RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
DisruptedGene model (rodent): PY17X_0407900; Gene model (P.falciparum): PF3D7_0307300; Gene product: end-binding protein 1 (EB1)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 22 May 2023, 16:37
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37208365
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherYang S, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, School of Life Sciences
Name InstituteFaculty of Medicine and Life Sciences, Xiamen University
CityXiamen, Fujian
Name of the mutant parasite
RMgm numberRMgm-5293
Principal name∆eb1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte formation, significant reduction in formation of male gametocytes (exflagellation); evidence for normal female gamete formation
Fertilization and ookineteNormal gametocyte formation, significant reduction in formation of male gametocytes (exflagellation); evidence for normal female gamete formation; reduced ookinete formation
OocystNo oocyst and sporozoite formation
SporozoiteNo oocyst and sporozoite formation
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of EB1

Protein (function)
End binding proteins (EBs) are the conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. In the paper evidence is presented that  Plasmodium EB1 is a unique orthologue distinct from the canonical eukaryotic EB1. It is not expressed in asexual blood stages but is expressed in the nucleus of gametocytes. Both in vitro and in vivo assays revealed that Plasmodium EB1 lost MT plus-end tracking but gained MT-lattice affinity. 

Normal growth/multiplication of asexual blood stages. Normal gametocyte formation, significant reduction in formation of male gametocytes (exflagellation); evidence for normal female gamete formation. No oocyst and sporozoite formation.

Additional information
Evidence is presented for:

- Complementation of P. berghei EB1 with P. falciparum EB1 (RMgm-5295).

EB1 expression: EB1 was expressed at the sexual stage gametocytes but not at asexual blood stages (including the ring, trophozoite, and schizont). EB1 was also detected at midgut oocysts and salivary gland sporozoites. EB1 was present in both gender gametocytes with higher expression level in males. Notably, EB1 were detected at the nucleus of gametocytes (see mutants expressing HA-tagged, GFP-tagged and 4Myc-tagged EB1; RMgm-5296RMgm-5297RMgm-5298).

- EB1 localization in males: Before activation of male gametocytes to produce the male gametes (eflagellation), EB1 was diffused in the male nucleus. At 1 minute post activation (mpa), EB1 formed a nucleus-associated intense focus, which was elongated across the nucleus at 2 mpa and further split into two foci at the opposite at 3 mpa. After another two rounds of elongation and splitting, eight discrete EB1 foci were formed and associated with the octoploid nucleus or the haploid separated nucleus. The EB1 signals were reduced to a level below detection in the exflagellated gametocytes at 15 mpa. The EB1 localization pattern coincides with the mitosis dynamics in male gametogenesis, and evidence is presented that EB1 associated with spindle. After gametocyte activation, EB1 signals overlapped with the spindle miccrotubules (MTs) throughout three rounds of spindle elongation and splitting. Throughout male gametogenesis EB1 did not associate with the axonemes, supporting its nuclear localization.

- EB1 role in chromosome replication/segregation: To quantitatively analyze genome replication, the eb1 gene was disrupted in a P. yoelii reporter strain DFsc7 (RMgm-4477) expressing GFP and mCherry in male and female gametocytes respectively (see mutant RMgm-5294). Flow cytometry analysis of male gametocytes at 8 mpa detected a comparable increase in DNA content in both parental DFsc7 and mutant DFsc7;Δeb1 parasites, indicating normal genome replication. Spatial separation of the haploid-set chromosomes in the nucleus at 8 mpa was observed in 49% (n=60) of WT male gametocytes but only in 10% (n=60) of Δeb1, indicating defective chromosome segregation in the absence of EB1. Only 22% (n=150) of Δeb1 male gametes were nucleated when released or releasing from gametocytes, significantly lower than WT male gametes (67%, n=150). These results indicate that EB1 depletion does not affect genome replication but impairs chromosome segregation to daughter male gametes.

- EB1 spindle location (1): Two double-tagged strains eb1::6HA;sas4::4Myc (RMgm-5299); and eb1::6HA;nup313::4Myc (RMgm-5300) were generated by tagging endogenous SAS4 (basal body protein; PF3D7_1458500; spindle assembly abnormal protein 4, putative) and Nup313 (nuclear pore  protein; PF3D7_1446500; nucleoporin NUP313, putative) with 4Myc from the eb1::6HA parasite. U-ExM revealed the spindles adjacent to the basal bodies with no overlapping between EB1 and SAS4 in activated male gametocytes.  EB1 partially overlapped with the Nup313-labeled nuclear pores, with EB1 at the inner side of nuclear envelope. These results fit with current understanding of spindle localization in Plasmodium male gametogenesis. Thus EB1 could be used to distinguish the spindle MTs from the axoneme MTs during male gametogenesis, especially at the later stage of this process.

- EB1 spindle location (2): The roles of MT-binding domains in spindle localization of PyEB1 and in chromosome segregation during male gametogenesis was investigated. The genomic sequence encoding EB1 N-terminal tail domain (ΔTrunc1, RMgm-5301), CH domain (ΔTrunc2; RMgm-5302), linker domain  (ΔTrunc3, RMgm-5303) and CH+linker domain (ΔTrunc4, RMgm-5304) respectively, were deleted from the parental parasite eb1::6HA. Domain truncation had no remarked effect on the protein level of PyEB1 in the truncated strains. Truncation of either CH or linker impaired spindle localization of PyEB1 (parasites with EB1 spindle localization: 80.3% in eb1::6HA, n=67; 5.3% in ΔTrunc2, n=80; 34.7% in ΔTrunc3). CH- or linker-truncated EB1 was distributed in the nucleoplasm, which is further confirmed by U-ExM analysis. Truncation of both CH and linker completely ablated PyEB1 spindle localization. Therefore, both CH and linker, two independent MT-binding domains, are critical for spindle localization of PyEB1. Consistent with it, loss of single CH or linker impaired chromosomes segregation in the gametocytes at 8 mpa while loss of both domains additively abrogated chromosomes segregation. Truncation of N-terminal tail seemed to have no effect on spindle localization of PyEB1 but resulted in the decreased chromosomes segregation, suggesting that the N-terminal tail is not required for MT binding but functions in chromosomes segregation. 

EB1 spindle/kinetochore attachment (1): Since EB1 deficiency caused abnormal spindle, we speculated a defective spindle-kinetochore attachment in the EB1-null  male gametocytes after activation. To investigate it, we tagged the outer kinetochore protein Ndc80 (PY17X_1116900; PF3D7_0616200) with a 4Myc in the eb1::6HA parasite (see mutant RMgm-5305)  and obtained a double-tagged parasite clone eb1::6HA;ndc80::4Myc (DTS1). IFA showed that Ndc80 was expressed in all proliferative stages of parasite, consistent with previous results. In gametocytes both EB1 and Ndc80 were diffused at nucleoplasm. After activation Ndc80 was concentrated to the foci and dynamically co-localized with EB1 throughout gametogenesis under confocal microscopy. Co-immunoprecipitation detected EB1 interaction with Ndc80 in activated gametocytes, but not in non-activated gametocytes, further confirming the spindle-kinetochore attachment in gametocytes after activation. U-ExM analysis of the activated DTS1 male gametocytes distinguished Ndc80 positioning relative to EB1 under high resolution. Nearly all individual Ndc80 foci were associated with spindles throughout male gametogenesis. The Ndc80 foci displayed an unusual lateral localization to spindle MTs, distinct from the end-on attachment of kinetochores with spindle MTs in the bipolar mitosis.

EB1 spindle/kinetochore attachment (2): To confirm lateral positioning of kinetochores to spindle MTs, three other kinetochore proteins SPC24 (PY17X_1444800; PF3D7_1227600), SPC25 (PY17X_1364500; PF3D7_1345900), and AKiT1(PY17X_0624000;PF3D7_0723800) were included. SPC24 and SPC25, similar to Ndc80, are the subunits of the outer kinetochore complex NDC80. AKiT1 is a Plasmodium inner kinetochore protein. Three double-tagged strains eb1::6HA; spc24::4Myc (DTS2; RMgm-5306), eb1::6HA; spc25::4Myc (DTS3; RMgm-5307), and eb1::6HA; Akit1::4Myc (DTS4;RMgm- 5308) were generated from the eb1::6HA parasite. Similarly as Ndc80, the SPC24, SPC25, and AKiT1 displayed lateral localization to spindle MTs throughout the endomitosis via IFA and U-ExM analysis. These results indicates that kinetochores establish connection with spindle from the beginning of endomitosis and maintain the connection throughout endomitosis. In addition, the EB1-decorated spindle MTs are associated laterally with kinetochores. 

-  EB1 spindle/kinetochore attachment (3):To investigate whether EB1 is required for the spindle-kinetochore attachment, the eb1 gene was removed in the DTS1 strain, obtaining the EB1-null mutant DTS1;Δeb1 (RMgm-5309). EB1 disruption seemed to have no effect on Ndc80 protein level in male gametocytes. However, the activated DTS1;Δeb1 male gametocytes exhibited significantly reduced spindle-clustering of kinetochore Ndc80 foci along rounds of endomitosis compared to the parental DTS1. Consistently, U-ExM revealed that the Ndc80 dots lost spindle-clustering and were dispersed throughout the nucleoplasm in the activated DTS1;Δeb1 male gametocytes. To further confirm the defective spindle for this kinetochore attachment in the absence of EB1,  the eb1 gene was removed in the strains DTS2 and DTS3, respectively, and  similar defects in the spindle-kinetochore were observed for attachment during male gametogenesis for the EB1-null parasites DTS2;Δeb1 (RMgm-5310) and DTS3;Δeb1 (RMgm-5311). This indicates that EB1 regulates spindle-kinetochore attachment during the endomitosis of male gametogenesis.

- (Lack of) EB1 - Ndc80 interaction: Attempts to disrupt the ndc80 gene (PY17X_1116900; PF3D7_0616200) in the P. yoelii parasite failed, indicating an essential role of Ndc80 in asexual blood stage development (see RMgm-5312).
A promoter swap method was used to replace 801 bp of endogenous ndc80 promoter sequence in the double tagged strain DTS1 with that (1826 bp) of clag1 gene (PY17X_1402200; PF3D7_0220800; cytoadherence linked asexual protein 2; CLAG2, RhopH1A), whose transcripts are expressed in asexual stages, but absent in gametocytes and mosquito stages. Correct modification in the resulting mutant parasite ndc80kd (RMgm-5313) was confirmed by PCR. The promoter replacement allowed Ndc80 expression in asexual blood stages at a level comparable with that of parental strain DTS1, but significantly reduced Ndc80 expression in gametocytes either before or after activation. Ndc80 depletion did not appear to affect spindle formation after gametocyte activation. EB1 expression and localization showed no difference in activated gametocytes between DTS1 and ndc80kd, indicating that EB1 does not rely on Ndc80 for spindle localization. However, the ndc80kd parasites significantly decreased the activated male gametocytes showing typical chromosome segregation at 8 mpa, consistent with the notion that NDC80 functions in the spindle-kinetochore attachment. The ndc80kd parasite produced fewer nucleated male gametes in vitro compared to the parental strain DTS1. In mosquitoes fed with ndc80kd parasite-infected mouse blood, no oocysts were detected in mosquito midguts. These results indicate that spindle localization of EB1 is independent of Ndc80. However, Ndc80 depletion in gametocyte causes a chromosome segregation defect, resembling EB1 deficiency in male gametogenesis.

- Role of S15 phosphorylation  in EB1 localization/function: Previous phosphoproteomic studies have revealed phosphor-regulation in the mitosis and microtubule-related proteins during male gametogenesis. Serine 15 (S15) in the N-terminal tail of EB1 was independently detected to be phosphorylated in both P. berghei and P. falciparum gametocytes after activation. S15 residue is conserved among Plasmodium species. To investigate, whether S15 phosphorylation plays a role in EB1 protein localization or function, the S15 was replaced with Alanine (A) of endogenous EB1 in the DTS1 RMgm-5305 parasite using the CRISPR-Cas9 method, generating the mutant clone S15A (RMgm-5314). S15A substitution had no effect on EB1 protein level. An antiserum targeting a synthesized EB1 antigen peptide containing phosphorylated S15, recognized an immunoblot band from cell lysates of the activated gametocytes but not the non-activated gametocytes of the DTS1 strain. In contrast, no band was detected in the activated S15A gametocytes. These results confirmed EB1 S15 phosphorylation in male gametogenesis of P. yoelii, similarly as reported in P. berghei and P. falciparum. The S15A gametocytes showed spindle localization of EB1 after activation in both confocal microscopy and U-ExM, indicating that S15A substitution does not affect the spindle MT-binding of EB1. Notably, the spindle attachment of Ndc80 was detected in 68% of activated S15A male gametocytes compared to the DTS1 strain (94%). Co-immunoprecipitation also detected the reduced association between Ndc80 and EB1 in activated gametocytes of S15A compared to DTS1. These results indicated that S15 phosphorylation is critical for EB1 function in the spindle-kinetochore attachment. Consistent with it, the S15A male gametocytes after activation displayed less chromosome segregation and produced fewer nucleated male gametes in vitro.

From the Abstract of the paper:
End binding proteins (EBs) are the conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. In the paper evidence is presented that  Plasmodium EB1 is a unique orthologue distinct from the canonical eukaryotic EB1. It is not expressed in asexual blood stages but is expressed in the nucleus of gametocytes. Both in vitro and in vivo assays revealed that Plasmodium EB1 lost MT plus-end tracking but gained MT-lattice affinity. This MT-binding feature of EB1 is contributed by both the CH domain and the linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission of parasite. EB1 is localized at the nucleoplasm of male gametocytes. Upon gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout three rounds of endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment was observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice affinity has evolved to fulfill the spindle-kinetochore lateral attachment in male gametogenesis

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0407900
Gene Model P. falciparum ortholog PF3D7_0307300
Gene productend-binding protein 1
Gene product: Alternative nameEB1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR/Cas9 plasmid pYCm was used for genomic modification (Zhang et al, 2014; Zhang et al, 2017).
To construct vectors for gene deletion, the 5’ and 3’ genomic fragments (400 to 800 bp) at the target gene were amplified as the left and right homologous templates respectively and inserted into the pYCm vector.
To construct vectors for gene tagging, the 5’- and 3’- flanking sequences (400 to 800 bp) at the designed insertion site of target genes were amplified as the left and right homologous templates respectively. DNA fragments encoding 6HA, 4Myc, GFP and mScarlet were inserted between the homologous templates in frame with the coding sequence of target gene. For each modification, at least two small guide RNAs (sgRNAs) were designed using the online program EuPaGDT. Paired oligonucleotides for sgRNA were denatured at 95 °C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6