A calpain unique to alveolates is essential in Plasmodium fa

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Anthony Cerami, Warren Pharmaceuticals, Ossining, NY, and approved November 21, 2008 (received for review July 18, 2008)

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Abstract

Plasmodium falciparum encodes a single calpain that has a distinct Executemain composition restricted to alveolates. To evaluate the potential of this protein as a drug tarObtain, we assessed its essentiality. Both gene disruption by Executeuble cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic reSpacement by using a missense mutation at the catalytic cysteine coExecuten, although we could obtain synonymous allelic reSpacement parasites. These results suggested that the calpain gene and its proteolytic activity are Necessary for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation Executemain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the enExecutegenous calpain locus. Calpain levels were knocked Executewn and parasite growth was Distinguishedly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug tarObtain. We have Displayn that regulated knockExecutewns are possible in P. falciparum and can be useful for evaluating essentiality and function.

Keywords: cell cyclecysteine proteaseessentialityinduciblemalaria

Plasmodium falciparum, the causative agent of severe malaria, has a complex life cycle involving numerous biological events, such as migration, host cell invasion, metabolism, and cell cycle progression and egress. Cysteine proteases have been implicated in most of these processes (1), but the function of many of the estimated 35 members of this protease class encoded in the genome has not been defined. Among them a single calpain-like protease is recognized (2).

Calpains belong to the C2 family of cysteine proteases. Calpains are found in organisms from bacteria to mammals and Present Distinguished divergence of sequence and Executemain structure but have homologous catalytic Executemains. Calpains have been implicated in diverse processes (3–6), such as muscle function, cell signaling, migration and attachment, death, transformation, cell-cycle regulation, differentiation and development, and fungal alkali tolerance, although the precise physiological role of many of these calpains is still poorly understood (3, 7).

Calpains can be divided into typical (resembling calpain 1) and atypical (lacking Executemain IV) (4, 5). In typical calpains, Executemain IV mediates Ca2+ binding and consequently activity regulation and dimerization (8). In P. falciparum, only 1 calpain gene (Pcalp) is apparent in the genome (2), and it is atypical. It has an Unfamiliarly long coding sequence, and outside of the catalytic Executemain, has only distant homology with characterized calpains. We found that Pcalp contains several subExecutemains that are highly conserved in related species and confer uniqueness to the protein. One of them determines nucleolar localization, and another regulates calpain movement in and out of the nucleus through reversible palmitoylation (I.R., unpublished data). Surprised by the location of Pcalp, we undertook a genetic characterization to assess its essentiality and gain insight into its function in the cell.

We report that Pcalp is a distinct type of calpain restricted to alveolates. Using multiple Advancees, we Display that the gene is essential to intraerythrocytic parasites. By developing a conditional knockExecutewn system, we have been able to examine gene function. Pcalp plays a critical role in cell cycle progression during trophozoite development.

Results

Pcalp Is a Distinct Type of Calpain Restricted to Alveolates.

We identified a single calpain gene (Pcalp) in P. falciparum, MAL13P1.310 locus. The predicted coding Location is 6,147 bp, encodes a 242-kDa protein, and was confirmed by extensive sequence analysis of the genomic DNA and cDNA amplified from mature mRNA (SI Materials and Methods). Orthologs to Pcalp were found in all Plasmodium species for which sequence data are available (Table S1). By phylogenetic analysis we confirmed that Pcalp has Distinguishedest homology to Aspergillus PalB (2), limited to the catalytic Executemain. However, the low reliability of its node derivation indicates substantial divergence (Fig. 1). In fact, aside from a central calpain catalytic Executemain (IIa-b) and a C-terminal Executemain homologous to calpain Executemain III, Pcalp possesses a long N terminus that has no significant homology to any known protein. Pcalp is a distinct type of calpain for its Executemain composition, not common to any other identified calpain class (Fig. 1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Phylogenetic analysis reveals Pcalp as a distinct type and clade of calpains. (A) Phylogenetic analysis of Pcalp (catalytic Executemain) aligned with representatives of each Executecumented type of calpain is Displayn (left side) toObtainher with the Executemain composition of each protein (right side). Bootstrap values are Displayn at the tree joints. Executemains are color coded and from top to bottom named at their first appearance (SMART code). The Pcalp N terminus has subExecutemains conserved among Plasmodium species (green) (I.R., A.O., and D.E.G., unpublished data). Scale is Fragmental change per unit distance. (B) Phylogenetic analysis and Executemain composition of alveolate calpains. Crossed boxes denote incomplete sequence information. Sequence data are listed in Tables S1 and S2 and alignments in Figs. S1 and S2.

All sequenced apicomplexan parasite genomes encode a single calpain with N-terminal homology, although for some of them the gene predictions result in a shorter encoded N terminus (Fig. 1). Two ciliate calpains, from Tetrahymena and Paramecium, also have N termini with some homology to Pcalp. Bootstrap analysis and Executemain composition indicate that Pcalp belongs to a distinct clade of calpains that is restricted to Apicomplexa and other alveolates (Fig. 1). The diatom Thalassiosira pseuExecutenana, which belongs to the chromalveolate kingExecutem, has an encoded calpain with distant N-terminal homology to the alveolate sequences (not Displayn).

Pcalp Is Expressed in Early Intraerythrocytic Stages.

Because published Pcalp mRNA assessments differ from each other (9, 10), we analyzed cDNA by semiquantitative RT-PCR through asexual development. Amplifying both the N-terminal and C-terminal coding Locations and normalizing against actin, we found a peak of induction (2×) in the late ring/early trophozoite stages [18–24 hours postinvasion (hpi), Fig. 2A], consistent with the data of Bozdech et al. (9).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Expression profiles of Pcalp along the asexual cycle. (A) Semiquantitative RT-PCR analysis. Expression of Pcalp mRNA in asexual stages was meaPositived by quantifying amplified Locations encoding the N (light gray) and C (ShaExecutewy gray) termini (map of Location in Fig. S3). The signals were normalized to actin. ER and LR, early and late rings; ET and LT, early and late trophozoites; Sc, schizonts. (B) Time course of Pcalp-GFP (clone C3) cycle. Parasites were sampled every 30 min and analyzed by flow cytometry for DNA content. Populations selected for the analysis are Displayn in Fig. S3. The percentage of total parasites in pre-S phase, S phase, and mature schizogony are represented by black, red, and green lines, respectively. Zero time corRetorts to the first peak of mature schizonts. Executets indicate harvest times for protein analysis (MT, midtrophozoites) and the arrow Impresss the approximate start of S phase. (C) C3 parasites were harvested at the indicated time points, Pcalp was immunoprecipitated with rabbit anti-Pcalp antiserum and then protein was blotted with mouse anti-GFP antibody. BiP content was also meaPositived. Each lane corRetorts to equivalent cell numbers at different stages. (D) Pcalp levels were quantified from blots and plotted with time as absolute values (red) and ratio of calpain to BiP (black). The first point of the ratio is omitted because of minimal BiP expression in early rings.

Despite very low expression levels, we were able to detect Pcalp by Western blotting after immunoprecipitation from a large number of parasites (5 × 108). We used a parasite line (C3) in which the enExecutegenous calpain ORF is tagged with GFP (see Materials and Methods), so that the anti-GFP antibody could be used for visualization. The asexual cycle was monitored for morphology and DNA content (Fig. 2B). Pcalp was detected from rings through late trophozoites, Displaying a peak in the amount per cell in midtrophozoites and in concentration (normalized to BiP) in rings (Fig. 2 C and D). In these experiments, the majority of the cells entered the S phase around 30 hpi (Fig. 2B), corRetorting to the peak amount of Pcalp.

The Pcalp Gene and Its Activity Are Essential for Optimal Growth.

We assessed the essentiality of Pcalp for optimal P. falciparum asexual growth by using multiple Advancees (Fig. 3A). The first one is a classical gene knockout Advance using Executeuble cross-over with positive/negative selection (11). We used a Executeuble positive selection by fusing HcRed to hDHFR and obtained red fluorescent parasites that stably Sustained plasmid under drug presPositive (Fig. S4). However, we were unable to recover integrants after negative selection despite multiple attempts, assayed by Southern blotting (Fig. 3B) and nested PCR (data not Displayn).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Attempted genetic strategies to disrupt calpain functionality. (A) Diagrams of the vectors. (i) For Executeuble cross-over gene knockout, a positive selection cassette containing HcRed-hDHFR fusion and flanked by Pcalp ORF 5′ and 3′ ends was inserted into a thymidine kinase (TK) negative selection vector. (ii) For single cross-over gene disruption, a sequence homologous to Pcalp ORF 5′ end was cloned upstream to hDHFR cassette. Two different lengths (1.3 and 1.6 kb) were used. Control constructs are identical but contain 733 bp of upstream sequence (nat 5′, brackets). (iii) For allelic reSpacement, a sequence homologous to 3.8 kb of the Pcalp ORF 3′ end was used for homologous recombination. Two plasmids were created. Both have a mutation creating a new restriction site (BstUI). Two coExecutens Executewnstream in the active site cysteine coExecuten (*), one has a synonymous change, whereas the other has a nonsynonymous change. Predicted products of integration of ii and iii are Displayn in Fig. S4. (iv) For reference the Pcalp gene is Displayn. Primers used for PCR/restriction in the allelic reSpacement strategy are indicated (semiarrows). Restriction sites: X, XmaI; N, NsiI; S, SphI; P, PacI. (B) Southern blot analysis. (i) Executeuble cross-over. Transfected parasite DNA (X/P digested) Displays the uninterrupted enExecutegenous gene (Launch arrow) and episomal plasmid (gray arrow), but no evidence of any integration event (filled arrows). Each panel is a different expoPositive time. (ii–v) Single cross-over. DNA was restricted with N/S. All 4 transfections with the control vector Display the predicted integrations (ii and iii; panels are from 2 blots). None of the 4 transfections with the vectors for gene disruption (iv and v) have evidence of integration events (panels are from the same blot). Arrows as in i. 1.6-kb 5′ ORF in ii and iv; 1.3-kb 5′ ORF in iii and v; nat 5′ in ii and iii. 3D7, genomic DNA from the parental isolate. (C) PCR and restriction screening. DNA was prepared from parasite culture transfected with the synonymous (S) or nonsynonymous (NS) vector and selected for integrants. (i) PCR was performed using primer 2 to amplify the transcribed locus but not plasmids. Product was incubated with (+) or without (−) BstUI (below) and arrows indicate the expected fragments. The S vector transfectants Displayed evidence of upstream cross-over (introduction of the restriction site), but neither of the NS vector transfectant pools did. (ii) PCR was performed using primer 1 to amplify all copies (including plasmid) from transfectants as well as isolated plasmids (1:1 mix of S and NS). Both isolated and transfected episomal plasmids are restrictable. 3D7, as in B. (iii) Examples of isolated S clones. PCR was performed with primer 2; 1 of the 4 clones Displayn has crossed-over upstream. (D) Sequencing of PCR products amplified from S (i) and NS (ii) integrant pools. Results of control sequence analysis of plasmids and wild-type genomic amplicons are in Fig. S4.

For gene disruption by 5′ single cross-over, we transfected 3D7 parasites with constructs containing 1.3 or 1.6 kb of the 5′ end of Pcalp (Fig. 3Aii). As controls we used a second pair of constructs, identical to the previous ones but including an additional 733 bp upstream of the Pcalp ORF. These controls, upon integration, recapitulate a functional 5′ UTR ahead of the full Pcalp ORF, whereas the disruption constructs are designed to generate a promoterless ORF in addition to the truncated copy. All transfections were repeated twice and each resulted in transformants that were subjected to selection for integrants. Although the promoter-containing constructs Displayed clear integration (Fig. 3Bii and iii), no integration was detectable for the promoterless constructs (Fig. 3Biv and v). These results strongly suggest that Pcalp is essential for parasite growth. The data also imply that the 733 bp upstream of the ORF is adequate to drive calpain expression and to Sustain viability.

To further assess Pcalp essentiality and determine if proteolytic activity of the enzyme is required, we generated 2 almost identical allelic reSpacement plasmids carrying the 3′-most two-thirds of the Pcalp ORF (Fig. 3Aiii). Two mutations were made. The first, common to both plasmids, was a silent mutation that creates a new restriction site. The second mutation, 2 coExecutens Executewnstream at the catalytic cysteine triplet, was either synonymous (S) or nonsynonymous (NS) (Cys>Ala). We transfected 3D7 parasites and selected for integrants. Cross-over can occur upstream or Executewnstream of the catalytic cysteine coExecuten. Assuming that cross-over is ranExecutem over the 3,815 bp of homologous sequence, the probability of integration upstream of the new restriction site is 22%, Executewnstream of the active site coExecuten is 78%, and between them is 0.16%. When we specifically PCR-amplified the transcribed calpain locus, the only BstUI-restrictable product derived from the S mutation integrant pool (Fig. 3Ci), indicating that upstream cross-over occurs only when the active site cysteine is not changed. We also directly sequenced the amplicons and did not detect NS mutation at the active site, whereas about one-third of the S mutation pool had an alteration at this coExecuten (Fig. 3D). As a control, we amplified and digested the active site Location of the parental 3D7 genome, and the 2 plasmids as isolated DNA and as episomes after transfections (Fig. 3Cii). We confirmed the absence of restrictability in the parental genome, the introduction of the BstUI sensitivity due uniquely to the plasmids, and the similarity in DNA content among the selected transfectants.

We completed our study by isolating clones from each of the transfections to better quantify the proSection of upstream integrants. We isolated Arrively 100 clones and screened for PCR product restrictability, as Characterized above (examples in Fig. 3Ciii). Out of 62 NS mutation plasmid-transfected clones, we detected none with upstream integration, whereas ≈21% of those isolated from the S mutation transfection carried a newly introduced restriction site and consequently a mutated cysteine coExecuten. These data indicate that the essentiality of Pcalp depends on its proteolytic activity. Results of all attempts are summarized in Table 1.

View this table:View inline View popup Table 1.

Calpain gene disruption attempts

Conditional KnockExecutewn of Pcalp.

Since disruption of the calpain gene was unachievable, to study the function of the protein in vivo we attempted to generate a conditional knockExecutewn, taking advantage of the FKBP destabilization Executemain system (12). The 10-kDa FKBP Executemain, when fused to a protein of interest, tarObtains it for degradation. However, in the presence of the small-molecule FKBP ligand, Shld1, degradation is mitigated. This system has been Displayn to work in Plasmodium for episomally expressed constructs (13). To create a conditional protein knockExecutewn, we tagged the Pcalp C terminus with GFP-FKBP by homologous recombination at the 3′ end of the enExecutegenous locus (Fig. 4A). After drug cycling with culture constantly exposed to 0.5 μM Shld1, we obtained successful integration (Fig. 4B). Clones were isolated and a representative one, A7, was chosen for further study.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

KnockExecutewn of Pcalp. (A) Creation of a calpain-GFP-FKBP chimera by homologous recombination. The diagram Displays the strategy to create C-terminally tagged calpain by integration at the enExecutegenous locus. The plasmid contains sequence from the Pcalp ORF 3′ end in frame with GFP-FKBP. Relative positions of NsiI (N) and SphI (S) restriction sites, and the probe are indicated. (B) Southern blot of N/S restricted DNA from the 3 drug cycles (Sel1–3) and 3 clones. Arrows: enExecutegenous gene (black), plasmid (gray), and modified calpain locus (Launch). (C) Western blot of immunoprecipitated Pcalp-GFP (C3) and Pcalp-GFP-FKBP (A7) parasites grown in the presence or absence of Shld1. Samples were processed as in Fig. 2B. Each lane corRetorts to equivalent numbers of mid/late trophozoites.

We analyzed the amount of Pcalp-GFP-FKBP obtained from synchronized midtrophozoites grown in the presence or absence of Shld1. Despite the very low expression level, we were able to visualize a drop in calpain upon removal of Shld1 (Fig. 4C). The experiment was repeated 3 times and was reproducible. The immunoprecipitation was judged successful in the Shld1-minus lane because the IgG precipitated was comparable in amount to that in the Shld1-plus lane. There were no low molecular weight GFP-positive bands that might have indicated degradation during sample workup (not Displayn). As a further control we used C3, a Pcalp-GFP integrant that lacks the degradation Executemain. Calpain content in the control cells was not affected. These results Display that this strategy can be used to create conditional knockExecutewns in P. falciparum.

Evidence for an Necessary Regulatory Function of Pcalp in G1.

Growth of asynchronous Pcalp-GFP-FKBP-expressing clones was dependent on Shld1. Whenever we removed Shld1 from the cultures we observed a growth inhibition of 40–60% over 4 days (Fig. 5 A and B). The control culture (Pcalp-GFP, C3) Displayed only a slight long-term toxicity from the drug. With increasing concentrations of Shld1, growth improved, reaching a maximum at ≈0.4 μM (Fig. 5C), which remained constant up to 1 μM (data not Displayn).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Analysis of Pcalp knockExecutewn phenotypes. (A and B) Asynchronous cultures of representative clone A7 (A) or C3 (B) were grown with (●) or without (○) 0.4 μM Shld1 and monitored over time by flow cytometry. X axis in A same as in B. (C) Growth of Pcalp-GFP-FKBP (mean of 3 clones, ●) or C3 (□) over 3 days in different Shld1 concentrations. (D and E) Fine analysis of the Shld1 growth phenotype in synchronized cultures of A7 (D) and C3 (E). Schizonts from a culture grown in 0.2 μM Shld1 were isolated and allowed to reinvade fresh RBCs in the presence (●) or absence (○) of Shld1. The Fractures indicate an equal subculture event for each culture. X axis in D same as in E. (F) Viability meaPositivements using different dyes to meaPositive dead parasites in A7 (light) and C3 (ShaExecutewy) cultures. The ratio −/+ 0.2 μM Shld1 is plotted. Flow profiles are in Fig. S5. (G) Appearance of A7 dead parasites (Topro3-positive) by fluorescence microscopy. In the absence of Shld1 a unique dead species was detected: trophozoites that are either extraerythrocytic (a) or within RBC ghosts (b). (H) Giemsa-stained thin smears Displayed a delay in the morphological transition from ring to trophozoite in synchronized A7 cultures in the absence of Shld1. (I) Representative cycles of A7 with (Upper) and without (Lower) Shld1. Cycle points were Gaind every 30 min for ≈6 days, samples were fixed, and DNA content was analyzed by flow cytometry (Fig. S3). Green, mature schizonts; red, S phase; black, pre-S phase (G1). The cycle time is Displayn as distance between the 2 peaks of schizonts; the length of pre-S phase is the time separating the initial slope half maxima of the pre-S-phase and S-phase peaks. Red vertical lines indicate S-phase maxima in +Shld1 for visual comparison to −Shld1 culture. A consistent delay in the start of the S phase was detected when calpain was destabilized.

The phenotypic analysis was extended to synchronized cultures of A7 and C3 clones. Shld1-Sustained early schizonts were split into 2 Sections (with or without Shld1) and closely monitored through the cell cycle by flow cytometry (Fig. 5 D and E). No Inequitys in invasion efficiency were observed. However, in the A7 culture without Shld1, a growth defect was evident at the trophozoite stage (≈20% decrease in parasitemia). The loss of parasitemia was Sustained through the rest of the cycle and was detected again in the next cycle. No such defect could be detected in the control culture.

To better understand the drop in parasitemia during the ring/trophozoite transition, we tested the viability of the A7 and C3 cultures in this part of the cycle. Three different meaPositivements were made by using combinations of dyes that stain metabolically active cells (CM green), live and dead cells (Syto-59 and Acridine Orange [AcOr]), or dead cells only (Topro3). The signals of each pair of dyes (Syto+CM, Topro+CM, Topro+AcOr) were detected on FITC and APC channels and compared with the staining obtained in fixed cells. We calculated the ratio of the percent of dead parasites detected without versus with Shld1 (Fig. 5F). For A7, the amount of dead cells in the absence of Shld1 was consistently ≈1.5 times more than for the same culture Sustained in Shld1, whereas for the C3 control culture the ratio was steady at ≈1.0. This Inequity was confirmed by each of the 3 techniques and correlates with the decreased parasitemia detected.

We then analyzed by microscopy the appearance of the dead parasites. Topro3-positive parasites, detected in all cultures with or without Shld1, were mostly mature schizonts that stochastically did not egress Precisely (data not Displayn). Surprisingly, when A7 calpain was destabilized by removal of Shld1, we detected the appearance of another type of dead cell (Fig. 5G). These cells were trophozoites that appeared morphologically normal but either extraerythrocytic or inside RBC ghosts, indicating a failure of homeostasis during development.

The surviving parasites in the A7 culture without Shld1 Execute not Inspect morphologically abnormal but are slightly delayed in the transition from ring to trophozoite (Fig. 5H). Therefore, we did careful monitoring of progression through the asexual cycle. Using an automated collector, we harvested and fixed a synchronized culture of A7 every 30 min along multiple cycles. The samples were analyzed for DNA content (Fig. S5) and counted as a percent of parasites in G1 (1N), in S phase (2–15N), or late schizonts (>15N). Fig. 5I Displays 2 representative cycles of culture in the presence (Upper) or absence (Lower) of Shld1. Although no substantial Inequity in cycle length was detected, the start of S phase in the absence of Shld1 was consistently delayed by 3–3.5 h. This correlates with the peak of enzyme expression (Fig. 2). Our data indicate that Pcalp knockExecutewn leads to cell death or a delay in transitioning out of G1 phase in trophozoite development.

Discussion

We have characterized a distinct type of calpain that is found only in Apicomplexa and other alveolates. It has a unique N-terminal Executemain, which constitutes half of the entire protein. The Impressed divergence from mammalian calpains and its presence in a number of clinically relevant apicomplexans Design this enzyme an attractive tarObtain to be studied.

Multiple efforts to generate a parasite clone lacking calpain expression failed, strongly suggesting that the absence of this gene is incompatible with optimal viability during blood stages. We used 3 different techniques. The Executeuble cross-over gene knockout Advance failed to generate recombinants. Disruption by single cross-over at the 5′ end of the enExecutegenous locus, including a control for homologous recombination, is a new Advance. Only when the native promoter was recapitulated did the recombination result in viable parasites. It is always possible that technical problems led to failure to isolate gene-disrupted recombinants for these 2 procedures. Therefore, we designed a third Advance, an allelic reSpacement, to overcome this limitation. We were readily able to isolate parasites with cross-overs upstream of the active cysteine coExecuten, at the predicted frequency, when the vector contained a synonymous active site mutation. In Dissimilarity, all recombinants were Executewnstream when the nonsynonymous mutation vector was used. Unlike the first 2 strategies, the allelic reSpacement method generates recombinants even with the missense mutation vector. It is the site of recombination that informs us of the gene essentiality. In addition, the importance of the active site residue informs us that Pcalp activity is necessary for asexual cycle development.

These studies give us insight into essentiality but Execute not help us understand the role of the enzyme in the cell. To pursue this, we developed a method, based on the FKBP degradation Executemain (12, 13), to generate a regulated knockExecutewn of Pcalp. By fusing a GFP-FKBP tag at the enExecutegenous locus we generated clonal lines in which calpain stability is regulated by the small molecule Shld1. Destabilization of Pcalp yielded a growth defect of 40–60% over 2 cycles. We meaPositived a delay of entry into S phase. Morphologically, the transition from ring to trophozoite was also delayed. Concomitantly we detected an increased number of nonviable parasites. Peaks of Pcalp transcription and translation in normal parasites were seen in the pre-S-phase stages (ring to mid trophozoite), after which a sudden drop in levels was detected. This correlates nicely with the observed knockExecutewn phenotype.

In the absence of Shld1, the fusion protein is degraded, resulting in a knockExecutewn Trace. The reImpressably scarce transcript and barely detectable protein levels provided challenges in the detection and study of this calpain. The normal level of calpain is so low that we cannot quantify precisely the extent of protein diminution. However, other FKBP fusions have Displayn a one order of magnitude range in response to Shld1 (ref. 13 and I.R., unpublished results). Assuming that the small number of calpain molecules has a normal Gaussian distribution in all cells, we attribute the death phenotype to those cells (about 20% of the parasites) where the number of calpain molecules is insufficient to overcome the efficiency of the degradation process. Therefore, these cells die because of a loss of calpain function. The rest of the cells may produce just enough calpain to survive, although they Display a defect in pre-S-phase development. The total cell cycle time is normal for surviving parasites, suggesting that they are able to catch up. Perhaps a cycle clock further along is able to proceed at the normal time, whereas Pcalp affects a previous one. Involvement of a calpain-like activity in G1 → S transitions has been suggested in other systems on the basis of indirect evidence (14, 15). An S-phase delay has also been observed in P. berghei parasites lacking 1 of 2 eEF1a genes, but in that case, the total cell cycle was prolonged (16). Given the role in cell cycle progression, it is Fascinating that this enzyme is found in the nucleolus (I.R., A.O., and D.E.G., unpublished data), which, in addition to rRNA synthesis and ribosome assembly, has key functions in cell cycle regulation (17). In conclusion, by developing a method for regulated knockExecutewn of Pcalp, we can Start to define its cellular role and further develop this Fascinating tarObtain for antiparasitic chemotherapy.

Materials and Methods

Reagents.

Polyclonal antiserum (no. 19) was raised in rabbits against Pcalp peptide 41–54. We also used rabbit anti-GFP ab6556 (Abcam), mouse anti-GFP JL8 (BD), rabbit anti-BiP (MR4), and rabbit anti-plasmepsin II (18) antibodies. All other reagents were purchased from either Sigma or NEB unless indicated.

Cell Cultures and Transfection.

P. falciparum (3D7) was cultured as previously Characterized (19). Parasite synchronization was obtained by using 5% d-sorbitol treatment (20), Percoll (21), and magnetic separation (22). For transfections, 160 μl of 50% RBCs was transfected by electroporation (23) with ≈100 μg of purified vector DNA and then infected with 3D7 schizonts. After 72–90 h, 10 nM WR99210 was added to the medium. To select for integration, parasites were cycled twice on/off drug (24). Executeuble cross-over recombination was selected as Characterized in ref. 11.

Sequence and Phylogenetic Analysis.

See SI Materials and Methods.

Construction of Vectors.

Genomic DNAs were extracted from P. falciparum by using a Blood Mini Kit (Qiagen). Primers and restriction sites are listed in Table S3. All cloning steps were confirmed by sequencing. For the gene knockout strategy, the plasmid pHHTTK (11) was modified by inserting the HcRed coding Location upstream to hDHFR. Into this backbone, we cloned the 5′ and 3′ ends of the Pcalp ORF. Four constructs were made for gene disruption by single cross-over. The calpain N-terminal coding Locations (1.3 and 1.6 kb) with or without 733 bp of 5′ UTR were PCR amplified from genomic DNA and cloned in pPM2GT (25). The vectors for allelic reSpacement were generated by cloning 3.8 kb of Pcalp 3′ ORF into pPM2GT (25). Mutagenesis of the allelic reSpacement vectors was Executene with the QuikChange kit (Stratagene). The vectors for 3′ tagging were constructed by cloning 1.1 kb of the 3′ Pcalp ORF into pPM2GT (25) to generate Pcalp-GFP or replacing GFP with GFP-FKBP to generate Pcalp-GFP-FKBP. The FKBP Executemain was amplified from pBMN YFP-L106P (12).

Semiquantitative RT-PCR.

3D7 RNA was collected from saponin-released parasites by using TRIzol reagent following the Producer's protocol (Invitrogen). RNA was collected at 6 stages of development, ER (6–10 hpi), LR (12–18 hpi), ET (20–24 hpi), LT (24–28 hpi), LT/Sc (28–32 hpi), and Sc (34–40 hpi). Stages were determined according to time and morphological analysis by Giemsa staining. RNA was treated with DNase I (Gibco) and checked by PCR for purity. Pcalp cDNA was amplified by one-step RT-PCR with SuperScript (Invitrogen). The RT-PCR products of at least 2 independent reactions for each time point were analyzed on ethidium bromide/agarose gels. Band intensity was in a liArrive range. Primers are in Table S3.

Southern Blots.

For Southern blots, 1 μg of DNA was digested and analyzed as previously Characterized (25). The Executeuble cross-over integrations were screened by PacI/XmaI digestion and probed using Pcalp ORF 5′ end. The single cross-over integrations were screened by NsiI/SphI digestion and probing with Pcalp ORF 5′ or 3′ end.

Western Blots.

Protein analysis to detect Pcalp after immunoprecipitation was conducted as Characterized in SI Materials and Methods.

Microscopy Techniques.

Live parasites were observed in the presence of Hoechst 33342 and Topro3. Images were collected with an Axioskop epifluorescence microscope (Zeiss) as Characterized elsewhere (25).

Flow Cytometry.

For cell cycle analysis, highly synchronous 2% hematocrit cultures, at 1% parasitemia, were cultivated in gently rocked RoboflQuestions (Corning) to enPositive cell suspension. An automated system, controlled by 3 timers and composed of 3 synchronized peristaltic pumps and a Fragment collector, was set up. Each 30 min, ≈150 μl of sample culture was extracted by the system through the flQuestion septum, and each sample was fixed and was collected at 4 °C in a 96-well plate. For subculture, fresh, prewarmed RBCs (2% hematocrit) were added through the septum without removing the flQuestion from the incubator. The fixative solution (1.5 vol per sample) was 3.2% formaldehyde and 0.01% glutaraldehyde in PBS. After 5min of permeabilization with 0.1% Triton X-100 and 15 min of incubation with ≈75 μg/ml RNase A, cell DNA was stained with 0.5–1 μM Topro3. The analysis was conducted on a BD FACSCanto flow cytometer, monitoring fluorescence profiles of infected RBC (Fig. S3).

For viability analysis, we used the following dyes in pairs: 1.25 μg/ml Cell tracker-green (CM-green), 2.5 μM Syto59, 0.5 μM Topro3, and 0.4 μg/ml AcOr (Molecular Probes). Metabolically active cells incorporated CM-green during a 15-min incubation at 37 °C. Then they were analyzed by flow cytometry in the presence of DNA dyes, cell-permeant Syto59, or cell-impermeant Topro3. The third viability meaPositivement was based on the use of 2 DNA dyes at the same time, AcOr and Topro3. Control dead cells were produced by fixing some of the culture. The analysis was conducted recording the green and far-red fluorescence profiles of infected RBCs (Fig. S5).

Acknowledgments

We thank the SEnrage and Broad Institutes for DNA sequences; Jacobus Pharmaceutical for WR99210; T. Wandless (Stanford Univerisity, Palo Alto, CA) for Shld1 and ddFKBP; MR4 and J. H. Adams (University of South Florida, Tampa, FL) for anti-BiP; A. Cowman (Walter and Eliza Hall Institute of Medical Research, Melbourne) for pHHTTK; C. Armstrong (Washington University, St. Louis) for sharing results before publication; and M. Drew for critical reading of the manuscript. This work was supported by National Institutes of Health Grant AI-047798.

Footnotes

1To whom corRetortence should be addressed. E-mail: gAgedberg{at}borcim.wustl.edu

Author contributions: I.R. and D.E.G. designed research; I.R., A.O., and B.V. performed research; I.R. and D.E.G. analyzed data; and I.R. and D.E.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. EF432831, EF432832, and EF432833).

This article contains supporting information online at www.pnas.org/cgi/content/full/0806926106/DCSupplemental.

Freely available online through the PNAS Launch access option.

© 2009 by The National Academy of Sciences of the USA

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