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Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle [1]

['Mohammad Zeeshan', 'University Of Nottingham', 'School Of Life Sciences', 'Nottingham', 'United Kingdom', 'Ravish Rashpa', 'University Of Geneva', 'Faculty Of Medicine', 'Geneva', 'David J. P. Ferguson']

Date: 2022-08 

Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

Funding: This work was supported by: MRC UK (G0900109, G0900278, MR/K011782/1) and BBSRC (BB/N017609/1) to RT; the BBSRC (BB/N017609/1) to MZ; the Francis Crick Institute (FC001097), the Cancer Research UK (FC001097), the UK Medical Research Council (FC001097), and the Wellcome Trust (FC001097) to AAH; the NIH/NIAID (R01 AI136511) and the University of California, Riverside (NIFA-Hatch-225935) to KGLR; the Swiss National Science Foundation project grant (31003A_179321) to MB and BBSRC (BB/N018176/1) to CAM. This research was funded in whole, or in part, by the Wellcome Trust [FC001097]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Here, we present a comprehensive genome-wide screen of all P. berghei kinesins, including additional analyses of previously studied kinesin-5, kinesin,-8B, and kinesin-8X [ 18 – 20 ], using gene-targeting approaches, live cell imaging, ultrastructure expansion microscopy and electron microscopy, and RNA-seq and ChIP-seq analyses. We examine the subcellular location of each kinesin using a protein endogenously tagged at the C-terminus with GFP, revealing a differential localisation of kinesins in mitotic and meiotic stages and a pellicular and polar location in certain invasive stages. Eight of the 9 kinesin genes are required only for parasite transmission through the mosquito vector, during the sexual and sporogony stages. Only kinesin-13 is likely essential during blood stage schizogony. An in-depth analysis of kinesin-13 and kinesin-20 during gametocyte and ookinete stages revealed distinct subcellular locations and functions in MT spindle assembly and formation, axoneme assembly, and cell polarity. Kinesin-20 was associated with a striking ring-like structure during zygote to ookinete differentiation and deletion of the kinesin-20 gene revealed a function in the morphology and motility of the ookinete. Kinesin-13 is expressed at all proliferative stages of the life cycle, and it associates with the kinetochore. A kinesin-13 genetic knockdown affected MT dynamics during spindle formation and axoneme assembly in male gametocytes, and subpellicular MT organisation in ookinetes. These findings help us understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.

In a recent bioinformatic analysis of kinesins in Apicomplexa, we found 9 kinesins encoded in the Plasmodium berghei genome, with members of 3 conserved kinesin subfamilies (kinesin-5, kinesin-8B, kinesin-8X, and kinesin-13); kinesin-4, kinesin-15, and kinesin-20; and 2 Apicomplexa-enriched kinesins: kinesin-X3 and kinesin-X4 [ 18 ]. Surprisingly, kinesin-5, kinesin-8X, and kinesin-8B were not essential for blood stage proliferation [ 18 – 20 ]. However, deletion of kinesin-5, which codes for a protein clearly colocated with the spindle apparatus in all proliferative stages, affected the production of infective sporozoites [ 19 ]. Kinesin-8X was required for endomitotic proliferation in oocysts, and kinesin-8B deletion resulted in a defect in axoneme biogenesis during male gametogenesis [ 18 , 20 , 21 ].

(A) Life cycle of Plasmodium spp. showing different proliferative and invasive stages in host and vector. (B) Live cell imaging showing subcellular locations of representative kinesin-GFP proteins (green) during various stages of the P. berghei life cycle. DNA is stained with Hoechst dye (blue). Scale bar = 5 μm. (C) A summary of expression and location of kinesins-GFP during different stages of P. berghei life cycle. (D) Summary of phenotypes resulting from deletion of different kinesin genes at various stages of life cycle. The phenotype was examined for asexual blood stage development (schizogony), exflagellation (male gamete formation), ookinete formation, oocyst number, oocyst size, sporozoite formation in oocyst, presence of salivary gland sporozoites, and sporozoite transmission to vertebrate host. N/D, not determined.

Plasmodium spp., the causative agents of malaria, belong to the phylum Apicomplexa. They are ancient haploid unicellular eukaryotes with several morphologically diverse proliferative stages during the complex life cycle in various cells, tissues, and organs of their vertebrate and invertebrate hosts ( Fig 1A ) [ 8 , 9 ]. In the mammalian host, the parasite proliferates within liver and red blood cells (RBCs) by repeated cycles of closed mitotic division retaining an intact nuclear membrane, with cytokinesis following the final nuclear division, in a process termed schizogony, to produce multiple infective haploid merozoites [ 10 ] ( Fig 1A ) . Some of these haploid parasites in the RBC arrest and commit to sexual development as gametocytes ( Fig 1A ) . Gametocytes develop no further into gametes unless ingested in a blood meal by a female mosquito (the invertebrate host). Male gametogenesis is very rapid and complete within 12 to 15 min after activation [ 11 , 12 ]. Within the nucleus, 3 rounds of DNA replication and chromosome segregation produce an 8N genome, which is followed by nuclear division and cytokinesis. At the same time, in the cytoplasm, axoneme assembly and maturation occur, leading to the formation of flagellate haploid male gametes in a process termed exflagellation [ 8 , 9 ]. The motile male gamete finds and fertilises the female gamete, and the resultant zygote differentiates through 6 distinct stages (I to VI) into a banana-shaped, invasive motile ookinete with a distinct apical polarity and conoid-associated proteins [ 8 , 13 , 14 ]. At the same time, in the first stage of meiosis, the DNA is duplicated and the now tetraploid ookinete develops over a 24-h period in the mosquito gut [ 8 , 14 ], before traversing the mosquito gut wall and forming an oocyst under the basal lamina. Within the oocyst, sporogony, which is a form of endomitosis, produces many haploid sporozoites [ 15 , 16 ]. Sporozoites are motile and invasive polarised cells that migrate to and invade the salivary glands, so that the bite of the infected mosquito injects them into the next mammalian host [ 17 ]. Overall, the complete life cycle of the malaria parasite is characterised by varied morphological differences in size and shape, together with various modes of cell division and proliferation ( Fig 1A ) .

Kinesins are microtubule (MT)-based motor proteins that use energy from the hydrolysis of ATP and function in various cellular processes including intracellular transport, mitotic spindle formation and chromosome segregation during cell division, and the organisation of cell polarity and cytoskeletal features associated with motility [ 1 , 2 ]. In eukaryotes, there are 14 to 16 kinesin subfamilies categorised according to the primary sequences of the motor domain, with similar biological roles also established by in vitro studies, and in vivo phenotypes for subfamily members [ 2 – 4 ]. Kinesin subfamilies that regulate MT dynamics, such as kinesin-8 and kinesin-13, are found in most eukaryotes including primitive and evolutionarily divergent eukaryotes [ 5 , 6 ]. Although there is an extensive kinesin literature with various bioinformatic and molecular investigations, information is sparse on these molecular motors in deep rooted pathogenic eukaryotes including Plasmodium spp. and other Apicomplexa, Giardia spp., and trypanosomes [ 6 ]. These primitive eukaryotes have a flagellate stage in their life cycle and may have a complex MT-associated cytoskeleton [ 7 ], indicating the importance of MT-based motor proteins in their development.

Results

Live cell imaging of Plasmodium kinesins reveals diverse locations during cell division, differentiation, and pellicle formation throughout the life cycle To investigate the expression and subcellular location of kinesins throughout the P. berghei life cycle, we generated transgenic parasite lines by single crossover recombination at the 3′ end of each gene to express a fusion protein with a C-terminal GFP-tag (S1A Fig). PCR analysis of genomic DNA from each line, using locus-specific diagnostic primers, indicated correct integration of the GFP sequence (S1B Fig). Immunoprecipitation assays using GFP-trap beads and mass spectrometry analysis of at least 6 kinesin-GFP proteins confirmed the presence of peptides of intact fusion protein in gametocyte lysates (S2A and S2B Fig). Each kinesin-GFP parasite line completed the full life cycle with no detectable phenotypic change resulting from the GFP tagging. We analysed the expression and subcellular location of these GFP-tagged proteins by live cell imaging at various stages of the life cycle. Taken together with the previously published results for kinesin-5, kinesin-8B, and kinesin-8X [18–20], we found that the 9 kinesins have a diverse pattern of expression, with distinct subcellular locations including the mitotic spindle, axonemes, the surface pellicle, and a polar distribution at various stages of the parasite life cycle (Fig 1B and 1C). Interestingly, only 2 kinesins, kinesin-5 and kinesin-13, were expressed throughout the parasite life cycle, including blood stage schizogony, and were located on the mitotic spindle in both asexual and sexual stages (S3 Fig). Kinesin-5GFP was restricted to the nucleus, while kinesin-13GFP had both a nuclear and cytoplasmic location (S3 Fig). Kinesin-8XGFP was also located on the nuclear spindle but only during the proliferative stages within the mosquito vector. Three kinesins (kinesin-8B, kinesin-15, and kinesin-X4) were expressed only during male gametogenesis with cytosolic locations (S3 Fig), and 2 kinesins (kinesin-20 and kinesin-X3) were first detected in female gametocytes with a diffuse location (S3 Fig). Their presence continues into the zygote and later stages of ookinete differentiation and sporogony with locations that are discussed in detail below. We also observed 2 kinesins at polar locations: kinesin-8X at the basal end of stage V to VI ookinetes and kinesin-13 at the apical end throughout ookinete development (S3 Fig). Overall, kinesin-5 and kinesin-8X are restricted to nuclear spindle and kinesin-13 is present in both nucleus and cytoplasm (Fig 1B and 1C). The Apicomplexa-enriched kinesin-X3 and kinesin-X4 are confined to ookinete and sporozoite pellicle and flagellar axoneme, respectively.

Genome-wide functional screen reveals that 8 out of 9 kinesins are required only for parasite transmission and not for blood stage proliferation Previously, we described the functional roles during mosquito stages of 3 kinesins, kinesin-5, kinesin-8B, and kinesin-8X, proteins that were not essential during blood stage development [18–20]. To study the function of the remaining 6 kinesins throughout the life cycle, we attempted deletion of each gene from P. berghei using a double crossover homologous recombination strategy as described previously [22] (S4A Fig). Successful integration of the targeting constructs at each gene locus was confirmed by diagnostic PCR (S4B Fig), except that kinesin-13 could not be deleted. PCR analysis of knockout parasites confirmed the complete deletion of these kinesin genes (S4B Fig), indicating that they are not essential during the asexual blood stage. kinesin-13, which could not be deleted despite several attempts, likely has an essential role during the asexual blood stage (Fig 1D). A recent functional profiling of the P. berghei genome [23] also supports an essential role for kinesin-13 during the blood stage. This previous study found that 5 kinesins (kinesin-4, kinesin-8B, kinesin-8X, kinesin-20, and kinesin-X4) are not essential for blood stage growth but provided no data for kinesin-5, kinesin-15, and kinesin-X3 [23]. Phenotypic analyses of the kinesin-knockout parasites, in comparison with the parental parasite (WTGFP), were carried out at various stages of the life cycle: in asexual blood stages, during male gametogenesis and the formation of exflagellation centres, during ookinete formation, in the number and size of oocysts, for the formation of sporozoites in oocysts and their migration to salivary glands, and for parasite transmission to the vertebrate host (Fig 1D). Taken together with previously published studies on kinesin-5, kinesin-8B, and kinesin-8X, only 2 knockout parasite lines (Δkinesin-8B and Δkinesin-15) showed a defect in the formation of male gametes (Figs 1D and S5A). Δkinesin-8B parasites produced no male gametes, as shown previously [20,21], while there was a significant decrease in male gamete formation in Δkinesin-15 parasites (Figs 1D and S5A). Next, we analysed the zygote to ookinete transition (round to banana-shaped cells) after 24 h following gametocyte activation. Three parasite lines (Δkinesin-8B, Δkinesin-15, and Δkinesin-20) produced no or reduced numbers of ookinetes (S5B Fig). Δkinesin-8B parasites produced no ookinetes, as expected because there were no male gametes to fertilise the female gametes (Figs 1D and S5B) [20]. Δkinesin-15 parasites produced significantly fewer male gametes, which would be expected to result in fewer ookinetes compared to WTGFP parasites (Figs 1D and S5B). In contrast, Δkinesin-20 parasites exflagellated normally, and, therefore, loss of this kinesin must have a direct effect on ookinete formation (S5B Fig). To assess the effect of kinesin gene deletions on oocyst development and infective sporozoite formation, 40 to 50 Anopheles stephensi mosquitoes were fed on mice infected with individual kinesin-knockout lines, and parasite development was examined. First, GFP-positive oocysts on the mosquito gut wall were counted at 7, 14, and 21 days post-infection (dpi). Three out of 8 kinesin-knockout lines showed defects in oocyst production; Δkinesin-8B parasites produced no oocysts as shown previously [20], while there was a significant reduction in Δkinesin-15 and Δkinesin-20 oocysts compared to WTGFP oocysts at 7 dpi and a further reduction by 14 and 21 dpi (S5C Fig). The adverse effects on ookinete production rather than a direct effect on oocyst development could explain this observation. Although there was no significant difference in the number of oocysts of other kinesin gene knockouts compared to WTGFP at 7 dpi, a significant reduction was observed for the Δkinesin-8X line at 14 dpi, which became more evident by 21 dpi (S5C Fig). Oocyst size was not affected in most of the lines that produced them; the only exception was Δkinesin-8X oocysts, which were approximately half the size of WTGFP oocysts at 14 dpi, and even smaller by 21 dpi (S5D Fig). Four out of 8 kinesin-knockout lines produced no or defective sporozoites; Δkinesin-8B and Δkinesin-8X produced no sporozoites, as reported earlier [18,20], while Δkinesin-15 and Δkinesin-20 lines had significantly reduced sporozoite numbers compared to control parental parasites (Figs 1D and S5E). These defects were mirrored in the salivary glands: For the Δkinesin-8B and Δkinesin-8X lines, no sporozoites were detected, as reported earlier [18,20], while Δkinesin-15 and Δkinesin-20 lines had a significantly reduced number. The Δkinesin-5 parasite produced significantly fewer infective salivary gland sporozoites (S5F Fig) as reported previously [19]. However, although several kinesin gene-knockout lines exhibited defects in sporozoite production and reduced salivary gland infection, these sporozoites were still infectious to the mammalian host as observed with successful infection of new hosts in mosquito bite back experiments (Figs 1D and S5G). In summary, for most of the kinesin gene-knockout P. berghei lines, there were clear developmental defects at specific stages of the life cycle within the mosquito vector.

Apicomplexa-enriched kinesins have discrete locations during pellicle formation (-X3) and axoneme assembly (-X4) during sexual development Previous bioinformatic analysis identified 2 divergent Plasmodium kinesins (kinesin-X3 and kinesin-X4) [5,18]; one of them (kinesin-X3) is restricted to the phylum Apicomplexa [18]. Kinesin-X4 is also restricted to Apicomplexa except that it is also present in the starlet sea anemone Nematostella vectensis [5]. The parasitic Apicomplexa are characterised by a specialised apical structural complex that coordinates the interaction with and penetration of host cells, and have a surface pellicle comprised of the plasma membrane and an underlying layer of fused flattened membrane vesicles of the inner membrane complex (IMC) with associated MTs [24,25]. To examine whether the kinesins are associated with these apicomplexan features, localisation by live cell imaging was performed. Kinesin-X3 and kinesin-X4 showed stage-specific expression during sexual development with a distinct location (S3 Fig). During zygote to ookinete differentiation, kinesin-X3 expression was restricted to one side of the cell in the early stages of development (stages I to III), suggesting an involvement in pellicle formation (Fig 2A). In later stages (stages IV to VI), the kinesin-X3 location became more distinct around the periphery of the ookinete. Monoclonal antibody (mAb) 13.1 conjugated with cy3 (red), which recognises the P28 protein on the surface of zygote and ookinete stages, stained these stages, and colocalised with kinesin-X3 (green) (Fig 2A), although kinesin-X3 was not present at the apical and basal ends of the developing ookinete (Fig 2A). The data suggest that kinesin-X3 is restricted to pellicle formation during ookinete and sporozoite stages in the mosquito. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Apicomplexa-enriched kinesins (kinesin-X3 and kinesin-X4) are located at the pellicle during ookinete differentiation and at axonemes in male gametogenesis, respectively, while nuclear kinesin (kinesin-8X) associates with the kinetochore at the centromere during male gamete formation. (A) Live cell imaging showing temporal location of kinesin-X3 (green) associated with pellicle formation (arrows) during zygote to ookinete transition (2–24 h after fertilisation). A cy3-conjugated antibody, 13.1, which recognises the protein P28 on the surface of zygote and ookinete stages, was used to track these stages (red). DNA is stained with Hoechst dye (blue). Scale bar = 5 μm. (B) Live cell imaging shows the association of kinesin-X4 (green) with axoneme marker kinesin-8B (red) during male gametogenesis. Note in later stages, axonemes (arrows) are labelled with both markers. DNA is stained with Hoechst dye (blue). Scale bar = 5 μm. (C) Live cell imaging showing the dynamics of kinesin-8XGFP (green) along with kinetochore marker NDC80Cherry (red) during male gametogenesis. DNA is stained with Hoechst dye (blue); scale bar = 5 μm. A diagrammatic representation of spindle/spindle pole and kinetochore is shown in upper panels. (D) Indirect immunofluorescence assays showing colocalisation of Pbkinesin-8X (red) and α-tubulin (green) in activated male gametocytes. Scale bar = 5 μm. (E) ChIP-seq analysis of kinesin-8X and NDC80 during gametocyte stage. Lines on top are division points between chromosomes and circles on the bottom indicate locations of centromeres. mpa, min post-activation. https://doi.org/10.1371/journal.pbio.3001704.g002 Using real-time live cell imaging of male gametogenesis, the expression and location of kinesin-X4 (green) was compared with that of axonemal protein kinesin-8B (red) located on basal bodies and axonemes [20]. Kinesin-X4 showed a diffuse cytosolic distribution during early stages of male gametogenesis (1 to 3 min post-activation [mpa]) but no strong signal on the basal body tetrads labelled with kinesin-8B (red) (Fig 2B). However, at 4 to 6 mpa, the kinesin-X4 signal distribution changed to resemble linear structures, which were maintained in the later stages (8 to 10 mpa) and showed colocalisation with kinesin-8B (Fig 2B). These data suggest that kinesin-X4 is located on axonemes together with kinesin-8B during flagella formation in Plasmodium spp.

Kinesin-8X and kinesin-5 are nuclear spindle kinesins associated with the kinetochore (NDC80) that bind centromeres In our previous studies, we showed that 2 kinesins, kinesin-5 and kinesin-8X, are associated with spindles and restricted to the nucleus during most of the life cycle stages [18,19]. To further examine the spatiotemporal dynamics of these kinesins during spindle formation, chromosome segregation, and axoneme biogenesis during male gametogenesis, we crossed parasite lines expressing kinesin-8XGFP and kinesin-5GFP with lines expressing NDC80-Cherry, a kinetochore protein in the nucleus, and kinesin-8B-Cherry, an axonemal protein in the cytoplasm, and compared protein location by live cell imaging (S6A Fig). Both kinesin-8X and kinesin-5 (green) were colocalised with NDC80 (red) suggesting a role in mitotic spindle function and chromosome segregation (Figs 2C and S6B). On the other hand, neither kinesin-5 nor kinesin-8X showed any overlap with cytosolic kinesin-8B (red) during male gametogenesis (S6C and S6D Fig) confirming their restricted location within the nuclear compartment. Kinetochores are multiprotein complexes assembled at the centromere of each chromosome, which mediate chromosome attachment to spindle MTs. Because kinesin-8X and kinesin-5 showed colocalisation with kinetochore protein NDC80, we analysed further the binding of these proteins at the centromere DNA. We performed ChIP-seq experiments with parasites undergoing gametogenesis (6 mpa), using kinesin-8XGFP and kinesin-5GFP tagged parasites and GFP-specific antibodies. Strong ChIP-seq peaks for each chromosome were observed with these kinesins, indicating their binding sites. Binding was restricted to a region close to the previously annotated centromeres of all 14 chromosomes [26] and identical to those identified in Plasmodium Condensin and NDC80 studies [8,27] (Figs 2D and S7). Together, live cell imaging and ChIP-seq analysis support the notion that kinesin-8X and kinesin-5 associate with kinetochores assembled at centromeres.

Global transcriptomic analysis of kinesin-13PTD parasites shows misregulation of transcripts for gene clusters involved in axoneme assembly and chromosome dynamics To examine the transcript level of other kinesins in kinesin-13PTD gametocytes, we performed qPCR for all the 9 kinesins and found that some, like kinesin-8B, kinesin-8X, kinesin-15, and kinesin-20, were down-regulated (Fig 6F). Since transcripts of other kinesins were affected, global transcription was investigated by RNA-seq analysis of kinesin-13PTD gametocytes at 0 and 15 mpa, representing times point before the start of male gametogenesis and just after male gamete formation (exflagellation), respectively. Kinesin-13 down-regulation in kinesin-13PTD gametocytes, relative to WTGFP gametocytes, was confirmed by RNA-seq analysis, showing the lack of reads for this locus (Fig 6G). In addition to reduced kinesin-13 expression, 34 other genes were significantly down-regulated and the expression of 152 genes was significantly up-regulated in kinesin-13PTD gametocytes before activation (at 0 mpa) (Fig 6H and S1B Table). Similarly, the expression of 22 genes was significantly down-regulated and the expression of 329 genes was significantly up-regulated in kinesin-13PTD gametocytes after 15 min activation (Fig 6I and S1C Table). Bioinformatic analysis of these differentially regulated genes revealed 2 important clusters of genes that were affected, including those coding for proteins involved in axoneme assembly, glideosome assembly, and chromosome dynamics (Fig 6J).

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