Summary

RMgm-134
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0824200; Gene model (P.falciparum): PF3D7_0923300; Gene product: perforin-like protein 3 | membrane attack ookinete protein, putative (PPLP3; Plasmodium perforin like protein 3; Membrane attack Complex; MAOP)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 19 February 2009, 21:22
  *RMgm-134
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) : 15520375
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/ResearcherK. Kadoto, M. Yuda
Name Group/DepartmentSchool of Medicine
Name InstituteMie University
CityMie
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-134
Principal namemaop(-)1; maop(-)2; maop(-)3
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 ookineteMutants showed normal exflagellation numbers and developed into mature ookinetes in vitro and in the midgut of A. stephensi mosquitoes. Ookinetes did not show any morphological differences from wild-type parasites. Mutant ookinetes cannot invade midgut epithelial cells as was shown by light microscopic analysis. Ookinetes cannot rupture the cell membrane of the midgut epithelial cell as was shown by electron microscopic analysis. Analysis of ookinete invasion/traversal showed that wild type ookinetes had invaded the midgut epithelium, and over half of them were attached to the basal lamina. The epithelial cells initially invaded by ookinetes were heavily damaged. They were characterized by high electron density staining, protrusion from the epithelium, absent or scant microvilli, and degeneration of cytoplasmic organelles such as mitochondria.
In contrast, mutant ookinetes were found neither in the cytoplasm of the midgut epithelial cell nor beneath the basal lamina. All of the mutant ookinetes were located outside the epithelium, and over the half of them adhered to the membrane of the epithelial cell, attaching their apical tip to epithelial cell surface. The epithelial cell surface where the ookinete attached was invaginated toward the cytoplasm, but no apparent breakage was observed in the extended cell membrane. The attached epithelial cells were kept intact, as shown by staining of normal electron density, dense microvilli, retention of the complex structure of the basal membrane labyrinth, and intact cytoplasmic organelles.
OocystNo oocysts are formed. Mutant ookinetes cannot invade and traverse midgut epithelial cells (see phenotype 'Fertilization and ookinete').
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MAOP (membrane attack ookinete protein; Membrane Attack Complex; MAC/perforin, putative; Plasmodium perforin like protein 3; PPLP3)

Protein (function)
The maop/pplp3 gene is a member of a small, conserved family of proteins encoding perforin-like proteins containing membrane-attack complex/perforin domains (MACPF)(Kaiser, K et al., 2004, Mol. Biochem. Parasitol. 133, 15-26). MAOP is specifically expressed in the ookinete and shows an micronemal location (micronemes).

Phenotype
The phenoype analyses indicate a role of MAOP in the invasion of the ookinetes of the midgut epithelial cells. Ookinetes can migrate to the gut epithelium and attach to the cell surface at the apical tip, but cannot enter the cytoplasm because of loss of the ability to disrupt the cell membrane.

Additional information

Other mutants
Mutants that lack expression of other members of the PPLP protein family:
RMgm-135, RMgm-137: Mutants lacking expression of SPECT2/PPLP1
RMgm-123: A mutant lacking expression of PPLP5
See also RMgm-92 for an unsuccessful attempt to disrupt PPLP4

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0824200
Gene Model P. falciparum ortholog PF3D7_0923300
Gene productperforin-like protein 3 | membrane attack ookinete protein, putative
Gene product: Alternative namePPLP3; Plasmodium perforin like protein 3; Membrane attack Complex; MAOP
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 parasitepbdhfr
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'-TCAGAGCTCGATTGTCATTATGGCTATTTTTCC-3'
Additional information primer 1
Sequence Primer 25'-TCAGGATCCTGCACCTAAATTGTCAAGTTTGTG-3'
Additional information primer 2
Sequence Primer 35'-GCGCTCGAATGAATCCCTTTTATATTTTCAC-3'
Additional information primer 3
Sequence Primer 45'-AAGGTACCTATCTCATTTCGCACTTATGATCC-3'
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6