From the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 RHHXGPRT 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).
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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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
(RHHXGPRT (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).
|
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.
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 RHHXGPRT 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.
|
Configuration of pHANA-0.5 under MPA Selection--
RHHXGPRT
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.
|
|
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 RHHXGPRT 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."
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 RH
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 RH
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 RH
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)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.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|