Summary

RMgm-43
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1003600; Gene model (P.falciparum): PF3D7_0404500; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
DisruptedGene model (rodent): PY17X_1003500; Gene model (P.falciparum): PF3D7_0404400; Gene product: 6-cysteine protein (P36)
Phenotype Liver stage;
Last modified: 13 February 2010, 23:51
  *RMgm-43
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 17517871
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 line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherM, Labaied, S.H.I. Kappe
Name Group/DepartmentDepartment of Pathobiology
Name InstituteUniversity of Washington
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-43
Principal namePy52/Py36-deficient parasites (CL1, CL2)
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 stageGliding motility and hepatocyte traversal in vitro (HepG2-CD81) of mutant sporozoites is similar to wild type sporozoites. Liver stage development is strongly impaired and parasites do not develop into the schizont stage. One hour after infection of the HepG2 cells, the number of intracellular p52/p36-deficient parasites showed a ~50% reduction compared to wild type sporozoites. Invaded sporozoites did not form a parasitophorous vacuole as shown by staining with antibodies against the UIS4 protein of P. berghei (PB100551.00.0), a parasitophorous vacuole membrane protein and demonstrated by electron microscope analysis. No schizonts were formed and sporadically small growth-arrested parasites were observed after 43h.
Infection of mice (BALB/c) by bite of infected mosquitoes and intravenous inoculation of high numbers of sporozoites (100.000) did not result in blood stage infection.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the P52 (P36p) and P36 protein.

Protein (function)
P52 (P36p) and P36 are members of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterised by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organisation throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.

Phenotype
P36 and p52  (p36p) are paralogous genes, located in tandem next to each other. Both genes have been disrupted  using a single DNA construct (double knock-out mutant). The phenotype analysis demonstrates a role of these proteins in liver stage development. Gliding motility, hepatocyte Invasion and traversal hepatocyte of mutant sporozoites is similar to wild type sporozoites. Inside the host hepatocyte development is arrested very soon after invasion.

Additional information
P. yoelii 'attenuated sporozoites' lacking expression of P36/P52 can induce sterile (protective) immunity in mice against challenge with wild type sporozoites by immunization of mice (BALB/c, C57Bl/6) with the mutant sporozoites.
Infection of mice by intravenously injection of high numbers of 'single knock-out' P. berghei mutants lacking either P52 (P36p) or P36 can result in a blood stage infection in a small number of mice ('breakthrough parasites'). No such breakthrough parasites were observed in the double knock-out P. yoelii mutant lacking expression of both proteins.

Other mutants
P. berghei mutants have been generated that lack expression of P52 (P36p) (RMgm-40, RMgm-41, RMgm-44).
P. berghei mutants have been generated that lack expression of P36 (RMgm-45; RMgm-353). A P. berghei mutant (RMgm-42) has been generated that lacks the expression of both P36p (P52) and P36.
In P. falciparum a mutant has been generated lacking P52 (PFD0215c; van Schaijk et al., (2008) PloS ONE 3(10):e3549). The 'arrested' phenotype of liver stage of this P. falciparum mutant in primary human hepotocytes is similar to the phenotype of P. berghei and P. yoelii mutants lacking expression of P52 (P36p).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1003600
Gene Model P. falciparum ortholog PF3D7_0404500
Gene product6-cysteine protein
Gene product: Alternative nameP36p; Pb36p; P52
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 modificationIn this mutant line the genes encoding P36 (PB000892.00.0) and P52/P36p (PB000891.00.0) have been disrupted (double knock-out mutant) using a single DNA construct.
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 15'-AATTGGTACCCAAATTAGTGCATGTATACAAGTAT-3'
Additional information primer 1Py52-5'RepF
Sequence Primer 25'-ATATCCTGCAGGCAAACGGTAATAGTGGACATCAT-3'
Additional information primer 2Py52-5'RepR
Sequence Primer 35'-AATTGGATCCTGAAATAGATGCATATCCTGGG-3'
Additional information primer 3Py36-3'RepF
Sequence Primer 45'-AATTACTAGTGTATGAATTGCGTGAGAAATGC-3'
Additional information primer 4Py36-3'RepR
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1003500
Gene Model P. falciparum ortholog PF3D7_0404400
Gene product6-cysteine protein
Gene product: Alternative nameP36
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 modificationIn this mutant line the genes encoding P36 (PB000892.00.0) and P52/P36p (PB000891.00.0) have been disrupted (double knock-out mutant) using a single DNA construct.
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 15'-AATTGGTACCCAAATTAGTGCATGTATACAAGTAT-3'
Additional information primer 1Py52-5'RepF
Sequence Primer 25'-ATATCCTGCAGGCAAACGGTAATAGTGGACATCAT-3'
Additional information primer 2Py52-5'RepR
Sequence Primer 35'-AATTGGATCCTGAAATAGATGCATATCCTGGG-3'
Additional information primer 3Py36-3'RepF
Sequence Primer 45'-AATTACTAGTGTATGAATTGCGTGAGAAATGC-3'
Additional information primer 4Py36-3'RepR
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6