region was cut out of the 1.9-kb parental sequence using flanking EcoRI and KpnI sites (KpnI derived from the multicloning site of pANA) and ligated into the pANA plasmid (pANA-0.5). The pANA and pANA-0.5 vectors were electroporated into RH and examined for EM activity in the absence of selective pressure as described above using pACYC184 to normalize for variations in DNA extractions and electroporations (see "Experimental Procedures"). Fig. 2A shows that the number of colony-forming units/parasite arising from DNA of the pANA-0.5 transformants is maintained at a higher level than the number recovered from those electroporated with the pANA vector. As there is no detectable difference in activity when pANA-0.5 was compared with a pANA-based vector carrying the parental 1.9-kb fragment (data not shown), we conclude that this 500-bp sequence confers the episomal stability accredited to pEM1. Although the pANA vector carrying the EMS did not stabilize the vector at the same levels as seen with the parental pEM1 clone (see Figs. 1 and 2A), we suggest that this variance is a result of the ~6.8-kb difference in size between the two vectors as discussed below.


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Fig. 2.   Comparison of the stability and methylation state of pANA and pANA-0.5 passaged through Toxoplasma. A demonstrates the stabilizing activity of the EMS in vectors free of other sequences from T. gondii while grown in drug-free medium. The potential variability in DNA extraction and subsequent E. coli transformation were normalized using the pACYC184 vector containing a tetracycline resistance marker and distinct origin of replication for compatibility with the pUC19-based vectors used in pANA and pANA-0.5. An equal amount of this vector was spiked into each of the cell pellets prior to DNA extraction. The difference between the number of tetracycline-resistant colony-forming units obtained after the extraction procedure to the number detected from the spike alone was used to normalize each transformation event. B demonstrates the methylation state of pANA and pANA-0.5 isolated from the second and third passage as determined by differential digestion with DpnI and DpnII. DpnII resistance is acquired by the methylation of GATC sites by the bacterially derived dam methylase activity. The inverse is true for DpnI resistance. Thus, the conversion of dam-methylated vectors from a DpnII- to DpnI-resistant state implies the active replication and modification of the DNA by the dam(-) parasites.

The complete sequence for this 500-bp EMS has been determined (GenBank accession #AF009625). No significant sequence homology was detected when compared with the available data bases, and there were no conserved motifs apparent in comparisons with known autonomous replicating sequences and centromeres. Although Southern blot data indicate that the 500-bp sequence is found in a single copy in the genome (data not shown), this assay may not detect a small conserved motif that could be more abundant. The other four constructs isolated (pEM2 to pEM5) were not used to probe genomic DNA because the critical regions responsible for EM activity have not been identified to generate specific probes. Since the insert size of these constructs is >= 7 kb, of which it is suspected only a small portion actually confers episomal maintenance, finding multiple bands would be meaningless as one or more may result from the hybridization of repetitive elements carried in the genomic insert (22). Southern blots of PvuII-digested DNA from all five of the vectors isolated carrying EM activity (pEM1-pEM5) were individually probed with the 500-bp EMS or the pEM2-pEM5 constructs to determine if there are sequence similarities between the individual EMSs. No cross-hybridization between any of the five probes (outside of the common pUC19 backbone) was detectable. Thus there is no substantial similarity in the sequences found within the library inserts. Until the critical regions in the other four plasmids (pEM2-pEM5) are identified and sequenced, we cannot exclude that they share some small, critical motif. Regardless, for our purposes, the 500-bp EMS identified here is of convenient size and possesses the activity desired.

Active Replication of the Episome by Toxoplasma-- To demonstrate active replication of the episomes, the methylation status of DNA recovered from Toxoplasma was examined using the dam methylase-sensitive restriction enzymes DpnI (only cuts DNA methylated by dam) and DpnII (will not cut dam-methylated DNA). Inasmuch as this methylase activity is not found in eukaryotic organisms, and the DNA used in all parasite transformations was from the dam(+) DH12 strain of E. coli, the loss of methylation (i.e. resistance to DpnI) would verify that the DNA was replicated by the parasite. Genomic and episomal DNAs isolated from the second and third passage from the experiment described above (data shown in Fig. 2A) were digested with either DpnI or DpnII and electroporated into E. coli. Fig. 2B shows that as early as the second passage, there is a clear difference in the number of colony-forming units arising from the digests between the EMS-bearing pANA-0.5 and parental vector. The activity of the enzymes in each reaction was confirmed by showing that the dam-methylated pACYC184 spike was virtually eliminated by the DpnI digest (<= 0.01% number of cfus obtained without digest) and not affected by the DpnII digest (data not shown). These data demonstrate the active replication of unselected episomes carrying the 500-bp EMS.

Interestingly, the DpnI resistance detected in the vector lacking the EMS (see Fig. 2B) suggests that a small number of these plasmids was replicated by the parasite. Although this appears to be a significant alteration of the methylation status when compared with the pACYC184 control, there is no way of controlling for differences in accessibility of the enzyme between the vectors recovered from the parasites and the pACYC184 spike. The different methylation profiles of pANA and pANA-0.5 at the second and third passage indicate that the difference in the number of replicated (DpnI-resistant) episomes is due either to the specific replication of pANA-0.5 episomes (ARS activity) or "nonspecific" replication of the constructs with the selective maintenance of those bearing the EMS (centromeric activity). Nonspecific replication refers to an unidentified ARS-like activity already present in the vectors analyzed without the requirement for Toxoplasma sequences. This form of episomal replication was observed in Leishmania (22) and may be responsible for the prolonged transient transformation observed in Toxoplasma (see below). However, the relative numbers of enzyme-resistant vectors and the comparison of the total number of episomes between the EMS ± vectors (see Fig. 2B) suggest that the constructs of the pANA-0.5 transformation are being actively converted from a DpnII-resistant phenotype to one that is resistant to DpnI. This would support the hypothesis that the activity ascribed to the EMS is one of elevating the replication competency of the plasmid in Toxoplasma (i.e. ARS-like activity).

Transformation Efficiency under MPA Selection-- The 500-bp EMS was subcloned into pHANA (pHANA-0.5) for a more quantitative analysis of transformation frequency under MPA selection in place of the slow kinetics of chloramphenicol activity. A total of 2 × 106 plaque-forming units of the RHDelta HXGPRT strain were electroporated with 10 µg of pHANA or pHANA-0.5, and 5 × 103 or 1 × 103 were immediately plaqued with or without MPA, respectively. An additional sample (5 × 104) of this electroporated population was grown under MPA selection to propagate the HXGPRT-transformed parasites as a passage flask for subsequent plaque assays. After sufficient time for plaque development, the parasites plated for the plaque assay were fixed and stained for analysis, while those grown in the passage flask (which had not yet completely lysed) were syringe-released, counted, and plated for a second round of plaque assay (± MPA) and passage in MPA(+) media. This procedure was repeated for five passages. After the second passage, the population corresponding to the pHANA transformation was not able to form plaques in drug-free medium, whereas the pHANA-0.5 population maintained a consistent percentage of viability throughout the selection (see Fig. 3). The inability to form plaques in drug-free medium after growth in MPA demonstrates that the plaques of the second passage were not viable at the time of harvesting the parasites. The difference in viability over time between the two populations demonstrates the transformation stability conferred by the EMS sequence on the pHANA vector. The delayed killing is most likely to be the result of the unstable transient transformation that was observed in Fig. 2A using pANA transformations without selection.


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Fig. 3.   Comparison of survival under MPA selection between pHANA and pHANA-0.5 in the RHDelta HXGPRT strain. The percentage of viable parasites obtained from RHDelta HXGPRT populations transformed with pHANA and pHANA-0.5 after growth in the presence of MPA is shown. Parasites grown under MPA selection (in parallel to the plaque assay) were syringe-released from host cells, counted, and plated for a plaque assay in the absence of drug pressure to determine the number of plaque-forming units surviving each passage. The percentage of viable parasites is derived from the number of plaque-forming units obtained divided by the total number of parasites plated for the assay.

Consistent with the above data, the percentage of MPA-resistant parasites gradually increased and leveled out at 35-40% in the pHANA-0.5 population, whereas the pHANA population dropped to zero at the third passage (data not shown). The observation that pHANA-0.5 transformants did not achieve more than 40% resistance to MPA after five passages under drug pressure was also seen in individual clones isolated from these populations that carry the pHANA-0.5 as an episome (data not shown). Since the RH strain demonstrates virtually 100% MPA resistance, wild type levels of HXGPRT should allow full survival. The decreased efficiency in the episomally transformed parasites may be due to the incomplete stability of this episomal DNA (see Fig. 2A). As many of these parasites would still be viable for plaque formation in the absence of MPA, the number of viable parasites is consistently higher than those maintaining sufficient levels of HXGPRT.

Configuration of pHANA-0.5 under MPA Selection-- RHDelta HXGPRT parasites, electroporated with 1, 10, and 100 µg of pHANA-0.5, were grown under MPA pressure and followed for five passages to determine the replication competence and copy number of episomes per parasite. Fig. 4A illustrates that by the third passage, all three of the transformations leveled out to approximately the same number of recoverable episomes/parasite. This convergence to a common copy number, despite the enormous difference in the amount of DNA used in the electroporation, implies tight regulation of the replication and/or segregation of the episomal DNAs. The observed control of episomal copy number may provide an alternative explanation to the incomplete MPA resistance observed in pHANA-0.5 transformed strains described above; if there is insufficient expression of HXGPRT activity from the DHFR-driven cassette in pHANA-0.5, a conflict with the tight control over the episomal copy number may inhibit the development of viable plaques under drug pressure. This hypothesis was not investigated further.


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Fig. 4.   Analysis of episomal stability and configuration under MPA selection for pHANA-0.5 in the RHDelta HXGPRT strain. A shows the rapid convergence of episomal copy numbers to a uniform level after transformation with varying amounts of DNA. After the fourth passage, the different transformed populations begin to lose the uniformity as individual transformants with a selective advantage are dominating the populations. B shows a Southern blot of EcoRI digests from DNA of the first five even-numbered passages of the population transformed with 10 µg of pHANA-0.5 and five individual clones carrying the same construct using a pANA probe. As suggested above, the observation of larger EcoRI fragments in the population at later passages indicates a selection for recombinants of pHANA-0.5 that have either integrated into the genome of RHDelta HXGPRT or recombined with other fragments of DNA in an episomal form. From five individual clones isolated from the second passage of a similarly treated population, clones 2, 3, and 5 appear to possess band(s) migrating at or near the expected rate for pHANA-0.5. Lanes 6 and 7 represent genomic DNA of the RHDelta HXGPRT strain without (negative control) and with (positive control) a spike of pHANA-0.5 vector, respectively.

The population electroporated with 10 µg was selected for an additional five passages to assay long term maintenance of the construct. Total genomic and episomal DNAs were digested with EcoRI from the even-numbered passages (2-10) and blotted for Southern hybridization using the ampicillin resistance marker of pANA as a probe (see Fig. 4B). As the EcoRI recognition site is unique in pHANA-0.5, the gradual increase in band size in later passages indicates that the episome integrated within the genome and/or recombined with additional DNA. Although only vector-size bands were detectable in the initial passages of the population, the evolution of these larger forms may have occurred early if there was a sufficient selective advantage for them over the unaltered pHANA-0.5. To determine if this recombinational activity occurred early or if it was a late event resulting from conformational instability of the episome, five MPA-resistant clones were isolated after the second passage of a population of RHDelta HXGPRT parasites transformed with 10 µg of pHANA-0.5. After 31-35 days of continuous culturing under MPA selection, DNA was extracted from the clones and electroporated into DH12 for the recovery of episomes. Of the five parasite clones, three were found to carry the construct episomally at a level consistent with the results obtained previously on a population scale (~5 × 10-3 episomes/parasite). Fig. 4B shows a Southern hybridization of the DNA from these clones digested with EcoRI and probed with the ampicillin resistance marker used above. The three episome-carrying clones possess band(s) co-migrating with the starting plasmid indicating that under prolonged growth, the introduced DNA can be maintained in episomal form, although these data do not address multimerization (see below). Thus, the observation that the prolonged maintenance of the population of transformed parasites shows predominantly heterogeneous, high molecular weight bands is more likely due to selection of rare clones that have a growth advantage than to inherent tendencies for the episome to integrate or acquire extraneous sequences.

To achieve an accurate determination of the episomal copy number, one of the three clones was chosen for Southern blot analysis using a DHFR probe to compare the copy number of pHANA-0.5 (using the DHFR sequences flanking the HXGPRT gene) to the endogenous single copy gene (7). Since we could not exclude the possibility that the DNA extraction procedure normally used could enrich for either the episomal or genomic sequences, parasites of this clone were embedded in an agarose plug prior to lysis and digestion for Southern blot analysis. The restriction endonuclease AvaII was used so that bands corresponding to the endogenous copy (4.3 kb) and the episomal copy (3 kb) that are complementary to the probe could be resolved. The blot was probed with the 5'-upstream sequence of DHFR, and after phosphorimaging analysis, an average copy number of 2 to 3 episomally derived DHFR sequences per genome equivalent was determined (data not shown). To examine the possibility of concatamerization, DNA extracted from this clone was partially digested with EcoRI and blotted for hybridization to a probe generated from the pANA backbone. Fig. 5 shows the resolution of the episome to a doublet of approximately equal intensity where one band co-migrates with pHANA-0.5 and the other slightly faster, indicative of a small deletion. Upon transforming E. coli with the episome of this clone, only constructs with RsaI restriction digest patterns indistinguishable from pHANA-0.5 were isolated (data not shown). This suggests that upon transformation of the DNA into E. coli, the concatamer is resolved to a monomeric form by the bacterial DNA recombination machinery (i.e. RecA). Concatamerization was also observed upon the analysis of 10 cfus isolated from the DNA of the selected population after 20 passages (77 days post-electroporation). A Southern blot of the RsaI-digested DNA, probed with the HXGPRT cassette and 500-bp EMS sequences, indicated that of the 10 cfus, one was indistinguishable from pHANA-0.5 whereas the rest consisted of at least four distinct groups of recombinants with two carrying more than one copy of both the HXGPRT cassette and the EMS sequences (data not shown). Since these episomes were not resolved by E. coli to the monomeric forms, we assume that the recombinational forms are more complex than simple concatamers. The above data suggest that there is a selection for recombination to enhance episomal stability and/or elevate the level of HXGPRT expression to better survive the MPA pressure.


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Fig. 5.   Comparison of episomal configurations between a clone of RHDelta HXGPRT stably transformed with pHANA-0.5 and a pHANA-0.5-spiked untransformed control by Southern blot analysis. Lane 1 corresponds to RHDelta HXGPRT genomic DNA completely digested with EcoRI for 2 h. Lanes 2, 4, 6, and 8 correspond to DNA recovered from 2 × 107 parasites of the RHDelta HXGPRT parental strain that was spiked with pHANA-0.5 at a concentration equivalent to 5 copies per parasite and digested for 0, 5, 30 min, and 2 h, respectively. The dominant bands in lanes 2 and 4 migrate at the rate of a nicked, circular form of pHANA-0.5 as established in previous blots. This band disappears as digestion is brought to completion, and the linearized version, migrating at 5.2 kb, appears in its place. Although the super-coiled form has been observed on previous blots, it appears that the majority of the spike DNA was nicked prior to electrophoresis as the super-coiled form is not detectable in this exposure. Lanes 3, 5, 7, and 9 correspond to similar amounts of DNA from clone 2 pHANA-0.5 (see Fig. 4B) digested with EcoRI for 0, 5, 30 min, and 2 h, respectively. The partial digest was used to illustrate the concatameric nature of the construct in the transformed strain as compared with the monomeric form of the spike in control lanes. Bands in lanes 3, 5, and 7 (indicated by A, B, and C) are believed to correspond to the nicked circular, linear, and supercoiled forms of the dimer present in this clone, respectively (see text). The band migrating above band A (the nicked-circular form of the dimer) in these lanes was also observed in the control lanes (lanes 2, 4, and 6) in longer exposures. Although the origin of this band is unknown, there does not appear to be a correlation of its presence and the concatamerization of pHANA-0.5 in clone 2. Lane 9 shows the dimer apparently consists of two versions of pHANA-0.5, one intact and the other with a small deletion.

Complementation of HXGPRT-deficient Mutants-- One of the potential uses of the EMS is in creating libraries for efficient complementation. To test this, a chemically mutagenized HXGPRT-deficient point mutant (TXR-2) was transformed with a genomic library in the pANAE vector (pANA carrying the EMS outside the multicloning site, see "Experimental Procedures") and selected for MPA resistance. This should result in the selection for plasmids carrying the HXGPRT gene. Although the same procedure was performed on the RHDelta HXGPRT strain, the point mutant serves as a superior test for the stability of the episome carrying large stretches of homologous DNA without the advantage of deletions in the loci of interest. An additional library was generated in the pANA vector (i.e. lacking the 0.5-kb EMS) to evaluate any differences that may be attributed to the possession of the EMS in the library backbone. The mutants were electroporated with 100 µg of each library (~17 pmol) and immediately plated on 100-mm Petri dishes of human foreskin fibroblasts for plaque development under drug pressure. As an accurate count of plaque-forming units requires the fixation and staining of the monolayer, duplicate plates for each transformation were required to allow the isolation of viable clones from a second plate. Dishes destined for plaque isolation were overlaid with media containing MPA and agar as described under "Experimental Procedures."

Following plaque development, the transient transformation frequency of TXR-2 was determined to be 22 and 9 MPA-resistant plaques per 106 plaque-forming units with the pANAE and pANA libraries, respectively. From nine plaques that were picked from the pANAE-transformed population, four were successfully expanded with one under constant MPA pressure and three grown in drug-free medium. Of the six pANA plaques picked for expansion, one was obtained in drug-free medium and one in the MPA-containing medium. The four clones added to drug-free medium were grown for 3.5 weeks before a final passage under MPA selection to recover those maintaining HXGPRT expression. Genomic and episomal DNAs from each of the six MPA-resistant clones were extracted and used to transform E. coli; five of these clones were found to carry episomal copies at numbers comparable with those observed in the pHANA-0.5 transformants (data not shown). The one odd clone, from the pANAE library grown without selection, had very few episomes competent for E. coli transformation. Of the few colony-forming units recovered from this clone, plasmid extraction was considerably less efficient, and three different restriction patterns were observed using PvuII. Southern blot analysis of the distinct constructs isolated from this clone show one carrying the EMS migrating at a rate suggestive of extensive recombination and a second that does not carry the EMS but does possess a faint band migrating at 1.7 kb which hybridized to an HXGPRT probe (data not shown). Although this would suggest there are at least some episomal copies carrying the HXGPRT gene in the isolate (see below), the state of the complementing DNA was not further investigated due to the instability in E. coli.

In the five different strains possessing competent shuttle vectors, there appear to be three distinct inserts as judged by PvuII digestion. An additional distinct clone was obtained from a RHDelta HXGPRT transformation with the pANAE library. These four distinct recombinants are shown in Fig. 6A. All of the recombinants recovered from the TXR-2 transformation were also obtained from the complementation of RHDelta HXGPRT that was under constant selection with MPA. Both the cut and circular forms of the episomes in DNA preparations from isolated parasite clones migrated at rates equivalent to the constructs passed through E. coli, indicative of a monomeric, supercoiled conformation (data not shown). The isolated constructs ranged from 9 to 11 kb and were replicated by the parasite as determined by the absence of methylation using DpnI and DpnII (data not shown). As these constructs are maintained in a monomeric form without obvious recombinatorial alterations, the proposed selection for larger recombinant configurations observed with pHANA-0.5 does not appear to be dependent upon multiple copies of the EMS. While the selective advantage observed in these recombinant pHANA-0.5 constructs was not further investigated, we suggest that larger DNAs (>= 10 kb) may have an elevated stability in a circular, episomal form. The published sequence of the HXGPRT gene (17) predicts a PvuII fragment of 1.7 kb. Fig. 6B shows that all four of the distinct library inserts have a fragment of this size that hybridizes to a probe of the HXGPRT open reading frame. Thus, the pANAE-based library can indeed be used to complement a mutant and identify the affected gene at the expected efficiency of ~10-4 using an average insert size of 6 kb.


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Fig. 6.   Analysis of four distinct constructs maintained in HXGPRT(-) strains of RH as episomes isolated from genomic libraries by MPA selection. A shows a PvuII digest of four representatives of the constructs isolated from the libraries (lanes 3-5 and 7), their respective parental vectors (lane 2, pANAE and lane 6, pANA), and genomic DNA isolated from RH (lane 9). Lane 1 has the size markers (1-kb DNA ladder) and lane 8 is blank. A single band migrating at 1.7 kb is found common among the four isolated DNAs. B is a Southern blot of this gel illustrating the hybridization of the 1.7-kb band to a 700-bp HXGPRT probe. Lanes 3-8 were exposed for 30 min. A longer exposure of the genomic digest in lane 9 (24 h) verifies that the endogenous HXGPRT gene co-migrates with the common 1.7-kb band isolated from these constructs. The episome corresponding to HPT-E1 (lane 3) was isolated from RHDelta HXGPRT grown under constant drug pressure. The recombinants in lanes 4, 5, and 7 were isolated from the TXR-2 strain and obtained after growth in medium containing constant drug pressure (lane 5), without drug pressure (lane 4), or both in the presence and absence of drug (lane 7) as described in the text.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Genomic sequences of Toxoplasma were selected for the ability to stabilize episomal vectors in the parasite without selective pressure. One of the five selected genomic sequences was arbitrarily chosen and reduced to a fragment of 500 bp (EMS) that still possessed the stabilizing activity. As one of the primary goals of this project was to develop a shuttle vector for the use of complementation, we used a chemically mutagenized HXGPRT(-) mutant and the RHDelta HXGPRT mutant to determine if the HXGPRT gene could be recovered from genomic libraries. The number of transient transformants from both libraries agrees with the expected value of 1 × 10-4 using an average insert size of 6 kb given the size of the parasites' genome. Episomes recovered from these MPA-resistant parasites were found to be monomeric, supercoiled, and replicated by the parasite as judged by DNA methylation using DpnI and DpnII.

We were surprised to find parasites successfully complemented with the EMS(-) library that maintained the construct in an episomal form. Unlike pHANA, this construct does not appear to require the EMS to be maintained episomally. One explanation for this result could be an EMS-like sequence relatively close to the endogenous HXGPRT gene. Evidence supporting this hypothesis is the limited repertoire of Sau3AI fragments carrying the HXGPRT recovered from clones of separate transformation events. Whereas at least three distinct recombinants were recovered from the pANAE library, only one was obtained from the pANA library in both the RHDelta HXGPRT and TXR-2 mutant transformations. The predicted high frequency of Sau3AI sites in the genome and the >20-fold redundancy of the pANA library would suggest that if an EMS-like sequence is unnecessary, we would have obtained a larger number of viable clones carrying a variety of fragments encompassing this genetic locus. The predicted map of this locus suggests that the clone isolated from the EMS(-) library is biased to one side of the genomic fragment, also suggestive of the proposed phenomenon. Finally, when the construct isolated from pHPT-A1 is compared with the pHANA vector in the transformation of RHDelta HXGPRT under the selective pressure of MPA, a similar level of stability (~3 × 10-3 cfu/parasite) was observed over four passages as found using pHANA-0.5 (data not shown). The isolation and characterization of this proposed EMS-like element was not pursued in this report.

With the addition of this episomal stabilizing sequence to the current molecular toolbox in Toxoplasma, we expect to be able to further our understanding of the events critical to the invasion, intracellular replication, and differentiation of this parasite. Since the HXGPRT gene is able to be used both as a positive and negative selectable marker under the pressure of MPA and 6-thioxanthine, respectively (17), we expect this episomal shuttle vector will also be useful in testing whether a given gene is essential by asking if parasites can survive under conditions which select against the HXGPRT gene. This is a critical technique when handling haploid organisms.

    ACKNOWLEDGEMENTS

The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr. David Roos: T. gondii host strain RH(EP)Delta HXGPRT. We thank Adrian Hehl, Laura Knoll, and the rest of the Boothroyd laboratory for helpful suggestions.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AI 21423 and AI 30230, the University of California AIDS Research Program, and a fellowship from the Howard Hughes Medical Institution (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 415-723-7984; Fax: 415-723-6853.

1 The abbreviations used are: ARS, autonomous replicating sequences; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; EMS, episomal maintenance sequence; kb, kilobase pair(s); bp, base pair(s); DHFR, dihydrofolate reductase; MPA, mycophenolic acid; cfu(s), colony-forming unit(s); EM, episomal maintenance.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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