RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4823
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0112100; Gene model (P.falciparum): PF3D7_0613800; Gene product: AP2 domain transcription factor, putative
Details mutation: T-to-C transition at position 5468 (serine (S) into phenylalanine (F))
Phenotype Asexual bloodstage;
Last modified: 19 June 2020, 22:23
  *RMgm-4823
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32076635
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone PbNK65-NYU
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherAkkaya M, Pierce SK
Name Group/DepartmentLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases
Name InstituteNational Institutes of Health
CityRockville
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4823
Principal namePbNK65(F) parasites
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageEvidence is presented that:
PbNK65F infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell–specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated PBANKA_0112100, in which T-to-C transition at position 5468 is introduced. This mutation results resulted in a change of a serine (S) into phenylalanine (F).

Protein (function)
A member of the apicomplexan homolog of plant Apetela2 (ApiAP2) transcription factor (TF) family.
There are 26 members of the ApiAP2 TF family, and despite their similarities, each has unique features in terms of their DNA recognition motifs and their expression patterns during the life cycle of Plasmodium, suggesting distinct functional properties. PBANKA_011210 (referred to here as ApiAP2) is a Pb ApiAP2 family member predominantly expressed in schizonts in the blood stage of the parasite infection in mice and appears to be essential as Pb parasites, in which the gene encoding ApiAP2 was knocked out, were not viable.
Recent comparative genetic screening of rodent Plasmodium strains revealed a non-synonymous SNP (a T-to-C transition at position 5468) in the first DNA binding domain of the ApiAP2 gene. This SNP resulted in a phenylalanine (F) in PbANKA and PbSP11 strains and a serine (S) in PbNK65 and PbK173 strains at amino acid position 1823 of the ApiAP2 protein. Given that this polymorphism is located in the ApiAP2 DNA binding domain and that the biochemical differences between phenylalanine, a  hydrophobic aromatic amino acid, and serine, a hydrophilic amino acid, we postulated that these two genes may encode functionally different ApiAP2 proteins.
Here, we provide evidence that the two polymorphic forms of ApiAP2 do bind to different DNA motifs, resulting in altered transcriptional profiles, and that infections of mice by PbNK65F  versus PbNK65S  result in markedly different host immune responses, one that is protective and one that is not.

Phenotype
Evidence is presented that:
PbNK65F  infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell–specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies.

Additional information
From the Abstract:
'Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite Plasmodium berghei (Pb) NK65 allowed infected mice to mount a T helper cell 1 (TH1)–type immune response that controlled subsequent infections. As compared to PbNK65S, PbNK65F  parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65F  infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell–specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0112100
Gene Model P. falciparum ortholog PF3D7_0613800
Gene productAP2 domain transcription factor, putative
Gene product: Alternative name
Details of the genetic modification
Short description of the mutationT-to-C transition at position 5468 (serine (S) into phenylalanine (F))
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor editing the AP2 (PBANKA_0112100) gene in Pb NK65NYU, replacing serine-1823 with phenylalanine (S1823F), we used the CRISPR-Cas9 system (32). A guide sequence of 20 nucleotides (5′-GCTGAATTAAAACCCCAAAG-3′), with protospacer adjacent motif (5′-AGG-3′), was selected by manual curation for targeting the Cas9 endonuclease to result in the desired editing (5467 TCT to TTT) in the AP2 gene. The 900 nucleotides of synthetic sequence with the mutated guide region and the desired SNP (5467 TCT to TTT) were subcloned in the pYC plasmid using Nco I and Xho I restriction enzyme sites. The resulting plasmid, pYC_NK65AP2ANKA, was used for the transfection of the Pb NK65NYU parasites
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