Summary

RMgm-123
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0711600; Gene model (P.falciparum): PF3D7_0819200; Gene product: perforin-like protein 5 (PPLP5, Plasmodium perforin-like protein 5; Membrane Attack Complex)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 23 March 2011, 15:17
  *RMgm-123
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) : 17367780
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherA. Ecker, R.E. Sinden
Name Group/DepartmentDivision of Cell and Molecular Biology
Name InstituteImperial College
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-123
Principal nameΔpplp5 clone 1; Δpplp5 clone 2
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 ookineteNormal numbers of ookinetes are produced. Most (>99%) of the ookinetes do not develop into oocysts. Ookinetes also failed to infect mosquitoes when ookinetes were cultured in vitro and fed to mosquitoes via membrane feeding.
OocystNo or very few oocysts are formed. Most (>99%) of the ookinetes do not develop into oocysts. However, the block in infection was not absolute, since a single oocyst was observed in two of 50 dissected mosquitoes in one experiment. Moreover, in another experiment a single sporozoite was observed in salivary gland dissections.
SporozoiteNo or very few oocysts are formed. Most (>99%) of the ookinetes do not develop into oocysts. However, the block in infection was not absolute, since a single oocyst was observed in two of 50 dissected mosquitoes in one experiment. Moreover, in another experiment a single sporozoite was observed in salivary gland dissections.
Despite the low numbers of sporozoites, infected mosquitoes were able to transmit parasites to C57BL/6 mice.
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PPLP5 (Membrane Attack Complex; MAC/perforin, putative; (perforin-like protein 5)

Protein (function)
The pplp5 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).

Phenotype
Phenotype analyses indicate a role during during invasion and traversal of the ookinetes through cells of the midgut wall. To test whether bypassing the midgut would rescue the mutant phenotype, cultured ookinetes were injected directly into the mosquito hemocoel.
Hemocoel injection completely restored mosquito infectivity of ookinetes as determined by counting of salivary gland sporozoites, indicating that indeed the block in infection is specifically due to the inability of  ookinetes to cross the midgut epithelium. At 24 hour after infection of mosquitoes ookinetes were found attached in large numbers to the apical side of the midgut where they persisted (in decreasing numbers) until 48 h post-infection. No signs of cell invasion were observed in these guts. Ookinetes were stuck within the microvilli layer but had successfully crossed the peritrophic matrix.

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-134: A mutant lacking expression of PPLP3/MAOP
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_0711600
Gene Model P. falciparum ortholog PF3D7_0819200
Gene productperforin-like protein 5
Gene product: Alternative namePPLP5, Plasmodium perforin-like protein 5; Membrane Attack Complex
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 ApaI and BamHI
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′-TTGGGCCCGTTGAATATGCATAGACAACATC-3′
Additional information primer 1AE27A
Sequence Primer 25′-CCAAGCTTTCACAAATATAGGCTACTCTTGC-3′
Additional information primer 2AE27B
Sequence Primer 35′-TGAATTCTCATATTGAATAGGCCTTATATC-3′
Additional information primer 3AE27C
Sequence Primer 45′-GGGGATCCTTTATCACTTCATATCCCAATAC-3′
Additional information primer 4AE27D
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6