A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development

Abstract

Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain 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 endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly 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 target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.

Fig. 1.

Phylogenetic analysis reveals Pcalp as a distinct type and clade of calpains. (A) Phylogenetic analysis of Pcalp (catalytic domain) aligned with representatives of each documented type of calpain is shown (left side) together with the domain composition of each protein (right side). Bootstrap values are shown at the tree joints. Domains are color coded and from top to bottom named at their first appearance (SMART code). The Pcalp N terminus has subdomains conserved among Plasmodium species (green) (I.R., A.O., and D.E.G., unpublished data). Scale is fractional change per unit distance. (B) Phylogenetic analysis and domain 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.

Fig. 2.

Expression profiles of Pcalp along the asexual cycle. (A) Semiquantitative RT-PCR analysis. Expression of Pcalp mRNA in asexual stages was measured by quantifying amplified regions encoding the N (light gray) and C (dark gray) termini (map of region 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 shown 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 corresponds to the first peak of mature schizonts. Dots indicate harvest times for protein analysis (MT, midtrophozoites) and the arrow marks 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 measured. Each lane corresponds 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.

Fig. 3.

Attempted genetic strategies to disrupt calpain functionality. (A) Diagrams of the vectors. (i) For double 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 replacement, 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 codons downstream in the active site cysteine codon (*), one has a synonymous change, whereas the other has a nonsynonymous change. Predicted products of integration of ii and iii are shown in Fig. S4. (iv) For reference the Pcalp gene is shown. Primers used for PCR/restriction in the allelic replacement strategy are indicated (semiarrows). Restriction sites: X, XmaI; N, NsiI; S, SphI; P, PacI. (B) Southern blot analysis. (i) Double cross-over. Transfected parasite DNA (X/P digested) shows the uninterrupted endogenous gene (open arrow) and episomal plasmid (gray arrow), but no evidence of any integration event (filled arrows). Each panel is a different exposure time. (ii–v) Single cross-over. DNA was restricted with N/S. All 4 transfections with the control vector show 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 showed 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 shown 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.

Fig. 4.

Knockdown of Pcalp. (A) Creation of a calpain-GFP-FKBP chimera by homologous recombination. The diagram shows the strategy to create C-terminally tagged calpain by integration at the endogenous 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: endogenous gene (black), plasmid (gray), and modified calpain locus (open). (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 corresponds to equivalent numbers of mid/late trophozoites.

Fig. 5.

Analysis of Pcalp knockdown 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 breaks indicate an equal subculture event for each culture. X axis in D same as in E. (F) Viability measurements using different dyes to measure dead parasites in A7 (light) and C3 (dark) 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 showed 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 acquired 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 shown 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.