RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-134

Malaria parasite	P. berghei
Genotype
Disrupted	Gene 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 modification	The 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
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	K. Kadoto, M. Yuda
Name Group/Department	School of Medicine
Name Institute	Mie University
City	Mie
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-134
Principal name	maop(-)1; maop(-)2; maop(-)3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Mutants 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.
Oocyst	No oocysts are formed. Mutant ookinetes cannot invade and traverse midgut epithelial cells (see phenotype 'Fertilization and ookinete').
Sporozoite	Not tested
Liver stage	Not 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

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0824200

Gene Model P. falciparum ortholog

PF3D7_0923300

Gene product

perforin-like protein 3 | membrane attack ookinete protein, putative

Gene product: Alternative name

PPLP3; Plasmodium perforin like protein 3; Membrane attack Complex; MAOP

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

pbdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

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 1	5'-TCAGAGCTCGATTGTCATTATGGCTATTTTTCC-3'
Additional information primer 1
Sequence Primer 2	5'-TCAGGATCCTGCACCTAAATTGTCAAGTTTGTG-3'
Additional information primer 2
Sequence Primer 3	5'-GCGCTCGAATGAATCCCTTTTATATTTTCAC-3'
Additional information primer 3
Sequence Primer 4	5'-AAGGTACCTATCTCATTTCGCACTTATGATCC-3'
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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