RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4192
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1318000; Gene model (P.falciparum): PF3D7_1454300; Gene product: SNF1-related serine/threonine protein kinase KIN
Details mutation: The T616 replaced by aspartic acid (KIN(T616D))
Phenotype Asexual bloodstage;
Last modified: 19 July 2017, 13:07
  *RMgm-4192
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) : 28678779
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherMancio-Silva L, Mota MM
Name Group/DepartmentInstituto de Medicina Molecular
Name InstituteFaculdade de Medicina da Universidade de Lisboa
CityLisboa
CountryPortugal
Name of the mutant parasite
RMgm numberRMgm-4192
Principal nameKIN(T616D)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageParasites expressing KIN(T616D) generated fewer merozoites and lower parasitaemia in mice
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of KIN, in which the T616, a conserved threonine residue in the activation loop, was replaced by aspartic acid.

Protein (function)
KIN is a putative serine/threonine kinase, with limited homology to the conserved family of sucrose non-fermenting 1 (SNF1) and AMP-activated kinases (AMPK) that regulate cellular energy homeostasis in yeast and mammalian cells, respectively. AMPK is a heterotrimeric complex comprising an α -catalytic subunit and two regulatory subunits β and γ. Although avian Plasmodium spp. genomes encode homologues to α, β and γ subunits, mammalian Plasmodium spp. seem to have lost the heterotrimeric complex and only retained KIN as a plausible AMPKα -related protein kinase. It is still possible that cryptic β and γ subunits exist but remain to be identified or that the poorly conserved N- and C-terminal extensions serve these functions. Homology of P. berghei and P. falciparum KIN to the AMPKα subunit is confined to the kinase domain, which includes a conserved threonine residue in the activation loop (T616 in P. berghei KIN), the phosphorylation of which is essential for SNF1 and AMPK activity, and replacement of this threonine by an aspartic acid residue has been validated as a phosphomimetic.

Phenotype
KIN(T616D) parasites generated fewer merozoites and lower parasitaemia in mice

Additional information
In this study they analysed blood stage/growth multiplication in mice kept under a dietary caloric restriction (CR) protocol (30–40% reduction in calorie intake, without changes in any dietary component, for 2–3 weeks before infection). Evidence is presented that KIN acts as a critical regulator that mediates sensing of nutrients and controls a transcriptional response to the host nutritional status.

Parasites lacking KIN (Δkin parasites) showed normal merozoite numbers in CR comparable to wild-type parasites in control experiments (normal diet), whereas wild type parasites produced fewer merozoites in CR. From day 4 onward, Δkin parasites outgrew wild-type in both dietary conditions, but presented lower parasitaemia in CR compared to AL mice, suggesting that additional parasite molecules may control growth at this point of infection.

See also mutant RMgm-521 lacking expression of KIN: this mutant showed a phenotype in salivary gland sporozoite production.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1318000
Gene Model P. falciparum ortholog PF3D7_1454300
Gene productSNF1-related serine/threonine protein kinase KIN
Gene product: Alternative name
Details of the genetic modification
Short description of the mutationThe T616 replaced by aspartic acid (KIN(T616D))
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitehdhfr
Promoter of the selectable markernot available
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