Shelph2, a bacterial-like phosphatase of the malaria parasite Plasmodium falciparum, is dispensable during asexual blood stage

Abstract

During the erythrocytic cycle of the malaria parasite Plasmodium falciparum, egress and invasion are essential steps finely controlled by reversible phosphorylation. In contrast to the growing number of kinases identified as key regulators, phosphatases have been poorly studied, and calcineurin is the only one identified so far to play a role in invasion. PfShelph2, a bacterial-like phosphatase, is a promising candidate to participate in the invasion process, as it was reported to be expressed late during the asexual blood stage and to reside within an apical compartment, yet distinct from rhoptry bulb, micronemes, or dense granules. It was also proposed to play a role in the control of the red blood cell membrane deformability at the end of the invasion process. However, genetic studies are still lacking to support this hypothesis. Here, we take advantage of the CRISPR-Cas9 technology to tag shelph2 genomic locus while retaining its endogenous regulatory regions. This new strain allows us to follow the endogenous PfShelph2 protein expression and location during asexual blood stages. We show that PfShelph2 apical location is also distinct from the rhoptry neck or exonemes. We further demonstrate PfShelph2 dispensability during the asexual blood stage by generating PfShelph2-KO parasites using CRISPR-Cas9 machinery. Analyses of the mutant during the course of the erythrocytic development indicate that there are no detectable phenotypic consequences of Pfshelph2 genomic deletion. As this lack of phenotype might be due to functional redundancy, we also explore the likelihood of PfShelph1 (PfShelph2 paralog) being a compensatory phosphatase. We conclude that despite its cyclic expression profile, PfShelph2 is a dispensable phosphatase during the Plasmodium falciparum asexual blood stage, whose function is unlikely substituted by PfShelph1.

Fig 1. Transgenic PfShelph2-HA₃ characterization.

(A) Scheme depicting the strategy used to engineer PfShelph2-HA₃ parasites. Plasmids pUF1-Cas9 and pL7-Shelph2*-HA₃ were transfected in Pf3D7 and selected by addition of DSM1 and WR drugs respectively. The red thunder represents Cas9 double strand break. Parasites were genotyped by PCR (WT and INT) using primers depicted as arrows in the figure. (B) PCR genotyping of two edited PfShelph2-HA₃ lines versus Pf3D7 parental strain. Control PCRs were performed on PfPRL gene (PF3D7_1113100) using primers MLa11 and MLa12. (C) Immunoblot of PfShelph2-HA₃ parasites on rings (R), trophozoites (T) or schizonts (S) extracts using anti-HA antibodies. Equivalent amounts of proteins were loaded per lane and verified using anti-Histone H3 antibodies. (D) IFA of PfShelph2-HA₃ parasites using anti-HA and anti-MSP1₁₉ antibodies at different stages of the RBC cycle, i.e. ring, trophozoite, schizont and merozoite. Scale bar, 2 μm.

Fig 2. Dual labeling of PfShelph2-HA₃ with rhoptry neck and exoneme markers.

IFA of PfShelph2-HA₃ parasites using anti-HA and (A) anti-PfRON4 or (B) anti-PfSUB1 antibodies on schizonts. Scale bar, 2 μm.

Fig 3. Successful disruption of Pfshelph2 gene and phenotypic characterization of PfShelph2-KO parasites in asexual blood stage.

(A) Scheme depicting the strategy used to generate PfShelph2-KO parasites. Plasmids pUF1-Cas9 and pL7-Shelph2-KO were transfected in Pf3D7 and selected by addition of DSM1 and WR drugs respectively. The red thunder represents Cas9 double strand break. Three clonal lines, namely B2, D3 and E1, were genotyped by PCR (LOCUS, 5’INT and 3’INT) using primers depicted as arrows in the figure. (B) PCR genotyping of three PfShelph2-KO lines versus Pf3D7. PCR 5’INT refers to 5’ integration, PCR 3’INT to the 3’integration, while PCR LOCUS shows the amplification of the whole shelph2 genomic locus. (C) RT-PCR of shelph2 mRNA in PfShelph2-KO lines versus Pf3D7. RNA samples were also used as PCR templates to verify the absence of contaminating genomic DNA. Control cDNA corresponds to the 880 bp amplification of PF3D7_1127000 from cDNA using primers MLa13 and MLa14, while PCR from gDNA is expected to amplify a 1850 bp fragment. (D) The intra-erythrocytic development of PfShelph2-KO lines B2, D3 and E1 was followed in comparison with Pf3D7. For each time point, smears were done in triplicate and 200 infected RBCs were counted per smear. The graph shows the ratio of each stage (R for ring, T for trophozoite and S for schizont) as a function of time post-invasion. Error bars represent standard deviation. This experiment was repeated 2 times independently. (E) The graph represents the number of merozoites produced per schizont in Pf3D7 or PfShelph2-KO lines. Tightly synchronized schizonts of about 40h were allowed to mature in presence of compound 2 for 4h. After one wash in complete medium, smears were done in triplicate and 50 infected RBCs were counted per smear. Error bars represent standard deviation. This experiment was repeated 3 times independently. (F) Parasite proliferation rate was measured in ring stage by FACS from one cycle to the next one. Results are shown as a percentage of Pf3D7 proliferation rate. Error bars represent standard deviation. This experiment was repeated 3 times independently.

Fig 4. PfShelph1 localization and gene transcript levels.

(A) Western-blot of Pf3D7, Pf3D7/GFP (empty vector) and Pf3D7/Shelph1-GFP parasite extracts. GFP alone is observed at 27 kDa, while Shelph1-GFP is expressed as a 66 kDa fusion protein. (B) Fluorescence microscopy of Pf3D7/Shelph1-GFP line. Scale bar, 2 μm. (C) Quantitative RT-PCR analysis was performed on cDNA from Pf3D7 and PfShelph2-KO parasites. The gene relative expression in PfShelph2-KO parasites is given following normalization to Pf3D7. PfPPKL and Pfshelph2 transcript levels were used as controls. Fructose bisphosphate aldolase was used as the reference gene. The graph represents the mean of two independent experiments, for which the data of the three PfShelph2-KO lines were pooled. Error bars represent standard deviation.