RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4876
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0205600; Gene model (P.falciparum): PF3D7_0107800; Gene product: double-strand break repair protein MRE11 (MRE11)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite;
Last modified: 28 September 2020, 11:03
  *RMgm-4876
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherGuttery DS, Tewari R
Name Group/DepartmentSchool of Life Sciences
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4876
Principal nameΔmre11
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
OocystThere were significantly fewer oocysts in Δmre11 lines, and the vast majority of these oocysts were significantly smaller than WT-GFP oocysts. In none of the Δmre11 oocysts was there any evidence of sporozoite development, and those of similar size to WT-GFP oocysts had patterns of fragmented GFP expression and reduced Hoechst DNA-staining. Investigation of the sex-cell lineage of this defect revealed that it could be rescued by crossing Δmre11 parasites with Δmap2 (male-defective) cells, suggesting that the function of MRE11 is inherited through the female.
The Δmre11 oocysts had a fully formed wall but the cytoplasm exhibited evidence of degeneration, with vacuolization and organelle breakdown, while the nuclei showed marked swelling of the nuclear membranes
Sporozoiteno sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MRE11 and expresses GFP under the constitutive eef1a promoter.
( bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.258657. this version posted August 24, 2020)

Protein (function)
Double-strand breaks in the DNA resulting from innate DNA replication errors or exposure to radiation and chemical mutagens activate the DSB repair (DSBR) pathway, facilitating repair via two distinct mechanisms: “error-prone” non-homologous end-joining (NHEJ) and “error-free” homologous recombination (HR). In Plasmodium, HR appears to be the predominant DDR pathway, with homologues of many key players of mammalian HR. A key initiator of the HR pathway is meiotic recombination 11 protein (MRE11), which in mammals is part of the MRN/X complex that includes MRE11, RAD50 and NBS1 (Nijmegen breakage syndrome 1, a homologue of Saccharomyces cerevisae Xrs2). This complex has a crucial role in DDR during mitosis, promoting HR between sister chromatids to repair mutations arising during DNA replication. MRE11 is not essential in Trypanosoma brucei bloodstream forms, but its deletion results in impaired HR, reduced growth and increased sensitivity to DNA double-strand breaks. Numerous proteins potentially involved in HR have been identified in P. falciparum, although NBS1 is not encoded in the genome. In a recent study, a single mre11 orthologue containing 2 incomplete but catalytically active MPP_MRE11 domains was identified in P. falciparum (Gene ID: PF3D7_0107800), and complementation experiments confirmed DDR activity in response to DNA damage. A role in DDR was confirmed when P. falciparum mre11 (along with its MRN partner rad50) was shown to be significantly upregulated in response to treatment to induce DNA damage with the alkylating agent, methyl methanesulfonate (MMS).

Phenotype
There were significantly fewer oocysts in Δmre11 lines, and the vast majority of these oocysts were significantly smaller than WT-GFP oocysts. In none of the Δmre11 oocysts was there any evidence of sporozoite development, and those of similar size to WT-GFP oocysts had patterns of fragmented GFP expression and reduced Hoechst DNA-staining. Investigation of the sex-cell lineage of this defect revealed that it could be rescued by crossing Δmre11 parasites with Δmap2 (male-defective) cells, suggesting that the function of MRE11 is inherited through the female. The Δmre11 oocysts had a fully formed wall but the cytoplasm exhibited evidence of degeneration, with vacuolization and organelle breakdown, while the nuclei showed marked swelling of the nuclear membranes 


Additional information
Evidence is presented that: 
Absence of PbMRE11 results in significant transcriptional downregulation in activated gametocytes of genes involved in ribonucleoprotein biogenesis, spliceosome function and iron-sulphur cluster assembly. 

MRE11 is known to regulate checkpoint signaling during meiosis in other eukaryotes, and since a normal (4N) DNA content was observed in mature ookinetes it is possible that it functions in the reductive division of meiosis in early oocyst development. A previous study analyzing the Plasmodium meiotic recombinase Disrupted Meiotic cDNA 1 (DMC1) showed deletion of this genes resulted in a similar phenotype to Δmre11 mutants, with significant reduction (up to 80%) in oocyst numbers, which were smaller compared to WT lines and transmission was completely ablated. However, unlike Δmre11 lines sporogony did occur but was slower and limited numbers of nuclei were observed; whereas in Δmre11 lines sporogony was completely ablated.

Mre11 mRNA is present in cells throughout the P. berghei life-cycle (except merozoites), based on single cell RNA-seq data, with the highest abundance in ookinetes/oocysts.

Analyses of a mutant expressing a C-terminal GFP-tagged version of MRE11 (RMgm-4877) showed the following:
MRE11-GFP was not present in asexual blood stage parasites, but was detected in the first sexual stages, more specifically in the nucleus of female but not male gametocytes. Following gametocyte activation, female, but not male, gametocytes continued to express the protein. After fertilization the protein remained associated with the nucleus throughout zygote/ookinete development; whereas in oocysts the expression was diffuse and in sporozoites it was focused at a single point adjacent to the nuclear DNA  

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0205600
Gene Model P. falciparum ortholog PF3D7_0107800
Gene productdouble-strand break repair protein MRE11
Gene product: Alternative nameMRE11
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe Δmre11 gene-knockout targeting vector was constructed using the pBS-DHFR plasmid, which contains polylinker sites flanking a Toxoplasma gondii dhfr/ts expression cassette, as described previously (53). A 611 bp fragment at the 5′ end of the mre11 sequence was generated from genomic DNA using PCR primers P0141 (5’-CCCCGGGCCCTTGTGCATACACATCAACAGATAA-3’) and P0142 (5’-GGGGAAGCTTATCCAAATCTGATAAGTAATTATCCA-3’) and inserted into pBS-DHFR using ApaI and HindIII restriction sites upstream of the dhfr/ts cassette. A 575 bp fragment generated with primers P0143 (5’-CCCCGAATTCGAATGAATTGAAGGATATCCCAG-3’) and P0144 (5’-GGGGTCTAGACTGTATTGGAGATGAATATTATGGA-3’) from the 3′ region of mre11 was then inserted downstream of the dhfr/ts cassette using EcoRI and XbaI restriction sites. The linear targeting sequence was released using ApaI/XbaI digestion of the plasmid.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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