1
Department of Veterinary Parasitology, University of Glasgow, Bearsden Road,
Glasgow, G61 1QH, UK
2
Department of Molecular Recognition, The Hannah Institute, Mauchline Road, Ayr
KA6, Scotland, UK
3
Adnan Menderes University, Faculty of Veterinary Medicine, Department of
Parasitology, Isikli, Aydin, Turkey
*
Author for correspondence (e-mail:
d.swan{at}vet.gla.ac.uk
)
Accepted April 27, 2001
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SUMMARY |
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Key words: Apicomplexan parasite, Host, parasite interaction, AT hook DNA-binding motif, Theileria annulata
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INTRODUCTION |
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Parasite infection of the host cell is associated with modulation of
leukocyte gene expression, including a number of genes encoding transcription
factors that are implicated in the control of cell division or apoptosis
(Dobbelaere et al., 1988;
Baylis et al., 1995
; Ole-MoiYoi
et al., 1993
; Botteron and
Dobbelaere, 1998
; Heussler et
al., 1999
). However, although
induction of host cell division is known to be Theileria dependent
(McHardy et al., 1985
), little
is known about how the parasite directly modulates leukocyte gene expression
or stimulates the host cell to divide.
It has been suggested that as the macroschizont differentiates into the
extracellular merozoite, parasite factors involved in host cell division may
be downregulated, resulting in the cessation of cell division (Carrington et
al., 1995). Thus, the
association between parasite and host cellular division would be uncoupled,
owing to the removal of the signal that initiates proliferation of the
infected lymphocyte. We have previously identified a small gene family whose
expression is downregulated during differentiation to the merozoite in T.
annulata (Swan et al.,
1999
). One member of this
family, TashAT2 encodes a gene product that bears a predicted AT hook
motif DNA-binding domain. Furthermore, experimental data suggest that TashAT2
is transported from the parasite to the host nucleus, implying a role in the
modification of host cell gene expression. We present further characterisation
of TashAT2 and two other members of the gene family, TashAT1
and TashAT3. All three genes form part of a cluster, encode AT hook
DNA-binding motifs (Johnson et al.,
1988
) and are very closely
related in sequence. We present evidence to suggest that TashAT3 and,
possibly, TashAT1 could be transported to the host cell nucleus and discuss
the possible implications.
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MATERIALS AND METHODS |
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Cloning and northern blot analysis
A fragment of TashAT1 had previously been isolated from a
gt11 library of genomic DNA derived from merozoites of the D7 infected
cloned cell line (Swan et al.,
1999
). The TashAT1
fragment was used to screen a
DASHII library of D7 genomic DNA using
standard protocols. Two
DASHII overlapping clones, based on their
restriction maps and hybridisation profiles, were isolated and a 13.4 kbp
region sequenced on both strands. DNA sequencing was performed on a Licor 4000
automated DNA sequencer according to the manufacturer's protocol. DNA and
protein analyses were performed using the GCG sequence analysis package
(Devereux et al., 1984
).
RNA from in vitro cultured cells and from purified T. annulata
(Ankara) piroplasms was isolated by the Triazol reagent, according to the
manufacturer's instructions (Sigma). Sporozoite RNA was isolated from T.
annulata (Ankara)-infected Hyaloma anatolicum ticks as described
by Williamson et al. (Williamson et al.,
1989). RNA was size
fractionated by electrophoresis through a formaldehyde-agarose gel and
analysed by Northern blotting as previously described (Shiels et al.,
1994
). Hybridisation was
carried out overnight at 65°C according to the method of Church and
Gilbert (Church and Gilbert,
1984
).
Generation of fusion protein and immunoblotting
A 362 bp fragment of TashAT1 starting 63 bp from the first
putative translational start site was PCR amplified using ampliTaq polymerase
and the primers; 5'-tttaggatccgtaaaatttgcttcttcc-3' and
5'-gaaggaattctggtggaattttaataaa-3'. The PCR product was then
subcloned into the vector pGEX-2TK (Pharmacia), expressed in Escherichia
coli strain JM109 as a glutathione-S-transferase (GST) fusion
protein and purified on a glutathione-sepharose column using the Pharmacia
protocol. Antisera (anti-TashAT1/3) to the fusion protein were raised in New
Zealand White rabbits and immunoblotting was carried out as described by Swan
et al. (Swan et al., 1999).
Signal was detected by ECL using the method provided by the suppliers (Pierce
and Warriner).
DNA-binding analysis of TashAT1
A gt11 library of D7 genomic DNA or purified
gt11 clones
were induced to express fusion protein under standard conditions.
Nitrocellulose filters were laid on top of the developing plaques, which were
then incubated at 37°C for 4 hours. The filters were removed from the
plates, washed briefly in TNE 50 (10 mM TrisCl pH 7.5, 50 mM NaCl, 1 mM EDTA,
0.1% NP40), then incubated overnight in blocking buffer (2.5% dried milk, 25
mM Hepes (pH 8.0), 1 mM DTT, 10% glycerol, 50 mM NaCl, 1 mM EDTA, 0.1% NP40)
at 4°C. The filters were then rinsed at 4°C in 1x binding buffer
(1x BB; 25 mM Hepes pH 7.9, 3 mM MgCl2, 4 mM KCl) and the
expressed fusion protein denatured by incubation in 1xBB containing 6 M
guanidinium HCl, 1 mM DTT twice for 5 minutes. Renaturation was then carried
out by immersing the filters for 5 minutes in denaturation buffer, which was
sequentially diluted 1 in 2 with 1xBB, 1 mM DTT, four times. The
renatured filters were washed in 1xBB, 1 mM DTT, incubated in
1xBB, 5% dried milk for 30 minutes, rinsed in 1xBB, 0.25% dried
milk and washed briefly with HNE (10 mM Hepes pH 7.9, 50 mM NaCl, 1 mM EDTA, 1
mM DTT). Hybridisation was then carried out in HNE buffer containing 500 µg
ml-1 poly dIdC filtered through a 0.22 µm filter. Double
stranded concatenated probes were generated by ligating oligonucleotides
radiolabelled at the 5' end with 32P using T4 polynucleotide
kinase. The oligonucleotides were: CAT1,
5'atgcGCACACAATTTGTAGGGCGAC3'; CAT1m2,
5'atgcGCACACAATTACGAGGGCGAC3'; CAT2, 5'
atgcAGATAAACATGCACACAATTTGTA3'; or CAT3, 5'atgcAGGGCGAC3'
previously annealed to their respective complementary oligonucleotides. Each
oligonucleotide had a four base overhang at the 5' end, either atgc (or
gcat in the reverse complement) shown in lower case, for concatenation. Probes
were added to the filters and incubated at 4°C for 60 minutes, after which
the filters were given three 5 minute washes in HNE buffer, followed by
exposure to X-ray film.
DNA-binding analysis of TashAT2
The procedure for determining TashAT2 DNA-binding specificity was based on
the method used by Pollock and Treisman (Pollock and Treisman,
1990), but used
glutathione-sepharose instead of immunoprecipitation to bind the DNA-protein
complex. In brief, a double-stranded 76 mer oligonucleotide with a 26 base
pair random core flanked by two specific 25 base sequences (primer F and
primer R) was PCR amplified and labelled using 32P dCTP. A part of
TashAT2 containing the AT hook domain (amino-acid residues 296-541) linked to
GST as a fusion protein (GST-TashAT2) (Swan et al.,
1999
), was purified and
approximately 3 µg of GST-TashAT2 or 3 µg of purified GST alone used per
binding cycle. 100 µl of a 50% solution of glutathione-sepharose was washed
twice in phosphate buffered saline (PBS) before adding 100 µl of PBS
containing the fusion protein. The mixture was then rotated at room
temperature for 15 minutes and the beads washed three times in binding buffer
(10 mM Hepes pH 7.9, 25 mM KCl, 1 mM EDTA, 50 mM NaCl, 0.1% NP40 plus protease
inhibitors) to remove excess protein. 0.4 ng of radiolabelled probe was added
to the beads in 25 µl of binding buffer and rotated for 30 minutes at
4°C. The beads were washed in binding buffer three times, followed by
phenol/chloroform extraction. The DNA was then ethanol precipitated from the
aqueous phase, washed in 70% ethanol and resuspended in 20 µl of TE pH 8.0.
10 µl were taken for PCR amplification using primer F and primer R, and the
cycle of protein to DNA binding repeated four times. To test the ability of
the amplified sequences to bind GST-TashAT, an electrophoretic mobility shift
reaction (EMSA) was set up containing 10 µl of the radiolabelled DNA that
was purified during each cycle, 0.5 µg of protein, 1xBB, 5% Ficoll, 1
µg of poly dGdC:dGdC in a 40 µl reaction. The reaction mix was incubated
for 30 minutes at 4°C. Electrophoretic separation was then carried out on
a 4% polyacrylamide gel cast in 0.5xTBE and run in 0.5xTBE buffer.
A mobility shifted band from cycle 4 was excised, eluted into dH2O,
PCR amplified and the EMSA repeated. The resulting mobility shifted band was
excised, PCR amplified and subcloned into the vector pGEMT-easy for
sequencing.
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RESULTS |
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Immunofluorescence indicates the host nucleus as a possible location
for TashAT1/3
Antisera were initially raised against the N terminus of TashAT1; however,
it was subsequently determined that this sequence is also present in TashAT3.
Therefore, all immunofluorescence studies carried out with this antisera
cannot distinguish between TashAT1 and TashAT3. Immunofluorescence analysis of
COS7 cells transfected with TashAT2 showed no reactivity using anti-TashAT1/3
indicating specific detection of TashAT1/3 (data not shown).
When anti-TashAT1/3 was used in IFAT analysis of the T.
annulata-infected cells, D7B12 and D7
(Fig. 3C,D), immunoreactivity
was observed against the macroschizont, displaying a punctate pattern of dots
that may represent reactivity with or the region surrounding parasite nuclei,
and against the host cell nucleus (Fig.
3C,D). In D7B12 cells, host nuclear reactivity was very bright in
some cells, although a large variation in immunoreactivity was observed within
each cell population. There was no reactivity with the uninfected control,
BL20 (Fig. 3E), whereas with
T. annulata-infected BL20 cells (TBL20)
(Fig. 3B), faint reactivity
with the host nucleus was obtained. During a differentiation time course of D7
cells, anti-TashAT1/3 reactivity against the host nuclei diminished overall,
although some cells showed clear fluorescence
(Fig. 3E). These cells probably
represented undifferentiated macroschizont-infected cells as the
differentiation process is known to be stochastic (Shiels et al.,
1994).
|
Differential expression of TashAT mRNA
In order to examine the relevance of the TashAT cluster to T.
annulata-infected cells in general, we determined the RNA expression
profile of all three TashAT genes in a range of
Theileria-infected cell lines, and in the sporoblast/sporozoite and
piroplasm stages of the parasite life cycle. The in vitro infected cell lines
analysed were the T. annulata (Ankara)-infected cell line TaA2 and
cloned cell lines derived from TaA2 (D7, D7B12, C9, and E3); low and high
passage cell lines infected with T. annulata (Hidirseyh) and T.
annulata (Diyarbakir); and the bovine lymphosarcoma cell line, BL20 and
its T. annulata (Ankara)-infected counterpart, TBL20. mRNA species at
2.1 kb, 3.6 kb and 4 kb have been deduced to correspond to TashAT1, 2
and 3 respectively, using probes derived from each of the individual
TashAT genes, which gave a more specific signal (Swan et al.,
1999; D.G.S.,
unpublished).
Northern blots were probed with the entire radiolabelled TashAT3 gene, which, out of the three TashATs, has the best overall homology with the other two TashATs. TaA2 and the cloned cell lines derived from it expressed the three mRNA species detected at 4 kb, 3.6 kb and 2.1 kb, corresponding to TashAT2, TashAT3 and TashAT1, respectively (Fig. 4A). Each of the cloned cell lines had essentially the same expression profile as the parent cell line, TaA2. TashAT1 and TashAT2 mRNA levels were more abundant than those of the TashAT3 RNA species; except in the case of D7B12 where TashAT3 mRNA levels were higher, and TashAT2 mRNA levels decreased slightly (Fig. 4A). TashAT1 expression did not alter significantly and was the most highly expressed of the TashATs in TaA2 and the cloned cell lines derived from TaA2. In marked contrast, cell lines T. annulata (Hidirseyh), and T. annulata (Diyarbakir) expressed TashAT1 at low to barely detectable mRNA levels (Fig. 4A). TashAT2 and TashAT3 mRNA species were present in these cell lines, although the levels of TashAT3 mRNA was fainter. TashAT2, however, was the only TashAT message detected in TBL20s. The TashAT probe was also used in hybridisation analysis of T. annulata (Ankara) sporoblast/sporozoite RNA and piroplasm RNA (Fig. 4A). The results indicated that TashAT3 and TashAT1 were expressed by sporoblast/sporozoites, and at this stage of the parasite life cycle, the TashAT3 message was detected at the highest level (Fig. 4A). There was no signal detected in piroplasm RNA by any of the TashAT probes. Hybridisation of the same blots with the gene encoding the parasite large subunit rRNA (Fig. 4B) confirmed that the mRNA levels of all three TashAT genes can vary depending on the cell line and passage number.
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Immunoblot analysis using anti-TashAT1/3
To determine whether anti-TashAT1/3 reactivity against the host nucleus in
D7B12 cells was due to recognition of TashAT1, TashAT3, or both, immunoblot
analysis was carried out on extracts of whole cells and host or parasite
enriched nuclear fractions (Fig.
5). A faint band at 180 kDa and a stronger band at 66 kDa were
specifically detected in whole cell extracts, relative to pre-immune serum, by
anti-TashAT1/3. Partitioning of host and parasite nuclear fractions, showed a
clear enrichment of the 180 kDa band in the host nuclear fraction, while the
66 kDa band was detected at increased levels in the parasite-enriched nuclear
fraction. Assessment of the origin of these polypeptides, host or parasite,
was performed by analysis of an uninfected BL20 extract. Faint recognition of
a band at 66 kDa, along with a band of 125 kDa, indicated that the
anti-TashAT1/3 antisera crossreacted with bovine-derived polypeptides. It was
concluded that the most likely candidate for a parasite-derived molecule
specifically detected by the antisera was the 180 kDa polypeptide. From the
predicted size of the ORFs, this polypeptide is unlikely to be encoded by
TashAT1 but could be derived from TashAT3.
|
TashAT polypeptides bind specifically to AT rich DNA
To test TashAT2 for DNA binding, a recombinant protein representing the AT
hook region and an upstream basic region of TashAT2 fused to the GST gene
(GST-TashAT2) was used in DNA-binding assays with a radiolabelled randomised
double-stranded oligonucleotide. Cycles of binding followed by PCR
amplification were carried out in an attempt to enrich for DNA with affinity
for GST-TashAT2. Protein-DNA complexes obtained with the enriched DNA
sequences were visualised by mobility shift gel electrophoresis and after four
binding cycles, an increase in the DNA-protein complex was clearly visible
(Fig. 6A). The complex from
cycle 4 was eluted from the mobility shift gel, PCR amplified and a further
mobility shift (Fig. 6A)
selected for a more specific DNA-protein complex. DNA eluted from this complex
was PCR amplified and subcloned. The sequences obtained from the inserts of 12
subclones are shown in Fig. 6B.
Although the sequences are not the same, they are all AT rich and show general
similarities. Thus, there are three AT-rich regions discernible in each
sequence separated by one to four G or C nucleotides, and a high proportion of
the AT-rich regions contain the sequence ATTTA or TAAAT.
|
As part of a separate investigation into stage differentiation of T.
annulata, a gt11 library of D7 genomic DNA had been screened for
proteins that bind a DNA motif located upstream of the Tams1 gene,
which encodes the major T. annulata merozoite surface protein (Shiels
et al., 2000
). One of the
concatenated oligonucleotide probes, CAT1
(Fig. 7A), bound to a
gt11 clone that expressed a fragment of TashAT1 containing
the AT hook DNA-binding domain. To test whether this binding was sequence
specific and whether it related to the specific mobility shifts observed for
the Tams1 promoter (Shiels et al.,
2000
), and to determine which
regions of the probe were required for binding to the TashAT1 fusion protein,
the purified lambda clone was probed with three more concatenated
oligonucleotides (Fig. 7A).
CAT1M2 is the same as CAT1 but with a three base pair change, CAT3 is composed
of the GC 3' region of CAT1 and CAT2 overlaps with CAT1 with 10 extra
bases 5' and missing the GC-rich region of CAT3. This demonstrated that
concatenated CAT3 bound weakly to the TashAT1
gt11 clone in
comparison with the CAT1, CAT1M2 and CAT2 probes. Binding reactions using the
CAT1M2 (Fig. 7B, panel A) and
CAT3 (Fig. 7B, panel B) probes
are shown. Neither probe displayed affinity for a control
gt11clone
purified from the same library (Fig.
7B, panels C,D) that was shown to express recombinant polypeptide
by reactivity with bovine antiserum raised against the parasite (data not
shown).
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DISCUSSION |
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The AT hook domains of TashAT2 and TashAT1/3 are very similar, but there
are some notable differences. TashAT2 has three AT hook motifs, whereas
TashAT1/3 has four. Also present in TashAT1/3 are two small basic repeats,
similar in sequence to regions found in the HMG1/2 DNA-binding domains
(Landsman and Bustin, 1993).
It could be proposed, therefore, that TashAT1/3 has a stronger affinity for
DNA than TashAT2 and that their sequence specificities will be different.
The individual members of the TashAT family show remarkable
sequence conservation. In particular, TashAT1 is virtually identical
to the 5' part of TashAT3, and TashAT2 and
TashAT3 are very similar over a region beginning at the AT hook
domain. This suggests a very recent duplication event has occurred, possibly
owing to adaptation to in vitro cell culture. Southern blotting of DNA derived
from a range of in vitro cell lines and in vivo derived piroplasm DNA,
however, indicates the existence of all three gene copies (data not shown).
Therefore, it would appear that gene duplication has occurred in vivo and is
not unique to a few in vitro infected cell lines. Identical copies of genes
have been found in other apicomplexan parasites; there are two identical
copies of the elongation factor in Plasmodium knowlesi and
Plasmodium berghei (Vinkenoog et al.,
1998
), of the rhoptry
associated protein from Babesia bovis (Suarez et al.,
1998
) and of the Rop2
gene from Toxoplasma gondii (Beckers et al.,
1997
).
Northern blot analysis of various cell lines indicated that in general, TashAT2 was found to be the most consistently and highly expressed of the genes. This may be related to the previous detection of anti-TashAT2 reactivity in macroschizont-infected in vivo derived cells (D. G. S. and B. R. S., unpublished). Analysis of RNA from tick derived sporozoites showed differences in the TashAT expression profile. TashAT3 is the major TashAT RNA to be expressed in sporoblasts/sporozoites, while the RNA species representing TashAT1 was also detected. The relationship between these expression patterns and the functional role of TashAT1/3 is not clear but their primary role might be during sporoblastogenesis in the tick salivary gland. Alternatively, they could function to allow the establishment of the parasite following sporozoite invasion of the bovine leukocyte. Either of these roles could involve transport to the host cell nucleus and modulation of tick or bovine gene expression.
The levels of TashAT1 and TashAT3 mRNA were found to be
plastic across different cell lines. Thus, TashAT3 was less abundant
than TashAT2 in the majority of cell lines, but more abundant in the
D7B12 cell line. A higher level of TashAT3 expression in D7B12 cells
was supported by immunoblot and immunofluorescence data. In a similar fashion,
TashAT1 mRNA levels were extremely low in the non-cloned cell lines.
The reasons for this plasticity in expression are unclear but may be related
to the derivation of individual cell lines or clones. Furthermore, it is
possible that the host cell background can influence the expression of the
TashAT gene cluster, as expression levels were significantly lower in
the TBL20 line derived from sporozoite infection of previously immortalised
BL20 cells. One possible consequence of the plasticity of TashAT
expression is that it may relate to the observed differences in bovine gene
expression displayed by individual cell lines and different passages of the
same cell line or clone (Adamson et al.,
2000; Sutherland et al.,
1996
).
The presence of three TashAT genes in a small cluster, coupled to
the possible presence of TashAT2 and TashAT1/3 in a host nucleus, provokes the
idea that at least two (and possibly more) parasite genes are involved in
modulation of the host environment by altering the control of leukocyte or
tick cell gene expression. Whether these proteins target different subsets of
genes is unknown, but modulation of host cell environment could include
induction of host cell division, as rearrangements of the AT hook that
contains HRX gene fused to a variety of partners are common in several types
of leukaemia (Waring and Cleary,
1997). Furthermore, the
archetypal AT hook-containing protein, HMGI(Y) and the closely related HMGIC
are upregulated in proliferating, non-differentiated cells, and chromosomal
translocations involving HMGI genes are found in many types of neoplasia
(Hess, 1998
). Thus, AT hook
DNA-binding proteins play a very important role in the control of eukaryotic
gene regulation and proliferation. It is therefore not unreasonable to
speculate that the TashAT cluster performs similar tasks in
Theileria-infected cells.
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ACKNOWLEDGMENTS |
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