Summary

RMgm-102
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0304700; Gene model (P.falciparum): PF3D7_0207300; Gene product: serine repeat antigen 8 (ECP1, egress cysteine protease 1; PbSERA5)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 7 January 2010, 19:54
  *RMgm-102
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) : 16027235
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherA.S.I. Aly, K. Matuschewski
Name Group/DepartmentDepartment of Parasitology
Name InstituteHeidelberg University School of Medicine
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-102
Principal nameecp1(-)
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
OocystNormal production of oocysts. Sporozoites fail to egress from midgut oocysts. Within the oocysts the sporozoites seemed to be arranged in circles whereas in wild type oocysts the sporozoites are arranged in the typical radial fashion. Mutant sporozoites displayed a continuous circular movement around a central axis, in both clockwise and anticlockwise directions. In wild type oocysts, sporozoite bending and flexing is seen on rare occasions and in general, no motility can be observed in wild type oocysts. Mutant oocysts were more resistant to permeabilization by detergents (1% saponin) compared to wild type oocysts.
SporozoiteSporozoites fail to egress from midgut oocysts. No sporozoites were detected in the hemocoel or in the salivary glands of infected mosquitoes despite efficient infection rates and high numbers of oocyst sporozoites. Mechanically liberated sporozoites from oocysts show gliding motility. Infection of Sprague/Dawley rats by intravenous injection of large numbers of mechanically liberated sporozoites (100.000) did not result in blood stage infections.
Liver stageInfection of Sprague/Dawley rats by intravenous injection of large numbers of mechanically liberated sporozoites (100.000) did not result in blood stage infections.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of ECP1 (egress cysteine protease 1; PbSERA5, an ortholog of P. falciparum SERA8).

Protein (function)
The ECP1 protein belongs to a family of proteases that were termed serine repeat antigens (SERAs). Members of this distinct Plasmodium protease family  belong to papain-like cysteine proteases based on a central 30-kD protease domain. A hallmark of papain-family cysteine proteases is the presence of the catalytic triad with invariant cysteine, histidine, and asparagine residues and the oxyanion-hole glutamine residue. Presence of these residues in the ECP1 proteins indicates that they might function as proteases.

Plasmodium species possesses SERAs of two major groups, specified as 'cysteine-type SERAs' and 'serine-type SERAs'. Serine-type SERAs contain an active site serine residue instead of the canonical cysteine residue. The genomes of different Plasmodium species contain different numbers of SERA genes. P. falciparum possesses nine SERA protease genes whereas the P. berghei genome contains five. P. falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. In P. falciparum mutants have been generated lacking expression of SERA1, 2, 3, 4 , 7, 8 and 9 without a distinct phenotype in the blood stages (MvCoubrie J.E. et al,  2007, Infection and Immunity, 75:5565-74).

SERA genes of P. berghei are arranged in a tandem cluster that contains two serine-type SERAs (PbSERA1, -2) and three cysteine-type SERAs (PbSERA3, -4 and -5). 

Sequence homology and syntenic organisation indicate that PbSERA3, -4 (PB000352.01.0) and -5 (PB000649.01.0) are orthologs of P. falciparum SERA6, -7, -8.

Phenotype
The phenotype analyzes indicate a role of ECP1 in egress of mature, viable sporozoites from the mature oocysts. In addition, it may play a role during liver stage development since the oocyst-derived sporozoites were not infectious to the mammalian host.

Additional information
Although the substrate of ECP1 is not known yet, the phenotype analysis of the mutant suggests that CSP is a likely candidate for a direct or indirect downstream proteolytic processing event, particularly because it seems to be one of the dominant parasite-derived components lining the inner side of the oocysts. See alo mutant RMgm-68  which expresses a mutated form of CS (region II-plus). Sporozoites of this mutant also show a defect in egress from the oocyst.

Other mutants

Other mutants
RMgm-271: A mutant expressing GFP under the control of the 5'-UTR of PbSERA3 (an ortholog of P. falciparum SERA6)
RMgm-272: The mutant expresses in addition to the endogenous SERA3  (the ortholog of P. falciparum SERA6) a TAP-tagged form of SERA3 under control of its own 5'-UTR and the dhfr/ts 3'-UTR
RMgm-365: A mutant expressing the endogenous SERA1 protein fused to the mCherry red fluorescent protein
RMgm-366: A mutant expressing the endogenous SERA2 protein fused to the mCherry red fluorescent protein
RMgm-367: A knock-out mutant lacking expression of SERA1
RMgm-368: A knock-out mutant lacking expression of SERA2
RMgm-369: A double knock-out mutant lacking expression of SERA1 and SERA2

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0304700
Gene Model P. falciparum ortholog PF3D7_0207300
Gene productserine repeat antigen 8
Gene product: Alternative nameECP1, egress cysteine protease 1; PbSERA5
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid single 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 genePartial
Additional remarks partial/complete disruption The ecp1 genomic locus is targeted with an EcoRV-linearized integration plasmid containing 5' and 3' truncations of the ecp1 open reading frame and the dhfr/ts positive selectable marker. Upon a single crossover event, the region of homology is duplicated, resulting in two truncated, non expressed ecp1 copies in the recombinant locus.
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'-GGACTAGTGAGCATATAGAAAGCCATATTCAAC-3'
Additional information primer 1ECP1for
Sequence Primer 25'-GGACTAGTGAGCATATAGAAAGCCATATTCAAC-3'
Additional information primer 2ECP1rev
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6