The Basal Complex Protein PfMORN1 Is Not Required for Asexual Replication of Plasmodium falciparum

ABSTRACT Plasmodium falciparum, the Apicomplexan parasite that causes the most severe form of human malaria, divides via schizogony during the asexual blood stage of its life cycle. In this method of cell division, multiple daughter cells are generated from a single schizont by segmentation. During segmentation, the basal complex forms at the basal end of the nascent daughter parasites and likely facilitates cell shape and cytokinesis. The requirement and function for each of the individual protein components within the basal complex remain largely unknown in P. falciparum. In this work, we demonstrate that the P. falciparum membrane occupation and recognition nexus repeat-containing protein 1 (PfMORN1) is not required for asexual replication. Following inducible knockout of PfMORN1, we find no detectable defect in asexual parasite morphology or replicative fitness. IMPORTANCE Plasmodium falciparum parasites cause the most severe form of human malaria. During the clinically relevant blood stage of its life cycle, the parasites divide via schizogony. In this divergent method of cell division, the components for multiple daughter cells are generated within a common cytoplasm. At the end of schizogony, segmentation partitions the organelles into invasive daughter parasites. The basal complex is a ring-shaped molecular machine that is critical for segmentation. The requirement for individual proteins within the basal complex is incompletely understood. We demonstrate that the PfMORN1 protein is dispensable for blood stage replication of P. falciparum. This result highlights important differences between Plasmodium parasites and Toxoplasma gondii, where the ortholog T. gondii MORN1 (TgMORN1) is required for asexual replication.


RESULTS
PfMORN1 is a member of the Plasmodium basal complex. To study the localization of PfMORN1, we fused a spaghetti monster V5 (SmV5) (30) epitope tag to the carboxy terminus of PfMORN1 (Fig. 1A) at the endogenous locus. As expected, PfMORN1-SmV5 forms rings around nascent daughter cells in segmenting schizonts by immunofluorescence (Fig. 1B). The PfMORN1 ring is first visualized in early segmentation, enlarges during mid-segmentation, and constricts to a punctate spot at the end of segmentation-a localization pattern consistent with previously identified members of the basal complex (5,27). Together with previous coimmunoprecipitation data using PfCINCH (5), these results confirm that PfMORN1 is a bona fide member of the P. falciparum basal complex.
PfMORN1 is efficiently knocked out within a single asexual cycle. To interrogate the function of PfMORN1, we utilized the loxPint inducible knockout system (31). As noted above, we introduced a recodonized PfMORN1 coding sequence fused to the SmV5 epitope tag. This cassette was flanked by loxP sequences nestled in synthetic introns (loxPint), with the first loxPint site introduced 99 bp into the PfMORN1 coding sequence. This strain was generated in the 3D7-pfs47DiCre parasite line that expresses both halves of a dimerizable Cre recombinase (32). The resulting transgenic parasite strain was named PfMORN1 SmV5-loxPint (Fig. 1A). The addition of the small molecule rapamycin (rapa) causes dimerization of the two halves of the Cre recombinase enzyme, allowing efficient excision of the loxP-flanked DNA sequences. Thus, following the addition of rapamycin to PfMORN1 SmV5-loxPint parasites, the activated DiCre recombinase excises .90% of the PfMORN1 coding sequence and the SmV5 epitope tag (see below for efficiency of excision), leading to a functional PfMORN1 protein knockout.
Sorbitol-synchronized ring stage parasites were treated with 100 nM rapamycin to induce excision. At the schizont stage, genomic DNA (gDNA) was evaluated by PCR to confirm excision of the loxP-flanked sequence from the parasite genome. The locations of the primers used for PCR are shown in Fig. 1A. We paired primer 1, which binds in the 59 untranslated region (UTR) of PfMORN1 and upstream of the 59 loxPint site, with primer 2, which binds downstream of the 39 loxPint site (Fig. 1A). With this primer set, a lack of recombination results in an amplification product of approximately 3.5 kb, and successful excision results in a product of a reduced size, approximately 1 kb. We also paired primer 1 with primer 3, which binds within the flanked loxPint sites (Fig. 1A). With this primer set, a lack of recombination results in an amplification product of approximately 2 kb, and successful excision results in no PCR product. We collected gDNA from late schizonts maintained in the absence and presence of rapamicin ([2]/[1] rapa) from the ring stage of the same cycle. As a control, the wild-type 3D7 gDNA resulted in no PCR products, as expected since both reverse primers 2 and 3 sit within the genetically altered construct. In [2] rapa conditions of PfMORN1 SmV5-LoxPint parasites, we observe that primers 1 and 2 (primers 1&2) produce a 3.5-kb PCR product and primers 1&3 produce a 2-kb product, suggesting no excision occurred. In [1] rapa conditions, we observe primers 1&2 produce a 1-kb PCR product and primers 1&3 result in no PCR products, demonstrating that efficient excision occurs upon addition of rapamycin ( Fig. 2A).
To examine efficiency of PfMORN1 protein knockout, we collected late schizonts maintained on [2]/[1] rapa from ring stage of the same cycle and performed an immunoblot probing with an antibody against the V5 epitope. Endogenous PfMORN1 is predicted to be ;41 kDa, and SmV5 adds an additional ;44 kDa. The expected ;85-kDa band is present in the [2] rapa lysate and is undetectable in the [1] rapa lysate (Fig. 2B). In addition, we collected schizonts maintained [2]/[1] rapa from the ring stage of the same cycle stage for immunofluorescence assay with antibodies against V5 and the IMC-associated protein PfGAP45 (33). We identified schizonts with PfGAP45 signal, indicating they are actively segmenting, and calculated the percentage of actively segmenting schizonts with observed PfMORN1 (V5) staining. In [2] rapa conditions, we observe 99.2% 6 0.8% of actively segmenting schizonts with PfMORN1 staining. In [1] rapa conditions, we observe an average of 1.20% 6 0.49% of actively segmenting schizonts with PfMORN1 staining, a .98% reduction in PfMORN1 detection by immunofluorescence after addition of rapamycin (Fig. 2C). These results demonstrate efficient excision following rapamycin treatment.
PfMORN1 is not required for asexual proliferation of P. falciparum. To examine the consequence of PfMORN1 deficiency on asexual proliferation, we performed a flow cytometry-based growth assay over two complete intraerythrocytic development cycles. Rapamycin ([1] rapa) or dimethyl sulfoxide (DMSO) ([2] rapa) was added to rings during cycle 0. Parasitemia was assessed by flow cytometry, after staining with SYBR green I, upon initial seeding (cycle 0) and for the following two replicative cycles. Over two asexual cycles, the parasitemia of parasites under [1] rapa conditions were not significantly different from those under [2] rapa conditions (Fig. 2D), and both were similar to the control 3D7 wild-type strain.
To examine the impact of PfMORN1 deficiency on major subcellular structures, we collected schizonts maintained [2]/[1] rapa from the ring stage of the same cycle for immunofluorescence assay. We probed with antibodies specific for V5 and four other proteins that are markers of major subcellular structures within the parasite: PfAMA1 (micronemes) (34), PfBCP1 (basal complex) (5), PfGAP45 (IMC) (33), and PfRON4 (rhoptries) (35). In the absence of PfMORN1, PfAMA1 still exhibits apical localization that is typically observed prior to egress (Fig. 3A). Confirming that micronemal PfAMA1 staining appears very late into the segmentation process, in [2] rapa parasites, PfMORN1 (V5) staining is observed only near the basal end of segmenting parasites when PfAMA1 staining is present. Despite being a member of the basal complex, PfMORN1 deficiency has no impact on the localization of the basal complex member PfBCP1, which forms rings around segmenting parasites in the absence of PfMORN1 (Fig. 3B). Similarly, in the absence of PfMORN1, PfGAP45 still surrounds segmenting daughter parasites from the apical end down to the basal complex, observed as a ring in a single z-slice (Fig. 3C). Finally, in the absence of PfMORN1, PfRON4 still localized as punctate dots at the apical end of the forming merozoites (Fig. 3D), suggesting normal morphology of rhoptries. These results demonstrate that PfMORN1 is not required for asexual parasite proliferation and that PfMORN1-deficient schizonts (and fully formed merozoites) are morphologically normal.

DISCUSSION
The basal complex is essential for the asexual proliferation of P. falciparum (5) and T. gondii (18,19,21,26). Moreover, the basal complex is hypothesized to form a contractile ring that facilitates, or even mediates, cytokinesis of nascent daughter parasites (18,19,21,26). The methods of cytokinesis during the asexual stages of P. falciparum (schizogony) and T. gondii (endodyogeny) differ, and the requirements for individual components of the basal complex likely also differ (2). However, it remains unknown which members of this multiprotein complex are redundant and/or dispensable for the P. falciparum intraerythrocytic development cycle. Dissecting which members of the basal complex are essential is an important step toward understanding the molecular functions and mechanisms of the basal complex. In T. gondii, TgMORN1 has an important role in endodyogeny, specifically for proper cytokinesis of daughter parasites (18,21). In contrast, the current study demonstrates that PfMORN1 is dispensable for daughter cell cytokinesis during schizogony. In the P. falciparum genome-wide transposon mutagenesis screen, there were no piggyBac insertions in the PfMORN1 coding sequence (36). However, the P. berghei PlasmoGEM knockout screen predicted that PbMORN1 (PBANKA_0515200) was dispensable in the mouse model (37). The use of a rapid (i.e., single cycle) inducible knockout system likely provides strong protection against the development of compensatory mutations. Given the lack of phenotype following knockout, it is difficult to further determine the molecular function of this protein during schizogony. It is important to note that PfMORN1 may be essential for a different stage of the P. falciparum life cycle or more important for asexual replication in vivo. Further studies are needed to investigate these potentials roles of PfMORN1 in other environments.

MATERIALS AND METHODS
Reagents and antibodies. Primers were obtained from Life Technologies, restriction enzymes from New England Biolabs, and DNA polymerases from Clontech. Commercially available antibodies were obtained from Bio-Rad (mouse anti-V5, clone SV5-Pk2) and Immunology Consultant Laboratories (rabbit anti-V5, clone RV5-45A-Z). Other primary antibodies were generously provided by Julian Rayner at the Cambridge Institute for Medical Research (rabbit anti-PfGAP45) ( (38). The primary rabbit anti-PfBCP1 antisera has been described previously (5).
Plasmid construction. To create the loxPint PfMORN1 HDR plasmid, we synthesized a gene block with a codon-altered PfMORN1 sequence (gBlock from Integrated DNA technology). Oligonucleotides oJDD5204/oJDD5205 and oJDD5206/oJDD5207 were used to amplify this gene block and introduce a point mutation that removed a BsaI-cut site. These fragments were fused by overlapping PCR. The PF3D7_1031200 39 and 59 homology regions, respectively, were PCR amplified from genomic DNA with oligonucleotides oJDD5200/oJDD5201 and oJDD5202/oJDD5203. The SmV5 epitope tag was amplified with oligonucleotides oJDD5208/oJDD5224 from pRR92 (5). The drug selection cassette (loxPint-3'UTR-Cam 5'UTR-hDHFR-hrp2UTR) was amplified with oligonucleotides oJDD5225/oJDD3907. The pGEM plasmid backbone (after site-directed mutagenesis to remove existing BsaI sites) was amplified with oJDD5227/oJDD5228. The six pieces were ligated via golden gate cloning by 150 cycles with 1 cycle consisting of 5 min of digestion with BsaI-HFv2 and 5 min of ligation with T4 ligase to generate pCJM17. All oligonucleotide and synthesized gene block sequences are shown in Table S1.
Western blot analysis. Parasite protein pellets were collected from parental parasites and PfMORN1 SmV5-loxPint parasites in both [1] rapa and [2] rapa conditions. Proteins were extracted using 0.2% saponin in phosphate-buffered saline (PBS) with protease inhibitor, washed with PBS, and resuspended in Laemmli buffer. Samples were run on a 4 to 20% mini-Protean TGX stain-free gels (Bio-Rad).
The gel was imaged to analyze total protein loading and then transferred to a nitrocellulose membrane. The membrane was blocked in Li-Cor Odyssey blocking buffer, incubated with primary antibody (1:3,000 anti-V5) in PBS with 3% bovine serum albumin (3% BSA/PBS), and then incubated in secondary antibodies diluted in Tris-buffered saline with Tween 20 (TBST). The membrane was visualized on a Li-Cor Odyssey CLx imager. Uncropped Western blot and total protein staining images are provided in Fig. S1 in the supplemental material.
Flow cytometry analysis of parasite replication. 3D7 parental parasites and PfMORN1 SmV5-LoxPint parasites were synchronized as early rings using 5% (wt/vol) sorbitol and plated in triplicate at 0.25% parasitemia in 1% hematocrit in both [1] and [2] rapa conditions. One hundred microliters of each sample was collected in triplicate on days 0, 2, and 4 after plating. The cells were washed once with 0.5% BSA/PBS and then incubated for 20 min with 1:1,000 SYBR green. Cells were washed with 0.5% BSA/PBS and then resuspended in PBS. The proportion of infected cells was measured by flow cytometry using a BD FACSCalibur.
Immunofluorescence assay. Dried blood smears were fixed to slides in 4% paraformaldehyde for 10 min, washed three times with 1Â PBS, permeabilized with 0.1% Triton X-100 in PBS, and washed again three times with 1Â PBS. Slides were blocked in 3% BSA/PBS for 1 h at room temperature. Primary antibody was diluted into 3% BSA/PBS, and slides were incubated with primary at 4°C overnight. The primary antibodies and dilutions for primary antibodies were as follows: anti-V5 (1:500), anti-PfAMA1 (1:200), anti-PfBCP1 (1:250), anti-PfGAP45 (1:5000), and anti-PfRON4 (1:200). The slides were washed three times with 1Â PBS and were incubated for 45 min at room temperature with secondary antibodies diluted in 3% BSA/PBS. The slides were washed three times in 1Â PBS then incubated with Hoechst 33342 diluted in PBS for 10 min at room temperature. Slides were washed three times with 1Â PBS and mounted using Vectashield Vibrance. Cells were visualized on Zeiss LSM880 with Airyscan with 63Â oil immersion objective.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.