Summary

RMgm-135
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1006300; Gene model (P.falciparum): PF3D7_0408700; Gene product: perforin-like protein 1 | sporozoite micronemal protein essential for cell traversal (PPLP1; Plasmodium perforin like protein 1; Membrane attack Complex; SPECT2)
Phenotype Sporozoite; Liver stage;
Last modified: 4 May 2009, 20:03
  *RMgm-135
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) : 15659064
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/ResearcherT. Ishino, M. Yuda
Name Group/DepartmentSchool of Medicine
Name InstituteMie University
CityMie
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-135
Principal namespect2(-)1; spect2(-)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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNormal numbers of midgut- and salivary gland sporozoites are formed. Sporozoites have a strongly reduced infectivity to rats after intravenous injection of sprozoites and strongly reduced numbers of exoerythrocytic forms (EEFs) in frozen sections of rat livers were counted 24 h after sporozoite inoculations.
Liver stageSporozoites have a strongly reduced infectivity to rats after intravenous injection of sprozoites and strongly reduced numbers of exoerythrocytic forms (EEFs) in frozen sections of rat livers were counted 24 h after sporozoite inoculations.
Sporozoites formed normal numbers of EEFs in hepatoma cells (HepG2), indicating that they retain normal infection ability.
Sporozoites had completely lost cell passage ability as shown in the cell-wounding assay. Cell-passage activity was estimated by the number of cells wounded by sporozoite passage, which were identified by cytosolic labelling with FITC-conjugated dextran.
In Kupffer cell-depleted rats, the sporozoites had restored liver infectivity. Parasitaemia after inoculation of mutant sporozoites was remarkably increased in Kupffer-depleted rats, becoming similar to that after inoculation of wild-type sporozoites In addition, the numbers of EEFs formed in the liver were also similar between spect2-disruptants and the wild type.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SPECT2/PPLP1 (Membrane Attack Complex; MAC/perforin, putative; Plasmodium perforin like protein 1, PPLP1;  sporozoite protein essential for cell traversal, SPECT2)

Protein (function)
The spect2/pplp1 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). SPECT2/PPLP1 is specifically expressed in salivary gland sporozoites (not in midgut sporozoites) and has a micronemal location (micronemes).

Phenotype
The phenotype analyses indicate a rol of SPECT2/PPLP1 in traversal of the sporozoites through cells of the host. HepG2 traversal and not invasion was inhibited in the mutant sporozoites. Mutant sporozoites had restored liver infectivity in Kupffer cell-depleted rats. These results suggest that SPECT2/PPLP1 is necessary for breaching the sinusoidal cell layer via the Kupffer cells prior to hepatocyte infection (see also 'Additional information').

Additional information
Analysis of sporozoites lacking SPECT2/PPLP1 indicates a role of traversal through host cells in the dermis (for freely moving until endothelial barriers are reached and for resisting attacks by phagocytic cells) and in the liver sinusoids, presumably for resisting destruction by Kupffer cells (R. Amino et al., 2008, Cell Host & Microbe, 3, 88-96).

Other mutants
Mutants that lack expression of other members of the PPLP protein family:
RMgm-137: Another mutant lacking expression of SPECT2/PPLP1
RMgm-123: A mutant lacking expression of PPLP5
RMgm-134: A mutant lacking expression of PPLP3/MAOP
See also RMgm-92 for an unsuccessful attempt to disrupt PPLP4

See also RMgm-138 and RMgm-139 for mutants lacking the expression of SPECT which show a comparable defect in cel traversal capacity, whereas the capabilty to invade hepatocytes is not affected.
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1006300
Gene Model P. falciparum ortholog PF3D7_0408700
Gene productperforin-like protein 1 | sporozoite micronemal protein essential for cell traversal
Gene product: Alternative namePPLP1; Plasmodium perforin like protein 1; Membrane attack Complex; SPECT2
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'-CGCGAGCTCGCTAACACATAGCGAAACCATGTTGTC-3'
Additional information primer 1
Sequence Primer 25'-CGCGGATCCTATAATCGTCATAATCATCTTCATCA TCACC-3'
Additional information primer 2
Sequence Primer 35'-CCGCTCGAGAAAGATGAAGAACAAAATGAGCA TATAGATA-3'
Additional information primer 3
Sequence Primer 45'-CGGGGTACCGCCAATTGTGTATTTTATGC AGTTTGACT-3'
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6