Summary

RMgm-862
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0305700; Gene model (P.falciparum): Not available; Gene product: Not available (SERA1)
Phenotype Asexual bloodstage;
Last modified: 1 December 2013, 10:49
  *RMgm-862
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) : 23634205
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii YM
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherHuang, X; Preiser, PR
Name Group/DepartmentDivision of Molecular Genetics & Cell Biology
Name InstituteSchool of Biological Sciences, Nanyang Technological University
CitySingapore
CountrySingapore
Name of the mutant parasite
RMgm numberRMgm-862
Principal nameSERA1-ko
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGrowth,multiplication and virulence of blood stages comparable to wild type (WT) parasites although evidence is presented for subtle differences in growth between mutant and WT parasites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of serine repeat antigen 1 (SERA1) and expresses GFP under the constitutive eef1a promoter of P. berghei.
(gene model sera1 in P. yoelii YM: PYYM_0306000)

Protein (function)
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 and P. yoelii 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 and P. yoelii 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 PySERA3 (PY000293), -4 (PY02062) and -5 (PY02063) are orthologs of P. falciparum SERA6, (PF3D7_0207500) -7 (PF3D7_0207400), -8 (PF3D7_0207300)

P. yoelii encodes two members of the SERAser subfamily, PySERA1 and PySERA2.
SERA1 (PY00291) and SERA2 (PY00292) previously recognized as SERA3 and a putative cysteine protease respectively.

Phenotype
The phenotype analyses indicate that SERA1 does not have an essential role during blood stage development. Growth,multiplication and virulence of blood stages comparable to wild type (WT) parasites although evidence is presented for subtle differences in growth between mutant and WT parasites.

Additional information
See also below the P. berghei mutants lacking expression of SERA1, SERA2 and lacking both SERA1 and SERA2. These mutants showed a normal growth of blood stages under the conditions tested.

Evidence has been presented that transcription of sera1 and sera2 are upregulated in the lethal YM strain compared to the non-lethal YA strain of P. yoelii.

Analyses of mutants expressing GFP-tagged SERA1 (RMgm-864) and SERA2 (RMgm-865) it appears that both PySERA1 and 2 are expressed in the later stage of parasite maturation and that the proteases are located insight the parasitophorous vacuole (PV). However, it cannot be ruled out that the observed location in the PV is either due to the GFP tag preventing proper export or due to the fact that SERA are processed at the C-terminal end prior to activation which could result in the GFP tag remaining within the PV while the protease is exported further. However, no obvious free GFP was detected in the parasite schizont extract using anti-GFP antibody.

Other mutants
RMgm-863: A P. yoelii mutant lacking expression of SERA2
RMgm-864, RMgm-865: Mutants expressing C-terminal GFP-tagged versions of SERA1 and SERA2 respectively

P. berghei mutants:
RMgm-102: A mutant lacking expression of SERA8 (PB000649.01.0; PFB0325c), a cysteine-type SERA homologue (serine repeat antigen 8, SERA-8; ECP1, egress cysteine protease 1; PbSERA5)
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-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 PY17X_0305700
Gene Model P. falciparum ortholog Not available
Gene productNot available
Gene product: Alternative nameSERA1
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationNo information is provided on the primer sequences used to PCR-amplify the targeting sequences
Additional remarks selection procedureTransfected parasites were first selected through FACs sorting for the green fluorescent signal, and sorted parasites were subsequently diluted for cloning.
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
Additional information primer 1
Sequence Primer 2
Additional information primer 2
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