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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Int J Parasitol. 2012 Sep 27;42(11):969–974. doi: 10.1016/j.ijpara.2012.09.003

Tricks in Plasmodium’s molecular repertoire - escaping 3′UTR excision-based conditional silencing of the chloroquine resistance transporter locus

Andrea Ecker a, Rebecca E Lewis a, Eric H Ekland a, Bamini Jayabalasingham a, David A Fidock a,b,*
PMCID: PMC3517811  NIHMSID: NIHMS411000  PMID: 23023047

Abstract

In the human malaria parasite, Plasmodium falciparum, the major determinant of chloroquine resistance, P. falciparum chloroquine resistance transporter (pfcrt), likely plays an essential role in asexual blood stages, thus precluding conventional gene targeting approaches. We attempted to conditionally silence the expression of its ortholog in Plasmodium berghei (pbcrt) through Flp recombinase-mediated excision of the 3′untranslated region (UTR) during mosquito passage. However, parasites maintained pbcrt expression despite 3′UTR excision. Characteriz ation of these pbcrt mRNAs, by 3′rapid amplification of cDNA ends, identified several replacement 3′UTR sequences. Our observations demonstrate the astounding genetic plasticity of this parasite when faced with the loss of an essential gene.

Keywords: Plasmodium berghei, pfcrt, Sporozoite, Conditional knockdown, 3′ UTR, RACE

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt; PlasmoDB ID: MAL7P1.27) play a central role in resistance of the human malaria parasite, P. falciparum, to several antimalarial drugs, most notably chloroquine (Petersen et al., 2011; Roepe, 2011; Ecker et al., 2012; Summers et al., 2012). pfcrt encodes an integral membrane protein that localizes to the parasite’s digestive vacuole, the site of hemoglobin digestion and chloroquine action. Early secondary structure predictions and homology modeling predicted that PfCRT is a transporter (Fidock et al., 2000; Martin and Kirk, 2004), and recent biochemical and pharmacological studies lend support to the hypothesis that mutant PfCRT can transport chloroquine out of the digestive vacuole (Summers and Martin, 2010; Baro et al., 2011; Sanchez et al., 2011; Griffin et al., 2012; Papakrivos et al., 2012). Nevertheless, more than a decade after its discovery, the native function of PfCRT remains unknown. Failed gene targeting attempts in P. falciparum and the mouse malaria parasite, Plasmodium berghei, suggest that this function is essential, at least in the parasite’s asexual blood stages (Waller et al., 2003; Ecker et al., 2011). Intriguingly, PfCRT peptides have also been detected by mass spectroscopy in sporozoites (Florens et al., 2002), an extracellular stage that neither possesses a digestive vacuole nor digests hemoglobin.

To gain insight into the functional requirement for CRT across the parasite lifecycle, we made use of a conditional knockout (cKO) system recently optimized for P. berghei by the Ménard laboratory (Combe et al., 2009; Lacroix et al., 2011). This method is based on the Flp recombinase/Flp recognition target (Flp/FRT) site-specific recombination system from yeast, involving Flp recombinase-mediated recognition of two 34 bp FRT sites leading to excision of the intervening DNA (referred to as the FRTed sequence). For P. berghei, this cKO approach begins by replacing the endogenous 3′ untranslated region (UTR) of the targeted gene with a FRTed sequence comprising a 3′UTR sequence from the thrombospondin related adhesive protein (trap; PlasmoDB ID: PBANKA_134980) gene followed by the downstream human dihydrofolate reductase (hdhfr) selectable marker. This double crossover homologous recombination event is undertaken in parasite strains that harbor an integrated copy of Flp recombinase that is expressed under a mosquito stage-specific promoter such that passaging of these parasites to Anopheles mosquitoes initiates excision of the FRTed 3′UTR. This leaves the gene of interest with no 3′UTR, thereby destabilizing transcripts and generating a functional KO. The power of this cKO system was initially demonstrated by Combe et al. (2009) who used it to silence msp1 (essential for parasite invasion of erythrocytes) and uncovered a second role for this gene in the formation of merozoites in infected hepatocytes. More recently this system was applied to conditionally silence the parasite invasion molecules AMA1 and RON4, revealing that the former is important only for merozoite invasion of erythrocytes whereas RON4 is required for sporozoite invasion of hepatocytes (Giovannini et al., 2011). This system was also used to conditionally inactivate the P. berghei cGMP-dependent protein kinase (PKG), leading to the developmental arrest of late liver stage parasites and the demonstration that these stages can induce potent immunity against sporozoite challenge (Falae et al., 2010).

To conditionally silence the P. berghei ortholog of pfcrt (known as pbcrt; PlasmoDB ID: PBANKA_121950), we first engineered the pPbcrt-cKO transfection plasmid to replace the endogenous pbcrt 3′UTR with the FRTed trap 3′UTR and hdhfr marker (Fig. 1A). Briefly, the last 0.6 kb of the pbcrt coding sequence was PCR amplified using primers p1 + p2 (listed in Table 1); and the first 0.6 kb of pbcrt 3′UTR was amplified using primers p3 + p4. These fragments were cloned into p3′TRAP_hdhfr_FRT (Combe et al., 2009) via SphI and NotI (for the coding sequence) or HindIII and SphI (for the 3′UTR). Notably, our pPbcrt-cKO plasmid unintentionally lacked the first 12 nucleotides of the trap 3′UTR compared with the plasmid used by Combe et al. (2009) (see below). SphI-linearized plasmid was then electroporated into asexual blood stage parasites of two “deleter” strains, TRAP/FlpL or UIS4/Flp. These recipient strains express either Flp recombinase or the thermolabile version FlpL (Lacroix et al., 2011). In the TRAP/FlpL strain, recombinase expression is driven by the trap promoter, which becomes active in maturing oocysts and peaks in salivary gland sporozoites (Rosinski-Chupin et al., 2007). The UIS4/Flp deleter strain controls Flp expression using the uis4 promoter, which becomes highly upregulated in salivary gland sporozoites (Matuschewski et al., 2002).

Fig. 1.

Fig. 1

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Generation of Plasmodium berghei chloroquine resistance transporter (pbcrt) conditional knockout (cKO) parasites via 3′untranslated region (UTR) excision mediated by Flp/FRT site-specific recombination. (A) Schematic of the double crossover event between the linearized transfection plasmid and the endogenous pbcrt locus, resulting in the pre-excision pbcrt-trap 3′UTR locus and the post-excision pbcrt-no3′UTR locus. The structure of the 13-exon pbcrt gene is shown stylistically, with introns in grey and exons in white. The thick black line denotes the plasmid backbone. Flp, Flp recombinase; FRT, Flp recognition target; trap, thrombospondin related adhesive protein; hdhfr; human dihydrofolate reductase selectable marker. H, HindIII; N, NotI; S, SphI. (B) PCR analysis of the pbcrt locus pre- and post-excision. The non-excised locus was detected using primers p5 + p6 (that yield a 1.5 kb band). The excised locus, post mosquito passage, was detected using primers p5 + p7 (yielding a 0.86 kb product), and primers p1 + p7 (0.73 kb). These primer pairs can also amplify the non-excised locus, yielding 3.1 and 3.0 kb products, respectively, in the pre-excision parasites (the presence of the 0.6 kb band in those lanes and in the post-excision bulk blood stages was determined by sequencing to be off-target amplification of the mouse integrin α-8 gene). Absence of the wild-type pbcrt locus was confirmed in all recombinant parasites using primers p1 + p4 (that yield a 1.2 kb product in wild-type parasites; data not shown). These primers yielded a large (>>3 kb) product spanning the plasmid backbone in the pbcrt-no3′UTR locus in post-excision blood stage parasites (this product would theoretically also be present in sporozoites with an excised locus, but it is difficult to amplify a product of this size from the low yield of parasite genomic DNA that can be obtained from infected mosquitoes). Excision of the trap 3′UTR was not observed in blood stage parasites prior to mosquito transmission, but was evident in midgut and salivary gland sporozoites. Following retransmission to naïve mice, most but not all blood stage parasites had excised the FRTed trap3′UTR locus. The re-cloned post-excision parasites T9-rc and U4-rc harbor the excised locus (pbcrt-no3′UTR) exclusively.

Table 1.

List of primers used in this study.

Name Nucleotide Sequence Description
p1 5′ -ccGCATGCTACACCATTGTTAGTTGTATACAAGG Forward primer of pbcrt 3′ coding sequence; SphI site underlined
p2 5′ -ttGCGGCCGCTTATGCCCTTGATGTTTCTATAGAAG Reverse primer of pbcrt 3′ coding sequence; NotI site underlined
p3 5′-ccAAGCTTTTGATACAACATTTTTATTTCTTAAATGATTTTTG Forward primer of pbcrt 3′UTR;HindIII site underlined
p4 5′ -ccGCATGCCTCTCTATACATAGGCAAATAAGG Reverse primer of pbcrt 3′UTR; SphI site underlined
p5 5′-CATATGTGATAATTTACTTGCTTGC Forward primer of pbcrt upstream coding sequence
p6 5′-CTGGTGCTTTGAGGGGTGAGC Reverse primer from hdhfr selectable marker cassette
p7 5′-CAGGAAACAGCTATGAC Reverse primer specific to the plasmid backbone
p8 5′-GTTGGTTCGCTAAACTGCATC Forward primer for hdhfr qPCR
p9 5′-CTGTTTACCTTCTACTGAAGAGG Reverse primer for hdhfr qPCR
p10 5′-TGCAGCAGATAATCAAACTC Forward primer for hsp70 qPCR
p11 5′-ACTTCAATTTGTGGAACACC Reverse primer for hsp70 qPCR
p12 5′-CCTTATCTCATTATTAGATGCTTCTAC Forward primer for pbcrt RT-PCR
p13 5′-CCAATATTCTTGGTTTTCTTACAGC Reverse primer for pbcrt RT-PCR
p14 5′-TATGGGTCCAAGATATTGTAGTAATAA Forward primer for Plasmodium berghei ama1 RT-PCR
p15 5′-GAATTAGCTTTACCATAAATATCTGC Reverse primer for P. berghei ama1 RT-PCR
QT 5′-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC(T)17 Primer to generate cDNA for 3′RACE
QO 5′-CCAGTGAGCAGAGTGACG Outer primer for 3′RACE
QI 5′-GAGGACTCGAGCTCAAGC Inner primer for 3′RACE
SP6 5′-ATTTAGGTGACACTATAG pGEM vector-specific primer to sequence 3′RACE products
M13F 5′-GTAAAACGACGGCCAGT pGEM vector-specific primer to sequence 3′RACE products
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amal, apical membrane antigen 1; hdhfr human dihydrofolate reductase selectable marker; hsp70, heat shock protein 70; Pbcrt, Plasmodium berghei chloroquine resistance transporter; qPCR, quantitative PCR; RACE, random amplification of cDNA ends; RT-PCR, reverse transcription PCR; UTR, untranslated region.

Deleter strain parasites electroporated with the pPbcrt-cKO plasmid were inoculated into CD-1 (Charles River) mice and subjected to two rounds of in vivo drug pressure with the parasite DHFR-specific inhibitor WR99210 (de Koning-Ward et al., 2000). This agent selects for double crossover events that have inserted the FRTed trap 3′UTR and the WR99210-resistant human dhfr selectable marker into the pbcrt target locus (Fig. 1A). Integrants were detected by PCR and recombinant clones were obtained by limiting dilution in mice. This cloning yielded pbcrt CKO-TRAP/FlpL clone 9 and pbcrt CKO-UIS4/Flp clone 4, hereafter referred to as the clones T9 and U4. All procedures with CD-1 and C57BL/6J (Jackson Laboratories) mice were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Columbia University Medical Center, USA.

To test whether excision of the FRTed 3′UTR would occur as predicted, the pre-excision T9 and U4 clones were passaged through Anopheles stephensi mosquitoes. Both clones produced oocysts as well as midgut and salivary gland sporozoites at normal numbers and with apparently normal morphology (data not shown). We extracted genomic DNA from oocysts and salivary gland sporozoites (harvested from mosquitoes 20-25 days after ingestion of blood meals harboring pre-excision blood stage parasites) and assessed the pbcrt locus by diagnostic PCR and sequencing. PCR analysis of clone T9 is illustrated in Fig. 1B (analysis of the U4 clone yielded the same results; data not shown). These data confirmed that excision of the FRTed trap 3′UTR was occurring in midgut sporozoites and went largely to completion in salivary gland sporozoites (Fig. 1B; note that the p5 + p6 band specific for the pbcrt-trap3′UTR locus was present in the pre-excision blood stage population but was almost absent in salivary gland sporozoites).

We also allowed T9- or U4-infected mosquitoes to feed on naïve, anesthetized C57BL/6J mice in order to test whether pbcrt post-excision cKO sporozoites (harboring the pbcrt-no3′UTR locus; Fig. 1A) were infectious. Surprisingly, both the T9 and U4 post-excision parasites were readily transmitted following sporozoite inoculation, with all mice being blood smear-positive on days 5 (T9, three out of three mice) or 7 (U4, four out of four mice; these mice were not checked on day 5). For comparison, in the same experiment two out of two mice bitten by mosquitoes infected with the parental TRAP/FlpL deleter strain (used to generate the cKO T9 parasites) were smear-positive on day 7, but not on day 5. The majority of the transmitted T9 and U4 parasites appeared to possess the pbcrt-no3′UTR locus, although a small signal for the non-excised pbcrt-trap3′UTR locus was also detected by PCR (see p5 + p6 lane in post-excision bulk culture; Fig. 1B). We quantified the excision rate by SYBR Green I (Bio-Rad, USA) quantitative PCR analysis using an Opticon2 Real-Time PCR Detector (Bio-Rad). Primers p8 + p9 were directed to the hdhfr segment of the FRTed sequence, and primers p10 + p11 were directed to the reference gene hsp70 (PlasmoDB ID: PBANKA_091440). The percent excision was then calculated based on the ΔΔC(t). This analysis demonstrated a 99.3 ± 0.2% and 97.1 ± 0.1% loss of the FRTed 3′UTR in the transmitted T9 and U4 parasites, respectively (mean ± S.D., calculated from three mice per line; each sample was run in triplicate). We note that this high degree of excision is consistent with earlier reports (Combe et al., 2009).

To obtain a pure population of pbcrt-no3′UTR parasites lacking a 3′ regulatory sequence, we re-cloned the transmitted parasite populations by limiting dilution, yielding the post-excision blood stage clones, T9-rc and U4-rc. PCR analysis confirmed the complete absence of the FRTed trap 3′UTR (Fig. 1B; see absence of band with p5 + p6). These post-excision clones were passaged by i.v. injection into naïve mice for closer analysis of their replication fitness compared with the parental deleter strains TRAP/FlpL (for T9-rc) and UIS4/Flp (for U4-rc). For this experiment, 105 parasites were injected i.v. into naïve C57BL/6J mice (five mice per line in each of two separate experiments), and monitored their parasitemias daily by flow cytometry using the DNA dye SYBR Green I and the mitochondrial vital dye MitoTracker Deep Red (Ekland et al., 2011). All four parasite lines revealed essentially the same progression of infection: a rise in parasitemia until days 5-6 peaking at ~2%, followed by a modest decline for ~3-4 days prior to resurgence of the infection (coinciding with an increase in reticulocytes towards which P. berghei is tropic, data not shown; (Fidock et al., 2004)). Parasitemias attained 9-12% by day 11 in all lines, confirming that all lines displayed similar growth kinetics (Fig. 2A). Given that past failed KO attempts have suggested that PfCRT and PbCRT are essential, at least in the parasite’s asexual blood stages (Waller et al., 2003; Ecker et al., 2011), parasite survival following excision of the 3′UTR was surprising.

Fig. 2.

Fig. 2

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Post-excision parasites harboring pbcrt -no3′UTR (Plasmodium berghei chloroquine resistance transporter - no3′untranslated region) show no marked loss of fitness and express pbcrt transcripts and protein product. (A) Growth kinetics of post-excision (T9-rc and U4-rc) and parental strains (TRAP/FlpL and UIS4/Flp) propagated in mice. Parasitemias are shown as means ± S.E.M. calculated from groups of 10 mice per line. (B) Reverse transcriptase (RT)-PCR analysis of pre-excision T9 blood stage parasites and the post-excision clone T9-rc. pbcrt and P. berghei apical membrane antigen 1 (pbama1, PlasmoDB ID: PBANKA_091500) transcripts were detected from parasite cDNA using primers p12 + p13 (yielding a 0.74 kb band) and p14 + p15 (yielding a 1.0 kb band), respectively. + and - RT denote reactions with and without reverse transcriptase. (C) PbCRT expression was assayed by western blot hybridization with saponin-lysed protein extracts from the post-excision clones (T9-rc and U4-rc) and the parental deleter strains. Membranes were stained for PbCRT using a mouse monoclonal anti-PfCRT hybridoma supernatant (monoclonal antibody (mAb) CU1711.626 generated against the synthetic PfCRT peptide (KKMRNEENEDSEGELTNVDC), diluted 1:100; Covance, USA) followed by incubation with a horseradish peroxidase-conjugated sheep anti-mouse IgG (NXA931 diluted 1:10,000; GE Healthcare, USA), and then detected using a chemiluminescent substrate (Pico West; Thermoscientific, USA). Membranes were then re-probed with rat monoclonal anti-P. berghei AMA1 antibody (mAb 28G2 diluted 1:100; (Kocken et al., 1998)) followed by incubation with a horseradish peroxidase-conjugated goat anti-rat IgG (NA935V diluted 1:10,000; GE Healthcare) and detection by chemiluminescence (ECL Plus; GE Healthcare). Each lane contains protein extract from a separate mouse infected with the indicated parasite line. Differences in band intensity are likely to reflect stage-dependent variation in levels of PbCRT and AMA1 expression (from samples collected at different times) as well as variable protein loading.

We suspected that PbCRT expression might somehow be maintained in the pbcrt-no3′UTR parasites and extracted RNA and protein to assess whether this was indeed the case. pbcrt mRNA was readily amplified from both post-excision clones by reverse transcriptase (RT)-PCR (results for T9-rc and the pre-excision T9 clone are shown in Fig. 2B; similar results were obtained with U4-rc and are not shown). PbCRT expression was also upheld in the post-excision T9-rc and U4-rc clones (Fig. 2C). We cannot quantify levels of protein expression due to the possible presence of truncated forms, as discussed below.

We hypothesized that in order to maintain expression without a 3′UTR being present in the pbcrt genomic locus, parasites employed alternative 3′UTRs to stabilize pbcrt mRNAs and permit protein production. To identify any such sequences, 3′ rapid amplification of cDNA ends (3′RACE; Scotto-Lavino et al., 2006) was performed on the transmitted, recloned parasites from both deleter backgrounds (T9-rc and U4-rc). These 3′RACE reactions produced a single dominant band of just over 0.5 kb in T9-rc, and several bands of different length in clone U4-rc (ca. 0.25, 0.3-0.4, 0.55, 0.7 and 2.2 kb) (Fig. 3A). These products included the last 0.24 kb of pbcrt plus the alternative 3′UTRs. Most of the observed sizes were therefore consistent with the only previously characterized Plasmodium 3′UTRs (~0.45 kb for Plasmodium gallinaceum pgs28 (Golightly et al., 2000) and 0.16-0.25 kb for pfcpna (Wong et al., 2011)). The 3′RACE products were gel-extracted and ligated into a pGEM-T Easy vector (Promega, USA), and five and 14 pbcrt-specific independent plasmids were sequenced from the T9-rc and U4-rc reactions, respectively. Fig. 3B illustrates the identified sequences.

Fig. 3.

Fig. 3

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Post-excision re-cloned parasites with the pbcrt-no3′UTR locus (Plasmodium berghei chloroquine resistance transporter - no3′untranslated region) express pbcrt mRNA with a variety of alternative 3′ regulatory sequences. (A) 3′ rapid amplification of cDNA ends (RACE) products for T9-rc and U4-rc. mRNA was reverse transcribed using primer QT (Scotto-Lavino et al., 2006). In the first round of PCR amplification, pbcrt 3′ cDNA sequences (comprising the last 0.3 kb of coding sequence extending into the poly(A) tail) were amplified using primers p5 and QO. In the nested PCR (shown here), pbcrt 3′ cDNA ends (comprising the last 0.24 kb of coding sequence plus the 3′ regulatory sequence) were amplified using primers p1 and QI. A single dominant band was observed for clone T9-rc, and several bands for clone U4-rc. PCR products were gel-extracted, ligated into a pGEM-T Easy plasmid and sequenced with vector-specific primers SP6 and M13F (Table 1). (B) Schematic representation of alternative 3′UTRs observed in pbcrt mRNAs from post-excision pbcrt conditional knockout (cKO) parasites. The exon-intron structure of the 13-exon pbcrt coding sequence is shown stylistically as grey (intron) and white (exon) boxes. All but one of the observed patterns resulted in the production of PbCRT protein with a modified C-terminus. PCR and sequencing analyses indicated that these alternative 3′ regulatory elements were added during transcript processing, as the post-excision pbcrt-no3′UTR genomic locus lacked a proximal 3′UTR.

In 3′ RACE products from the U4-rc clone, we observed three different types of mRNA (Fig. 3B). First, in five pGEM-T Easy clones a poly(A) tail was directly attached to the pbcrt fully spliced coding sequence upstream of the stop codon. Two different attachment sites were observed (-10 and -77 nucleotides from the stop codon) with the resulting PbCRT protein lacking the last four or 26 C-terminal amino acids, respectively. Second, in seven pGEM-T Easy clones a poly(A) tail was attached to the plasmid backbone that remained in the pbcrt-no3′UTR locus, with attachment sites observed at either +112, +214 or +448 nucleotides from the stop codon. In this second set of sequences, therefore, the entire pbcrt coding sequence was retained, followed by the NotI restriction site used for cloning the pPbcrt-cKO plasmid, the single FRT site remaining after site-specific recombination, and 62, 164 or 398 nucleotides of plasmid backbone, respectively. These sequences were followed by the poly(A) tail. Third, in two pGEM-T Easy clones a poly(A) tail was attached to an unspliced pbcrt sequence upstream of its stop codon. This resulted in PbCRT being prematurely terminated with the last 58 C-terminal amino acids being replaced by six different amino acids. These RACE-PCR products were of mRNA origin, and not due to genomic DNA contamination, as no product was obtained in a control RACE-reaction that did not include reverse transcriptase.

For T9-rc, four of the five bacterial clones contained the same sequence (sequence A), with the fifth bacterial clone containing a very similar sequence (sequence B). In these sequences the 3′end of pbcrt was followed by an inverted trap 3′UTR followed by a poly(A) tail. Importantly, at the genomic level the T9-rc clone had the expected arrangement of the pbcrt-no3′UTR locus (Fig. 1A), as assessed by diagnostic PCR and sequencing. This suggests that the pbcrt mRNAs acquired the inverted trap 3′UTR sequence during the process of transcription. Of note, the P. berghei trap genomic locus is located on chromosome 13, as opposed to chromosome 12 for pbcrt. Interestingly, the trap gene is positioned in a tail-to-tail arrangement with PBANKA_134970 (non-annotated), with only 0.6 kb separating the two stop codons. It is therefore quite likely that the trap 3′UTR is bidirectional and the inverted sequence might therefore provide a functional 3′UTR when joined onto the 3′ end of the pbcrt coding sequence. Interestingly, this inverted trap 3′UTR was connected imperfectly to the pbcrt coding sequence, resulting in a modified PbCRT C-terminus. In sequence A, the last 15 PbCRT amino acids (NDSEAELTSIETSRA*; where * denotes the C-terminal end of the sequence) were replaced with YDHDYAKRAINPH*, while in sequence B the last three PbCRT amino acids (SRA*) were replaced with AMTMITPSAQLTLTKGNKSWSLGGPSIIFVS*.

It is worth pointing out that the anti-PfCRT monoclonal antibodies used herein are directed to a C-terminal amino acid stretch (residues 401-419; (Fidock et al., 2000)) that was lost from sequence A of T9-rc and from some of the U4-rc alternate mRNA species (Fig. 3B). Our western blots of clones T9-rc and U4-rc (Fig. 2C) are therefore qualitative and cannot provide quantitative assessment of the level to which PbCRT protein expression was preserved. It is clear, however, that at least some of the alternate 3′UTRs identified retain function, both in T9-rc and U4-rc parasites, such that PbCRT protein can still be produced. Of note, neither T9-rc nor U4-rc displayed any reduction in growth compared with the parental lines (Fig. 2A). Given that in P. falciparum a reduction of PfCRT levels by 30-40% (achieved through truncation of the 3′UTR) resulted in slower growth (Waller et al., 2003), the lack of a growth phenotype in our pbcrt-no3′UTR clones is further evidence for the ability of the aberrant 3′UTRs to maintain pbcrt expression.

Of note, we have recently learned that our pPbcrt-cKO plasmid design differed subtly from plasmids described in the earlier Flp/FRT reports (Combe et al., 2009; Lacroix et al., 2011) in that our plasmid was missing the first 12 nucleotides of the trap 3′UTR. This did not appear to affect expression of pbcrt prior to excision, nor did it impact excision of the FRTed sequence (see Fig. 1), which was found to be highly efficient at the genomic DNA level by quantitative PCR, and was verified by sequence analysis. This sequence difference therefore seems to be an unlikely explanation for the aberrant pbcrt mRNAs observed in the post-excision parasites. The lack of this short sequence stretch constitutes the only difference between our construct and plasmids used to successfully silence other genes (Combe et al., 2009; Lacroix et al., 2011), and as such provides a possible explanation for the production of aberrant pbcrt mRNAs observed in our post-excision parasites.

In summary, pbcrt expression was maintained following removal of its 3′UTR in independent clones derived from two different deleter strains. Several alternative 3′UTR sequences were identified (Fig. 3B), underscoring the astounding plasticity of this parasite at the nucleic acid level. Notably, following mosquito bite or direct inoculation with post-excision blood-stage parasites, mice became blood smear-positive with clones T9 and U4 with no delay compared with the parental deleter lines TRAP/FlpL and UIS4/Flp, respectively. This suggests that these modified pbcrt mRNAs are readily generated. We cannot be certain that all of these mRNAs result in the production of functional PbCRT protein. However, given the continued expression of PbCRT in both T9-rc and U4-rc, we are confident that at least some of these alternate 3′UTRs are able to sufficiently stabilize pbcrt mRNAs for successful translation into protein. These sequences may therefore provide insights into the necessary characteristics of endogenous Plasmodium 3′UTRs, which remain largely undefined (Horrocks et al., 2009). These findings also call for careful molecular analysis of cKO events based on 3′UTR excision, as Plasmodium may adopt alternative elements to overcome gene silencing. In addition, our observations demonstrate that the parasite is under enormous selective pressure to maintain pbcrt expression, consistent with this protein product having an important or essential function in the stages following the 3′UTR excision process.

Highlights.

  • We sought to conditionally silence the chloroquine resistance transporter to test its role across the Plasmodium lifecycle.

  • This used the Flp/FRT system to excise the 3′UTR during mosquito passage, leading to unstable, non-functional transcripts.

  • Despite successful excision, P. berghei progressed normally through the lifecycle by attaching alternative 3′UTR elements.

  • This study highlights the remarkable ability of Plasmodium at the genomic level, to overcome disruption of essential genes.

Acknowledgements

We would like to thank Robert Ménard [Institut Pasteur, France] for the deleter strains and the p3′TRAP_hdhfr_FRT plasmid and Jean Nonon at New York University, USA for providing mosquitoes. We also thank the reviewers for their very helpful comments. AE gratefully acknowledges funding from the Human Frontier Science Program in the form of a Long Term Fellowship. Funding for this work was also provided by the US National Institutes of Health (R01 AI50234, to DF).

Footnotes

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References

  1. Baro NK, Pooput C, Roepe PD. Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast. Biochemistry. 2011;50:6701–6710. doi: 10.1021/bi200922g. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Combe A, Giovannini D, Carvalho TG, Spath S, Boisson B, Loussert C, Thiberge S, Lacroix C, Gueirard P, Menard R. Clonal conditional mutagenesis in malaria parasites. Cell Host Microbe. 2009;5:386–396. doi: 10.1016/j.chom.2009.03.008. [DOI] [PubMed] [Google Scholar]
  3. de Koning-Ward TF, Fidock DA, Thathy V, Menard R, van Spaendonk RM, Waters AP, Janse CJ. The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome. Mol. Biochem. Parasitol. 2000;106:199–212. doi: 10.1016/s0166-6851(99)00189-9. [DOI] [PubMed] [Google Scholar]
  4. Ecker A, Lakshmanan V, Sinnis P, Coppens I, Fidock DA. Evidence that mutant PfCRT facilitates the transmission to mosquitoes of chloroquine-treated Plasmodium gametocytes. J. Infect. Dis. 2011;203:228–236. doi: 10.1093/infdis/jiq036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ecker A, Lehane AM, Clain J, Fidock DA. PfCRT and its role in antimalarial drug resistance. Trends Parasitol. 2012 doi: 10.1016/j.pt.2012.08.002. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ekland EH, Schneider J, Fidock DA. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. FASEB J. 2011;25:3583–3593. doi: 10.1096/fj.11-187401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Falae A, Combe A, Amaladoss A, Carvalho T, Menard R, Bhanot P. Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development. J. Biol. Chem. 2010;285:3282–3288. doi: 10.1074/jbc.M109.070367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol. Cell. 2000;6:861–871. doi: 10.1016/s1097-2765(05)00077-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fidock DA, Rosenthal PJ, Croft SL, Brun R, Nwaka S. Antimalarial drug discovery: efficacy models for compound screening. Nat. Rev. Drug Discov. 2004;3:509–520. doi: 10.1038/nrd1416. [DOI] [PubMed] [Google Scholar]
  10. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ. A proteomic view of the Plasmodium falciparum life cycle. Nature. 2002;419:520–526. doi: 10.1038/nature01107. [DOI] [PubMed] [Google Scholar]
  11. Giovannini D, Spath S, Lacroix C, Perazzi A, Bargieri D, Lagal V, Lebugle C, Combe A, Thiberge S, Baldacci P, Tardieux I, Menard R. Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa. Cell Host Microbe. 2011;10:591–602. doi: 10.1016/j.chom.2011.10.012. [DOI] [PubMed] [Google Scholar]
  12. Golightly LM, Mbacham W, Daily J, Wirth DF. 3′ UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum. Mol. Biochem. Parasitol. 2000;105:61–70. doi: 10.1016/s0166-6851(99)00165-6. [DOI] [PubMed] [Google Scholar]
  13. Griffin CE, Hoke JM, Samarakoon U, Duan J, Mu J, Ferdig MT, Warhurst DC, Cooper RA. Mutation in the Plasmodium falciparum CRT protein determines the stereospecific activity of the antimalarial Cinchona alkaloids. Antimicrob. Agents Chemother. 2012;56:5356–5364. doi: 10.1128/AAC.05667-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Horrocks P, Wong E, Russell K, Emes RD. Control of gene expression in Plasmodium falciparum - ten years on. Mol. Biochem. Parasitol. 2009;164:9–25. doi: 10.1016/j.molbiopara.2008.11.010. [DOI] [PubMed] [Google Scholar]
  15. Kocken CH, der Wel AM, Dubbeld MA, Narum DL, van de Rijke FM, van Gemert GJ, van der Linde X, Bannister LH, Janse C, Waters AP, Thomas AW. Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization. J. Biol. Chem. 1998;273:15119–15124. doi: 10.1074/jbc.273.24.15119. [DOI] [PubMed] [Google Scholar]
  16. Lacroix C, Giovannini D, Combe A, Bargieri DY, Spath S, Panchal D, Tawk L, Thiberge S, Carvalho TG, Barale JC, Bhanot P, Menard R. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nat. Protoc. 2011;6:1412–1428. doi: 10.1038/nprot.2011.363. [DOI] [PubMed] [Google Scholar]
  17. Martin RE, Kirk K. The malaria parasite’s chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol. Biol. Evol. 2004;21:1938–1949. doi: 10.1093/molbev/msh205. [DOI] [PubMed] [Google Scholar]
  18. Matuschewski K, Ross J, Brown SM, Kaiser K, Nussenzweig V, Kappe SH. Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J. Biol. Chem. 2002;277:41948–41953. doi: 10.1074/jbc.M207315200. [DOI] [PubMed] [Google Scholar]
  19. Papakrivos J, Sa JM, Wellems TE. Functional characterization of the Plasmodium falciparum chloroquine-resistance transporter (PfCRT) in transformed Dictyostelium discoideum vesicles. PLoS One. 2012;7:e39569. doi: 10.1371/journal.pone.0039569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Petersen I, Eastman R, Lanzer M. Drug-resistant malaria: molecular mechanisms and implications for public health. FEBS Lett. 2011;585:1551–1562. doi: 10.1016/j.febslet.2011.04.042. [DOI] [PubMed] [Google Scholar]
  21. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roepe PD. PfCRT-mediated drug transport in malarial parasites. Biochemistry. 2011;50:163–171. doi: 10.1021/bi101638n. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rosinski-Chupin I, Chertemps T, Boisson B, Perrot S, Bischoff E, Briolay J, Couble P, Menard R, Brey P, Baldacci P. Serial analysis of gene expression in Plasmodium berghei salivary gland sporozoites. BMC Genomics. 2007;8:466. doi: 10.1186/1471-2164-8-466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanchez CP, Mayer S, Nurhasanah A, Stein WD, Lanzer M. Genetic linkage analyses redefine the roles of PfCRT and PfMDR1 in drug accumulation and susceptibility in Plasmodium falciparum. Mol. Microbiol. 2011;82:865–878. doi: 10.1111/j.1365-2958.2011.07855.x. [DOI] [PubMed] [Google Scholar]
  25. Scotto-Lavino E, Du G, Frohman MA. 3′ end cDNA amplification using classic RACE. Nat. Protoc. 2006;1:2742–2745. doi: 10.1038/nprot.2006.481. [DOI] [PubMed] [Google Scholar]
  26. Summers RL, Martin RE. Functional characteristics of the malaria parasite’s “chloroquine resistance transporter”: implications for chemotherapy. Virulence. 2010;1:304–308. doi: 10.4161/viru.1.4.12012. [DOI] [PubMed] [Google Scholar]
  27. Summers RL, Nash MN, Martin RE. Know your enemy: understanding the role of PfCRT in drug resistance could lead to new antimalarial tactics. Cell Mol. Life Sci. 2012;69:1967–1995. doi: 10.1007/s00018-011-0906-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Waller KL, Muhle RA, Ursos LM, Horrocks P, Verdier-Pinard D, Sidhu AB, Fujioka H, Roepe PD, Fidock DA. Chloroquine resistance modulated in vitro by expression levels of the Plasmodium falciparum chloroquine resistance transporter. J. Biol. Chem. 2003;278:33593–33601. doi: 10.1074/jbc.M302215200. [DOI] [PubMed] [Google Scholar]
  29. Wong EH, Hasenkamp S, Horrocks P. Analysis of the molecular mechanisms governing the stage-specific expression of a prototypical housekeeping gene during intraerythrocytic development of P. falciparum. J. Mol. Biol. 2011;408:205–221. doi: 10.1016/j.jmb.2011.02.043. [DOI] [PMC free article] [PubMed] [Google Scholar]

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