RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5521
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1430900; Gene model (P.falciparum): PF3D7_1215100; Gene product: SUN domain-containing protein, putative (SUN1)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 19 November 2024, 12:21
  *RMgm-5521
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) : 39541984
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherSayers C, Billker O
Name Group/DepartmentThe Laboratory for Molecular Infection Medicine Sweden
Name InstituteUmeå University
CityUmeå
CountrySweden
Name of the mutant parasite
RMgm numberRMgm-5521
Principal namesun1-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteSun1-KO microgametocytes formed exflagellation centres when activated in vitro at the same rate as wild type. Sun1-KO ookinetes contained only half the DNA of wild type ookinetes, probably because most microgametes failed to deliver DNA to
the zygote. This was NOT the result of defective replication during gametogenesis, but mitotic spindles were missing from nuclei, suggesting SUN1 is important for the microgamete nucleus or its genome to connect to the axoneme.
Fertilization and ookineteMorphologically mature ookinetes are formed, but very few oocysts present on the midguts of infected mosquitoes. Sun1-KO ookinetes contained only half the DNA of wild type ookinetes, probably because most microgametes failed to deliver DNA to
the zygote. This was NOT the result of defective replication during gametogenesis, but mitotic spindles were missing from nuclei, suggesting SUN1 is important for the microgamete nucleus or its genome to connect to the axoneme.
OocystStrongly reduced oocyst formation. Morphologically mature ookinetes are formed, but very few oocysts present on the midguts of infected mosquitoes
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SUN1

Protein (function)
The gene encoding SUN1 was selected based on a (male-)fertilty mutant screen. This mutant showed normal motility score of male gametes of the mutant.
SUN1 is a protein with a carboxy-terminal SUN (Sad1p/UNC-84) domain, PBANKA_1430900. SUN domain proteins were present in the first eukaryote and typically span the inner nuclear envelope, where they form part of the LINC (linker of nucleoskeleton and cytoskeleton) complex that anchors the nucleus by connecting the nuclear lamina to the cytosolic cytoskeleton. The lack of nuclear lamins and canonical LINC complex components in apicomplexa led us to hypothesize that SUN1 has a non-canonical function in male gamete formation

Phenotype
Sun1-KO microgametocytes formed exflagellation centres when activated in vitro at the same rate as wild type. Morphologically mature ookinetes are formed, but very few oocysts present on the midguts of infected mosquitoes. Sun1-KO ookinetes contained only half the DNA of wild type ookinetes, probably because most microgametes failed to deliver DNA to
the zygote. This was NOT the result of defective replication during gametogenesis, but mitotic spindles were missing from nuclei, suggesting SUN1 is important for the microgamete nucleus or its genome to connect to the axoneme. Strongly reduced oocyst formation.

Additional information
Cellular events of microgametogenesis are well characterized by ultrastructure expansion microscopy (U-ExM) and staining for α-tubulin, the centrosome marker centrin, the general protein label N-hydroxysuccinimide (NHS)-ester highlights microtubule organising centres (MTOCs) and the axonemes and spindles that emanate from them. In expanded sun1-KO gametocytes the two tetrads of forming axonemes were positioned on top of each other, consistent with the absence of a mitotic spindle that would be required for their separation along the nuclear envelope. Often these structures appeared in the cytosol distant from the nuclear envelope, suggesting SUN1 may be required to capture the MTOCs onto the nuclear membrane

Analysis of a mutant expressing a C-terminal HA-tagged version of SUN1 (RMgm-5522) showed the following: SUN1-HA was not detected in asexual blood stages and female gametocytes but present in male gametocytes. SUN1-HA was associated with the nuclear envelope of expanded microgametocytes, and two minutes after activation some of the protein formed two rings, one at the centre of each windmill-like tetrad of budding axonemes that were present at the opposite poles of the first endomitotic spindle. Labelling centrin to mark the cytosolic part of the MTOCs corroborated the nuclear envelope localisation of SUN1-HA. In expanded sun1 KO gametocytes the two tetrads of forming axonemes were positioned on top of each other, consistent with the absence of a mitotic spindle that would be required for their separation along the nuclear envelope. Often these structures appeared in the cytosol distant from the nuclear envelope, suggesting SUN1 may be required to capture the MTOCs onto the nuclear membrane. Eight minutes after activation, when WT microgametocytes have completed their third endomitosis, the base of each axoneme was marked by a SUN1-HA ring located between the cytosolic and the nucleoplasmic portion of each bipartite MTOC, precisely where spindle microtubules would need to pass through a nuclear pore to connect a haploid set of chromosomes to the centriolar region that has given rise to the axoneme. At the same time point, sun1 KO microgametocytes also had elongated axonemes, which were however, emanating from MTOCs that remained connected, consistent with an absence of the mitotic events which in WT split the eight MTOCs and distribute them around the nuclear envelope

Evidence is presented for the following:
 SUN1 interacts with an allantoicase-like protein. Immunoprecipitation of SUN1-HA from gametocyte lysates eight minutes post-activation identified one of two allantoicase-like proteins encoded in the P. berghei genome, which we designated ALLC1 (PBANKA_1304400). A reciprocal pull-down of ALLC1-HA confirmed the interaction with SUN1. Both proteins also interacted with the essential endoplasmic reticulum chaperone BiP and a DDRGK-motif containing conserved Plasmodium protein expressed most abundantly in male gametocytes. While the disruption of the DDRGK-motif had no effect on fertility, allc1 is a male fertility gene, like sun1 (as determined in the male-fertility screen).

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1430900
Gene Model P. falciparum ortholog PF3D7_1215100
Gene productSUN domain-containing protein, putative
Gene product: Alternative nameSUN1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe PlasmoGEM PBANKA_1430900 (SUN1) KO and HA-tagging transfection vectors were individually transfected into the PbANKA Cl15 cy1 parasite background line. Pyrimethamine-selected parasites were genotyped by PCR.
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