Summary

RMgm-40
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1002200; Gene model (P.falciparum): PF3D7_0404500; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
Phenotype Liver stage;
Last modified: 12 February 2009, 13:19
  *RMgm-40
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) : 16103357
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherM.R. van Dijk, A.P. Waters, C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-40
Principal name274cl1.1; 434cl1
Alternative namep36p-
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, hepatocyte traversal and hepatocyte invasion in vitro (HepG2) of mutant sporozoites is similar to wild type sporozoites. Liver stage development is strongly impaired and parasites do not develop into the schizont stage and most invaded parasites cannot be detected anymore at 24 hours after infection. Inside the hepatocytes the formation of a parasitophourous vacuole is not detected at 15 and 24h after infections as shown by staining with antibodies against the parasithophorous vacuole membrane protein PbExp1 (HEP17).
Infection of mice (C57BL/6) by bite of infected mosquitoes did not result in blood stage infection. Only intraveneous inoculation of very high numbers of sporozoites (50.000) resulted in blood stage infection in a very low percentage of the mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the P36p protein.

Protein (function)
P36p is a member 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.
A paralogue of the p36p gene, p36 (PB000892.00.0; pbs36), is located in the genome next to p36p (in tandem).

Phenotype
The phenotype analysis demonstates a role of this protein 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. berghei 'attenuated sporozoites' lacking expression of P36p 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.

Host hepatocyte apoptosis is normally inhibited by wild type sporozoites upon invasion and establishment of the parasitophorous vacuole. The rapid disappearance of cells infected with the mutant sporozoites might be due to the inability of the mutant to prevent apoptosis of the host cell. The level of apoptosis in hepatocytes infected with mutant sporozoites was significantly higher than in hepatocytes infected with wild type sporozoites.

Disruption of the P. falciparum ortholog of P36p, P52 (PFD0215c) results in a comparable phenotype of the sporozoites that become similarly arrested during liver stage development (van Schaijk, B.C. et al., 2008, PloS ONE, 3:e3549)

 Other mutants
A P. berghei mutant has been generated that lacks expression of this protein and in addition expresses the reporter protein GFP constitutively throughout development (RMgm-41).
An independent P. berghei mutant has been generated that lacks expression of this protein (RMgm-44).
A P. berghei mutant (RMgm-42) has been generated that lacks the expression of not only this protein but also its paralogue P36 (PB000892.00.0; pbs36).
A P. yoelii mutant (RMgm-43) has been generated that lacks the expression of not only this protein but also its paralogue P36 (PY01341; p36).
In P. falciparum a mutant has been generated lacking this protein (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 the P. berghei mutant.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1002200
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 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 15'-CGATCGATGAATAATAGTAAATGATGAAACGTCG-3'
Additional information primer 1L903
Sequence Primer 25'-CCCAAGCTTAATTACGTCCCCTGGATATGC-3'
Additional information primer 2L904
Sequence Primer 35'-GGATATCTAGTGATGAGGATGAATCG-3'
Additional information primer 3L905
Sequence Primer 45'-CGCGGATCCAATGCTTGAATACATGTGG-3'
Additional information primer 4L906
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6