RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
DisruptedGene model (rodent): PY17X_1237700; Gene model (P.falciparum): PF3D7_0519500; Gene product: CCR4 domain-containing protein 1, putative (CCR4-1)
Phenotype Gametocyte/Gamete; Oocyst;
Last modified: 27 February 2019, 17:48
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) : 30703164
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/ResearcherHart KJ, Lindner SE
Name Group/DepartmentDepartment of Biochemistry and Molecular Biology, Center for Malaria Research
Name InstitutePennsylvania State University, University Park, State College
Name of the mutant parasite
RMgm numberRMgm-4588
Principal nameCCR4-1(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteReduced male gametocyte production; enhanced production of immature/female gametocytes.
Fertilization and ookineteNot tested
OocystReduced number of oocysts
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of CCR4-1 (and expresses GFP; promoter not known)

Protein (function)
In addition to transcript stabilization, translational control can also be accomplished by the degradation of transcripts. Degradation of mRNAs is typically initiated by deadenylases, which remove the protective poly(A) tail.
In many eukaryotes, the main complex responsible for deadenylation is the CAF1/CCR4/NOT complex, which also participates in transcriptional elongation, translational repression, and histone modification functions, and thus acts broadly upon gene expression.
To first assess the importance of the CAF1/CCR4/NOT complex in Plasmodium, we bioinformatically identified the genes for all members of the canonical CAF1/CCR4/NOT complex in Plasmodium, except for not3 and caf130. The absence of these two particular genes is not surprising, as these genes are also absent in some eukaryotes. In addition, we identified four CCR4 domain-containing proteins (PyCCR4-1, PyCCR4-2, PyCCR4-3, PyCCR4-4) that have homology to CCR4 deadenylases in other eukaryotes (e.g. yeast, human, mouse).
The typical domain architecture of CCR4-like proteins involves a Leucine Rich Repeat Region (LRR) and an Endonuclease/Exonuclease/Phosphatase (EEP) domain. The LRR mediates the interaction of CCR4 with CAF1 and the rest of the NOT complex, while the EEP domain contains active site residues required for deadenylation activity. Of these, we found that the overall length and sequence conservation within the EEP domain of PyCCR4-1 aligns most closely with the consensus CCR4 domain-containing proteins from model eukaryotes and humans. However, beyond the CCR4-EEP domain, there is no significant homology between other regions from PyCCR4-1, 2, 3, and 4 to each other, or to homologues from model species.

Reduced male gametocyte production; enhanced production of immature/female gametocytes. Reduced number of oocysts.

Additional information
From the paper:
'We obtained clonal gene-deletion mutants for all four CCR4 genes which revealed that deletion of any one of these genes individually was not lethal in asexual blood stages. Moreover, deletion of pyccr4-2, pyccr4-3, or pyccr4-4 resulted in transgenic parasites that behaved as wild-type in all life cycle stages with respect to parasite numbers, prevalence of mosquito infection, and developmental timing/completion throughout the Plasmodium life cycle. Thus, CCR4-2, -3, and -4 may play redundant roles with one or more of the other CCR4-domain containing proteins'.

'Here, it was found that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in gametocytes, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are ~4-fold less able to productively infect mosquitoes. We find that the deletion of the C-terminal portion of CAF1 in both Plasmodium yoelii and Plasmodium falciparum phenocopies the deletion of pyccr4-1. We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly upon mRNAs and indirectly to modulate transcript abundance'

Evidence is presented that:
- PyCCR4-1 associates with a canonical CAF1/CCR4/NOT complex
- The putative catalytic residues of PyCCR4-1 are required for its roles in gametocytogenesis and transmission
- Truncation of PyCAF1 phenocopies the deletion of pyccr4-1
- PyCCR4-1, PyCAF1, and PyNOT1 localize to discrete cytosolic granules
- PyCCR4-1 affects important gametocyte and mosquito stage transcripts

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1237700
Gene Model P. falciparum ortholog PF3D7_0519500
Gene productCCR4 domain-containing protein 1, putative
Gene product: Alternative nameCCR4-1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
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 markerunknown
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