RMgmDB - Rodent Malaria genetically modified Parasites

Back to search results

Summary

RMgm-5272
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1465200; Gene model (P.falciparum): PF3D7_1249700; Gene product: conserved Plasmodium protein, unknown function (LINUP, liver stage nuclear protein)
Phenotype Liver stage;
Last modified: 30 December 2022, 13:57
  *RMgm-5272
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherGoswami D, Vaughan AM
Name Group/DepartmentCenter for Global Infectious Disease Research
Name InstituteSeattle Children’s Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-5272
Principal namePy linup‾
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageSevere liver stage attenuation. in Pylinup− sporozoite infection with 1,000 sporozoites only one of ten mice became patent and this with delay (day twelve) and seven of twenty mice became patent when infection was done with 10,000 sporozoites with patency in the blood stage positive mice delayed to days seven through twelve. Five of ten mice became patent when using the 50,000 sporozoite dose and patency was delayed to days seven to nine. Highly susceptible BALB/cByJ mice infected with 50,000 wildtype sporozoites all became patent on day three after sporozoite infection, whereas only nine of twenty mice infected with Py linup− sporozoites became patent and the day to patency ranged from days eight to ten.
Liver stage size measurements revealed that at 24 hours post-infection, Py linup− liver stages were comparable in size to wild-type. However, at both 36 and 48 hours post-infection, Py linup− liver stages were significantly smaller than wildtype with the size differences being more pronounced at the later time point post-infection. At 48 hours post infection, differences in protein expression and DNA replication/segregation were evident in Py linup− liver stages when compared to wildtype liver stages. Specifically, the branching of the mitochondria and apicoplast was reduced and DNA replication as well as DNA segregation appeared significantly reduced. Furthermore, the extensive invaginations of the liver stage plasma membrane that precedes merozoite formation called cytomere formation was severely disordered in Py linup− liver stages. This was evident from the changed expression pattern of the parasite plasma membrane marker MSP1, which localizes to the late liver stage schizont plasma membrane.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of LINUP (liver stage nuclear protein)

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.12.13.519845

Protein (function) 

PY17X_1465200, is a single exon gene encoding a 746 amino acid protein and is conserved among Plasmodium species. The overall amino acid identity between the P. yoelii, P. falciparum and P. vivax syntenic orthologs was 40%, whilst amino acid similarity was 60%. Identity in a 122 amino acid stretch near to the N-terminus (amino acids 44-161) was 89%. In addition, comprehensive protein BLAST searches revealed that the gene has no orthologs in other Apicomplexa or any other eukaryote and is thus unique to Plasmodium. PY17X_1465200 contains a conserved N-terminal 24 amino acid nuclear localization sequence (NLS) in Py, Pf and Pv that was nearly identical in amino acid sequence among the three species.

Phenotype
Severe liver stage attenuation. in Pylinup− sporozoite infection with 1,000 sporozoites only one of ten mice became patent and this with delay (day twelve) and seven of  twenty mice became patent when infection was done with 10,000 sporozoites with patency in the blood stage positive  mice delayed to days seven through twelve. Five of ten mice  became patent when using the 50,000 sporozoite dose and patency was delayed to days seven to nine. Highly susceptible BALB/cByJ mice infected with 50,000 wildtype sporozoites all became patent on day three after sporozoite  infection, whereas only nine of twenty mice infected with Py linup− sporozoites became patent and the day to patency ranged from days eight to ten.

Liver stage size measurements revealed that at 24 hours post-infection, Py linup− liver stages were comparable in size to wild-type. However, at both 36 and 48 hours post-infection, Py linup− liver stages were significantly smaller than wildtype with the size differences being more pronounced at the later time point post-infection. At 48 hours post infection, differences in protein expression and  DNA replication/segregation were evident in Py linup− liver  stages when compared to wildtype liver stages. Specifically, the branching of the mitochondria and apicoplast was reduced and DNA replication as well as DNA segregation appeared significantly reduced. Furthermore, the extensive invaginations of the liver stage plasma membrane that precedes merozoite formation called cytomere formation was severely disordered in Py linup− liver stages. This was evident from the changed expression pattern of the parasite plasma membrane marker MSP1, which localizes to the late liver stage schizont plasma membrane.

Additional information
See RMgm-5273 for a mutant expressing a C-terminal mCherry-tagged version of LINUP. Analysis of this mutant showed the following: LINUPmCherry expression was only seen during mid-to-late liver stage development, which in Py has an approximate 50-hour duration. LINUPmCherry expression was not detected at 24 hours of liver stage development but was detected at both 36 and 48 hours the latter a timepoint when merozoites form during the final stages of exo-erythrocytic schizogony. LINUPmCherry localized to live stage nuclei together with parasite DNA and partially co-localized with the histone marker, histone 3 acetylated lysine 9 (H3K9) which marks areas of active gene expression.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1465200
Gene Model P. falciparum ortholog PF3D7_1249700
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameLINUP, liver stage nuclear protein
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 Deletion of Py LINUP (PY17X_1465200) was achieved using CRISPR/Cas9 technology. In brief, LINUP was deleted using double crossover homologous recombination and complementary regions of LINUP upstream and downstream of the open reading frame were ligated into plasmid pYC L2 to create pYC_LINUP. pYC_LINUP was transfected into the blood stage schizonts of Py 17XNL.
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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