Wild-type 3D7 WT parasites transfected with water not subjected A or subjected B to 72 h of pyrimethamine pressure; parasites transfected with pHC1 empty vector control C and pHC1G6 PD-
Trang 1bifunctional glucose-6-phosphate dehydrogenase )
6-phosphogluconolactonase
Almudena Crooke1,*, Amalia Diez1, Philip J Mason2,†and Jose´ M Bautista1
1 Department of Biochemistry and Molecular Biology IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Madrid, Spain
2 Haematology Department, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, UK
Malaria is a major health hazard in tropical and
sub-tropical areas around the world In Africa alone, every
year over a million children under the age of 5 years
die of malaria and around 300–500 million people are
infected by the parasite [1,2] Added to this, the
appearance of parasites resistant to antimalarial drugs
is on the increase and it is not proving easy to develop
an efficient vaccine against Plasmodium falciparum
There is thus a need for new therapeutic targets
The sequencing of the P falciparum genome [3–5] has revealed a large amount of molecular information This information, coupled to microarray mRNA ana-lysis [6,7] and specific expression proteomic anaana-lysis of the parasite’s developmental stages [8], is allowing the molecular exploration of new strategies to fight against malaria
Based on their equivalent functions in other organ-isms, the search for genes thought to be essential for
Keywords
antisense RNA; dsRNA; gene silencing;
glucose-6-phosphate dehydrogenase;
malaria; Plasmodium falciparum
Correspondence
J.M Bautista, Departamento de Bioquı´mica
y Biologı´a Molecular IV, Universidad
Complutense de Madrid, Facultad de
Veterinaria, Ciudad Universitaria, 28040
Madrid, Spain
Fax: +34 91 3943824
Tel: +34 91 3943823
E-mail: jmbau@vet.ucm.es
*Present address
Department of Biochemistry and Molecular
Biology IV, Universidad Complutense de
Madrid, Escuela de O ´ ptica, Madrid, Spain
†Present address
Division of Hematology, Department of
Internal Medicine, Washington University
School of Medicine, St Louis, USA
(Received 5 August 2005, revised 2 February
2006, accepted 10 February 2006)
doi:10.1111/j.1742-4658.2006.05174.x
The bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phospho-gluconolactonase (G6PD-6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus This study was designed to examine the effects of RNA-mediated PfG6PD-6PGL gene silencing in cultures of P falciparum on the expression of para-site antioxidant defense genes at the transcription level The highest degree
of G6PD-6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD-6PGL gene PfG6PD-6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation
In addition, an immediate transcriptional response was shown by thiore-doxin reductase suggesting that P falciparum G6PD-6PGL plays a physio-logical role in the specific response of the parasite to intracellullar oxidative stress P falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up-regulation of antioxidant genes Collectively, our findings point to an important role for this enzyme
in the parasite’s infection cycle The different characteristics of G6PD-6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies
Abbreviations
CT, cycle threshold; FeSOD, iron superoxide dismutase; G6PD-6PGL, glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase; GPx, glutathione peroxidase; GR, glutathione reductase; PPP, pentose phosphate pathway; TrxR, thioredoxin reductase.
Trang 2the functions of the malaria parasite and for structural
differences with respect to their human homologues, is
a research strategy aimed at finding potential specific
antimalaria targets [9–12]
P falciparumG6PD-6PGL is a bifunctional enzyme
exclusive to Plasmodium species [13] that probably
arose from the fusion of two genes in a common
ancestor [14] The deduced protein has a subunit
molecular mass of 107 kDa, in agreement with the
tetramer molecular weight calculated by size exclusion
chromatography [15] Its C-terminal half (residues
311–911) is clearly homologous to other described
G6PDs (with glucose 6-phosphate dehydrogenase
activity), though sequence similarity is interrupted by a
62 amino acid stretch with no similarity found to date
It has been nevertheless experimentally shown that this
62 amino acid insertion is essential for the activity of
the bifunctional enzyme [16] In contrast, the 310
amino acid protein sequence of the N-terminal region
clearly differs from most eukaryotic and prokaryotic
G6PDs, and shows 6-phosphogluconolactonase
activ-ity; thus G6PD-6PGL catalyses the first two steps of
the pentose phosphate pathway [13] The occurrence of
large insertion sequences that differ with respect to
their homologous proteins in other species has been
often observed in many gene products of P falciparum
and other Plasmodium species, but their structural
functions and origins are unknown [16,17]
In both the host and parasite, the pentose phosphate
pathway (PPP) is essential for neutralizing reactive
oxygen species during red blood cell infection with the
malaria parasite Accordingly, PPP activity is greatly
increased in infected red blood cells compared to
non-infected ones, and the parasite PPP is responsible for
82% of this activity [13,18]
Plasmodium falciparumG6PD-6PGL could therefore
be a potential therapeutic target not only because of
its structural characteristics that make it different from
its human equivalent, but also because of the
import-ance of this enzyme in the parasite’s intraerythrocyte
stage [16] The present paper describes the effects of
G6PD-6PGL silencing in P falciparum, confirming the
key role of this enzyme in the intraerythrocyte stage of
infection
Results
Effects of PfG6PD-6PGL gene silencing on growth
and parasite development
In a first attempt at silencing the G6PD-6PGL gene,
erythrocytes infected with ring-stage P falciparum 3D7
(pyrimethamine-sensitive clone) were electroporated
with pHC1G6 PD-AS (expressing antisense RNA) and the empty vector pHC1 as control In addition, silen-cing by dsRNA was also attempted by transfecting ring-stage parasites with a dsRNA–G6PD duplex using water and dsRNA–Rab5a as controls Figure 1 shows the parasite’s morphology in the different transfected cultures
After 24 h, all electroporated P falciparum cultures (wild-type, pHC1, pHC1G6 PD-AS, dsRNA-G6PD and dsRNA–Rab5a) showed a 77–83% reduction in parasitaemia, in agreement with previously reported data [19] As shown in Fig 1A, control cultures elec-troporated with water were apparently normal, with
Fig 1 Effects of PfG6PD-6PGL gene silencing on parasite develop-ment Parasites from different cultures were stained with Giemsa and examined by light microscopy at the indicated time points Wild-type 3D7 (WT) parasites transfected with water not subjected (A) or subjected (B) to 72 h of pyrimethamine pressure; parasites transfected with pHC1 (empty vector control) (C) and pHC1G6
PD-AS vectors (D) subjected to 96 h of pyrimethamine pressure; WT parasites transfected with water (E), dsRNA–Rab5a (dsRNA control) (F) and dsRNA-G6PD (G) 24 h post-transfection Black and red arrows point to pyknotic parasites and gametocytes, respectively.
Trang 3no pyknotic parasite forms or gametocytes appearing
throughout the entire protocol This indicates the
recovery of the parasites after electroporation, with no
loss in their capacity for multiplication, as the three
stages of the intraerythrocyte cycle were detected
In control cultures transfected with water but treated
with 100 ngÆmL)1 of pyrimethamine, pyknotic forms
appeared (Fig 1B) as a consequence of the complete
absence of live parasites after three days of
pyrimeth-amine pressure, demonstrating that sensitivity to
pyri-methamine in this strain is an adequate selection
method of identifying parasites transfected with pHC1
Unlike the case in electroporated control cultures
not exposed to pyrimethamine, in which mainly
ring-stage forms and few mature or stress forms such as
gametocytes were observed, parasites from both the
pHC1 and pHC1G6 PD-AS electroporated cultures
subjected to pyrimethamine pressure mainly appeared
to be at the trophozoite or gametocyte stage (Fig 1C–
D; Table 1) Although this effect is most probably
attributable to pyrimethamine acting on the
nontrans-fected parasite population [20,21], we cannot preclude
the possibility of some abnormal stage forms due to
the presence of the vector itself
As shown in Table 2, parasitaemia levels of the
pHC1 and pHC1G6 PD-AS parasites exposed to
pyri-methamine determined at 48 h, indicated that 23–25%
of the parasites had acquired resistance to
pyrimeth-amine mediated by the transfected vectors This
resist-ance decreased to 5–6% at 96 h without further
reduction
In the P falciparum cultures electroporated with
dsRNA or water (control culture), in the absence of
pyrimethamine pressure, similar parasitaemia levels
were observed in the course of one complete
intra-erythrocyte cycle (Table 3) Alterations to the cycle
were not observed in any of the dsRNA electroporated
cultures whose growth was synchronized for the entire
24 h (Table 3) In addition, as shown in Fig 1E–F, no morphological changes were observed in control cul-tures electroporated with water or with the duplex dsRNA–Rab5a In contrast, the cultures electro-porated with dsRNA-G6PD (Fig 1G) showed clear morphological changes in about 50% of the parasites, mostly abnormal trophozoites, whereas the morphol-ogy of the remaining 50% trophozoites was apparently normal
Quantification of mRNA expression in pHC1G6 PD-AS and dsRNA-G6PD transfected parasite cultures
Under pyrimethamine pressure, expression of the selectable marker and complementary mRNA strand from the G6PD-6PGL gene were determined by
RT-Table 1 Effect of pHC1G6 PD-AS on the stage-specific development of P falciparum The results of the parasitaemia, ring, trophozoite and gametocyte assays are the means and standard deviations of three independent experiments.
Culture
Incubation time with
a
To determine parasitaemia, about 10 000 erythrocytes were examined and the number of infected erythrocytes was reported as a percent-age of the total Stpercent-age-specific development was assessed by counting the fractions of rings, trophozoites and schizonts (asexual stpercent-ages).
No schizonts were detected at the indicated time points The fraction of gametocytes (sexual stage) was calculated as the percentage detected in 10 000 erythrocytes nd, not detected.
Table 2 Multiplication rates and efficiency of plasmid segregation
in pHC1 and pHC1G6 PD-AS transfected parasites The results of the parasitaemia assays represent the means and standard deviat-ions of three independent experiments.
Culture
Incubation time for pyrimethamine (h)
Parasitaemia (%) a
Segregation (%) b
a To determine parasitaemia, about 10 000 erythrocytes were examined and the number of infected erythrocytes was reported as percentage of the total.bThe level of parasitaemia after (48 and
96 h) and before (0 h) pyrimethamine pressure was used as a quantitative measure of plasmid segregation (expressed as a per-centage).
Trang 4PCR (Fig 2) Also, the effect of the presence of
intra-cellular pHC1G6 PD-AS was analyzed by quantitative
mRNA expression analysis of G6PD-6PGL and of
several key genes involved in defense against oxidative
stress after 48 or 96 h of pyrimethamine pressure:
glutathione reductase (GR), iron superoxide dismutase
(FeSOD), thioredoxin reductase (TrxR) and
glutathi-one peroxidase (GPx) Biochemical characterization of
recombinant PfGPx indicates that this enzyme has a
strong preference for Trx over GSH, and could be
considered a thioredoxin dependent peroxidase [22] At
48 h, a 60% reduction in G6PD-6PGL gene expression
was detected (Fig 3) This reduction also caused a
generalized down-regulation of the expression of the
other antioxidant genes analyzed The expression of
TrxR and GR, both NADPH dependent enzymes, was
reduced by four- and fivefold, respectively (Fig 3)
FeSOD and GPx, which remove the superoxide anion
and hydrogen peroxide, respectively, showed five- and threefold down-regulation After 96 h of incubation in the presence of pyrimethamine, levels of G6PD-6PGL expression were still reduced by 40%, while expression levels of the other antioxidant response genes were restored, returning to levels close to those recorded in cultures transfected with the control pHC1 vector However, it should be noted that, at 96 h, parasites transfected with pHC1 showed the up-regulation of most of the antioxidant genes except G6PD-6PGL compared with wild-type transfected parasites (Fig 4B) Thus, GR levels increased sixfold and TrxR, FeSOD and GPx doubled their wild-type levels This effect was also apparent for TrxR at 48 h (Fig 4A) Hence, we must carefully interpret this silencing through antisense RNA produced by pHC1, due to the effect per se that the presence of pHC1 has on the parasite antioxidant response
Table 3 Multiplication rate and stage-specific development of parasites transfected with dsRNA The results of the parasitaemia, ring and trophozoite assays are expressed as the means and standard deviations of three independent experiments nd, not detected.
a To determine parasitaemia, about 10 000 erythrocytes were examined and the number of infected erythrocytes was reported as percentage of the total Stage-specific development was assessed by counting the fractions of rings, trophozoites and schizonts No schizonts were detected at the indicated time points b Fifty percent of trophozoites detected in the dsRNA-G6PD cultures were abnormal (but not pyknotic).
200 bp
100 bp
200 bp
M
100 bp
M 1 2 3 4
Fig 2 High intracellular expression capacity of the pHC1G6 PD-AS vector Total RNA from WT and pHC1G6 PD-AS parasites were RT-PCR amplified and the products examined on ethidium bromide-stained agarose gels (A) Expression of the PfG6PD-6PGL gene noncoding strand: lane M, 100 basepair ladder molecular weight marker; lane 1, pHC1G6 PD-AS parasites subjected to 48 h of pyrimethamine pressure; lane
2, pHC1G6 PD-AS parasites subjected to 96 h of pyrimethamine pressure; lane 3, pHC1G6 PD-AS vector PCR product (positive control) (B) TgDHFR-TS gene expression: lane M, 100 basepair ladder molecular weight marker; lane 1, pHC1G6 PD-AS parasites subjected to 48 h of pyrimethamine pressure; lane 2, pHC1G6 PD-AS parasites subjected to 96 h of pyrimethamine pressure; lane 3, WT parasites (negative con-trol); lane 4, pHC1G6 PD-AS vector PCR product (positive control) RT-PCR amplification yielded bands of the expected size: 152 basepair (antisense PfG6PD-6PGL mRNA) and 160 basepair (TgDHFR-TS mRNA).
Trang 5In preliminary experiments performed on cultures
after 24, 48, 72 and 96 h of electroporation with
dsRNA, a transient effect was observed lasting not
longer than 48 h Aliquots for qRT-PCR analysis were
taken at 3 and 24 h after electroporating with
dsRNA-G6PD Three hours after electroporation,
G6PD-6PGL silencing at the mRNA level was 86%, while
FeSOD and GPx expression levels were normal
(Fig 5) Nevertheless, this diminished G6PD-6PGL
expression effect was accompanied by a sevenfold
up-regulation of TrxR and a threefold down-up-regulation of
GR (Fig 5) After 24 h, normal G6PD-6PGL
expres-sion levels were restored In parallel, the expresexpres-sion
levels of the other antioxidant genes, stabilized at
sim-ilar levels to those observed in electroporated cultures
without dsRNA (Fig 5)
To test this G6PD-6PGL specific knockdown by
dsRNA-G6PD, we then determined G6PD-6PGL
transcript levels in cultures transfected with dsRNA–
Rab5a Our results indicated no appreciable differences
in G6PD-6PGL expression between dsRNA–Rab5a or
wild-type parasites, thus confirming the specificity of
dsRNA-G6PD (data not shown)
Discussion
To assess the capacity of a gene or its product to act
as an antimalaria target, its role in the biology of the
parasite needs to be well established For this purpose,
several systems for the functional analysis of P
falci-parumgenes have been developed including gene
silen-cing by antisense RNA [23], or more recently, by
Fig 3 Effect of pHC1G6 PD-AS on parasite mRNA Quantifying
G6PD-6PGL, GR, TrxR, FeSOD and GPx mRNA levels by qRT-PCR
in parasites, transfected with pHC1 and pHC1G6 PD-AS vectors,
obtained at the indicated time points during the course of
pyrimeth-amine pressure Expression level data for each gene obtained from
parasites transfected with the pHC1G6 PD-AS vector were
normal-ized to the 18S rRNA signal (internal control) and the normalnormal-ized
values of control parasites (transfected with pHC1) were set at 1.
Error bars represent the standard deviations of the means obtained
in three replicate assays.
A
B
Fig 4 Oxidative stress gene up-regulation by the presence of pHC1 derived vectors Gene expression analysis by real-time RT-PCR of G6PD-6PGL, GR, TrxR, FeSOD and GPx from pHC1 and
WT water electroporated parasites at 48 and 96 h (pyrimethamine pressure was only applied to pHC1 transfected parasites) The norm-alized number of genome equivalents was determined using the
18 s rRNA gene as internal control In this experiment, a control culture under pyrimethamine pressure was included in parallel, with
no significant mRNA expression signal detected at 48 and 96 h Error bars represent the standard deviations of the means obtained
in three replicate assays.
Fig 5 Effect of the dsRNA-G6PD duplex on parasite mRNA (A) Quantifying G6PD-6PGL, GR, TrxR, FeSOD and GPx mRNA levels
by qRT-PCR in parasites transfected with dsRNA-G6PD, 3 h and
24 h after electroporation Expression level data for each gene obtained from parasites transfected with dsRNA-G6PD were nor-malized to the 18S rRNA signal (internal control) and the nornor-malized values of control parasites (transfected with water) were set at 1 Error bars represent the standard deviations of the means obtained
in three replicate assays.
Trang 6RNA interference [24,25] Antisense RNA has been
found in humans, mice, plants and protozoan parasites
such as P falciparum [26] The fact that endogenous
antisense RNAs are widespread in P falciparum,
sug-gests that they could be a natural gene expression
reg-ulatory mechanism [26,27] Recent studies suggest that
in P falciparum antisense RNA is synthesized by
RNA polymerase II [26]
In our model, P falciparum G6PD-6PGL was
silenced in vivo through antisense RNA by
construct-ing the vector pHC1G6 PD-AS accordconstruct-ing to a
previ-ously established strategy [23] This vector comprises
two expression cassettes, one to drive the expression of
a selectable marker, which in this case was the
Toxo-plasma gondii dihydropholate reductase thymidylate
synthase gene, and the other to allow expression of the
PfG6PD-6PGL gene noncoding strand (antisense
RNA) This noncoding strand transcription strategy to
produce antisense RNA has been recently successfully
used in other protozoan and mammalian cells [28–30]
In P falciparum, antisense RNA has also been
success-fully applied to silencing the PfCLAG9 gene [23],
inhibiting PfCLAG9 mRNA translation and
diminish-ing cytoadherence of the protein to melanoma cells (a
function associated with this protein) Since our
con-struct expresses high levels of the selection marker and
the antisense RNA strand, the in vivo activity of both
promoters (P falciparum calmodulin and Plasmodium
chabaudidihydropholate reductase) and the stability of
their mRNAs was observed under our experimental
conditions
Parasites transfected with the antisense
RNA-G6PD-6PGL vector showed reduced mRNA-G6PD-RNA-G6PD-6PGL
expression at 48 h and there was a simultaneous
reduc-tion in TrxR, GR, GPx and FeSOD transcripts After
96 h, G6PD-6PGL silencing persisted at a slightly
higher level, but the expression of the other four
anti-oxidant genes was restored This could be the combined
outcome of two effects: a loss or diminished number of
copies of the vector in some parasites due to low
segre-gation competence [31,32], and stress to the parasite
caused by the vector itself This last effect is suggested
by the up-regulation of these genes observed at 96 h in
control transfections with pHC1 (lacking an insert), as
indicated by reduced parasitaemia levels and increased
levels of mRNA for the antioxidant genes in the pHC1
transfected cultures at 96 h The discordant effects
detected at 96 h in cultures transfected with vectors
containing or lacking an insert indicate that after 48 h,
other interacting factors could modify the molecular
phenotypic effect of using this vector This is the first
report of the detection at the molecular level of the
toxic effect of a DNA vector in P falciparum
Although mechanisms of RNAi silencing in many species are not well understood, this technique has been used to study gene function in a great variety of organisms including Trypanosoma brucei, Drosophila melanogaster and a limited number of vertebrates [33– 35] Despite the fact that, so far, the genes encoding the required RNAi machinery have not been detected
in any of the currently available Plasmodium databases [36], RNAi silencing has been achieved in P falcipa-rum [24,25] Thus, it could be that the data reported for Plasmodium, as well as our results using dsRNA-G6PD, are the consequence of an antisense RNA rather than a direct RNAi effect However, it is also true that, to date, 60% of the genes predicted for
P falciparum have no known homologs, and we have
no clues as to their function [5]
Transfection of P falciparum with dsRNA-G6PD seems to be a gentle procedure in that no growth changes were observed in the cultures, allowing obser-vation of the molecular phenotypic effects of transfec-tion Besides the water-transfected parasite control, dsRNA–Rab5a was used as a second control, since the silencing target Rab5a belongs to a P falciparum multigene family [37] with overlapping functions, as occurs in other organisms [38,39] Accordingly, no morphological changes were observed in either type
of control cultures, while the cultures transfected with dsRNA-G6PD showed morphological alterations in about 50% of the trophozoites, suggesting that what
we are looking at is a genuine effect of inhibited G6PD-6PGL expression causing cell stress Moreover, this effect is appreciable at the stage of highest meta-bolic activity, the trophozoite stage In the early stages (3 h), dsRNA-G6PD was highly effective at silencing, decreasing mRNA levels by up to 86% Nevertheless, this silencing was transient, since after
24 h, mRNA levels had almost recovered It should
be noted that the G6PD-6PGL silencing experiment-ally produced at the ring stage corresponds in clinical isolates to the time at which the highest amounts of G6PD-6PGL transcripts are shown [42] Silencing of parasite G6PD-6PGL in rings caused cell stress, indu-cing the up-regulation of TrxR by sevenfold This induction of the thioredoxin system against oxidative stress has been previously described in Streptomyces coelicolor and Bacillus subtilus subjected to oxidative stress [43,44] An important role of thioredoxin is to reduce ribonucleotide reductase P falciparum TrxR is able to reduce thioredoxin, which together with per-oxyredoxins, transforms peroxides and also helps to reduce oxidized glutathione [9] If the lack of G6PD-6PGL produces lower reduction equivalents, increas-ing TrxR to counteract this effect would require
Trang 7NADPH from alternative sources, as has been
sugges-ted by other authors [45,46] Also it seems that at
this early developmental stage in which high
mRNA-G6PD-6PGL levels are normally expressed [46], the
G6PD-6PGL silencing consequence of a threefold
decrease in GR expression would modify the
equilib-rium of the oxidative stress cascade, by strongly
indu-cing TrxR Thus, TrxR induction would render
reduced thioredoxin, the ribonucleotide reductase
sub-strate, producing deoxynucleotides for normal parasite
replication [9,10,47], and de novo G6PD-6PGL
tran-scription would reestablish expression levels after
24 h
Experimental procedures
Vector construction and dsRNA design
The oligonucleotides 5¢xg6pd (5¢-CACTGATAAAATATT
ACTCGAGAAACCATTTGG-3¢) and 3¢xg6pd (5¢-GAC
TTGTTTTTCCTCGAGTTCCTTAAGTAAAGG-3¢)
con-taining XhoI sites (underlined) were used to PCR amplify a
960 basepair G6PD-6PGL fragment from P falciparum
3D7 (corresponding to GenBank nt 1803–2763, accession
number X74988) The resultant PCR fragment was excised
with XhoI and ligated into the XhoI site of plasmid pHC1
[48] to produce pHC1G6 PD-AS Parental pHC1 consists
of a dual cassette in which the mutated T gondii
dihydro-pholate reductase thymidilate synthase gene, conferring
resistance to pyrimethamine, is flanked by the P chabaudi
dihydrofolate reductase promoter and P falciparum
histi-dine-rich protein 2 terminator sequence The second
cas-sette expresses the inserted antisense mRNA driven by the
P falciparum calmodulin promoter and terminated by the
3¢-untraslated region of P falciparum heat shock protein 86
[48] The antisense direction of the G6PD-6PGL fragment
inserted with respect to the calmodulin promoter, was
con-firmed by plasmid DNA NdeI digestion Parental plasmid
pHC1, lacking the G6PD-6PGL fragment was used as a
transfection control Plasmid DNAs were purified (Plasmid
Maxi Kit, Qiagen, Chatsworth, CA, USA) from overnight
Escherichia colicultures
A 21 basepair dsRNA (sense: UACAUCAUGCACCAA
CGAAdTdT; antisense: UUCGUUGGUGCAUGAUGUA
dTdT) was designed for the target sequence (UACAUCA
UGCACCAACGAA) of the G6PD-6PGL gene,
follow-ing Dharmacon siDESIGN Center criteria (http://design
dharmacon.com/) In addition, a dsRNA corresponding to
the PfRab5a gene (GenBank accession number AE001399)
(target sequence: UAUGCAAGUAUUGUCCCAC; sense:
UAUGCAAGUAUUGUCCCACdTdT; antisense: GUGG
GACAAUACUUGCAUAdTdT) was also designed to use
as control All dsRNAs were obtained from Dharmacon
Research (Lafayette, CO, USA) in annealed and lyophilized
forms and were suspended in RNase-Dnase-free water before use
Parasite cultures and electroporation
P falciparum 3D7 (pyrimethamine-sensitive strain) was grown and double synchronized using standard procedures [49,50] Parasites (ring stage 8–10% parasitaemia) were transfected by electroporation with 100–150 lg of purified plasmid DNA or 40 lg of dsRNA as described [19] The parasites transfected with pHC1 or pHC1G6 PD-AS were subsequently cultured for 48 h in 75 cm2flasks, after which they were subjected to a selection pressure of 100 ngÆmL)1 pyrimethamine for a further 96 h The level of parasitaemia after and before pyrimethamine pressure was used as a quantitative measure of plasmid segregation (expressed as a percentage) The growth and development of each transfec-tion was monitored daily by Giemsa staining blood films Parasites transfected with dsRNA-G6PD or dsRNA– Rab5a were kept for 24 h in 75 cm2flasks with no selection pressure
Isolation of total RNA RT-PCR and quantitative RT-PCR
Total RNA was isolated from infected red blood cell cul-tures using the ABI Prism 6100 Nucleic Acid Prepstation (Applied Biosystems, Foster City, CA, USA) Isolated RNA was reverse transcribed to cDNA using the High-CapacitycDNA Archive Kit (Applied Biosystems) as des-cribed by the manufacturer using specific reverse primers
To confirm expression of the T gondii DHFR-TS gene (selectable marker) and antisense G6PD-6PGL mRNA syn-thesis from plasmid pHC1G6 PD-AS, cDNA was obtained using reverse primers (3¢Tgdhfr-ts, 5¢-TGTAGACATGC
GGAAGAAGTAC-3¢) for further PCR amplification by adding forward primers (5¢Tgdhfr-ts, 5¢-GAAGGAGCTG
CCTCGTCTGAG-3¢) For real-time transcript quantifica-tion by molecular beacons, cDNA was obtained using spe-cific reverse primers and qRT-PCR was performed in an ABI Prism 7000 Sequence Detector (Applied Biosystems) Sequence design of molecular beacons and primers for G6PD-6PGL, Fe-SOD, GR, TrXR, GPx and 18S-rRNA quantitative transcription analysis was performed according
to a previously published procedure [51]; these designs are provided in Table 4 The qRT-PCR involved 1 cycle each
of 50C ⁄ 2 min and 95 C ⁄ 10 min, followed by 45 cycles of
57C ⁄ 1 min, 95 C ⁄ 30 s, and 45 C ⁄ 29 s All analyses were run in triplicate An 18S-rRNA signal was used as endo-genous control to normalize mRNA relative expression [42,51–54] Cycle threshold (CT) was defined as the frac-tional PCR cycle number at which the fluorescent signal is
Trang 8greater than the minimal detection level Standard curves were prepared for all targets and endogenous references, using genomic DNA concentrations and their correspond-ing CT For each experimental sample, the amounts of target and 18S-rRNA were calculated from the standard curves Then the target amount was divided by the endo-genous reference amount to obtain a normalized target value To generate the relative expression levels, each normalized target value of interest was divided by the calibrator normalized target from the nontreated sample
Statistical analysis
Results are expressed as mean ± SD All the experiments were repeated on at least three different cultures Statistical analysis was performed using graphpad software (Prism 4.0) at P < 0.05 The standard deviation of the Cts values among triplicates was always < 0.30 CT
Acknowledgements
We are indebted to Nuria Trinidad for her excellent technical assistance with the parasite cultures and to Alan Cowman for his generous gift of the pHC1 Thanks are also due to two anonymous reviewers for their useful comments A.C was awarded a
predoctor-al fellowship by the Comunidad de Madrid This research was funded by grants PM1999-0049-CO2-01 and BIO2003-07179 from the Spanish MCYT
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