RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4166
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0605800; Gene model (P.falciparum): PF3D7_1207300; Gene product: LIMP protein, putative (LIMP)
Name tag: GFP
Phenotype Fertilization and ookinete; Sporozoite;
Last modified: 1 June 2017, 18:50
  *RMgm-4166
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28525314
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 cl15cy1
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherSantos JM, Janse CJ, Mair GR
Name Group/DepartmentParasitology, Department of Infectious Diseases
Name InstituteUniversity of Heidelberg Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4166
Principal nameLIMP::GFP
Alternative name2180cl1m4
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 ookinete production. In the ookinete LIMP::GFP highlighted, apart from faint surface staining, foci that correspond to crystalloids, transient ookinete stage-specific organelles that provide protein and lipid material for sporozoite formation in the oocyst
OocystNot tested
SporozoiteNormal oocyst and sporozoite production.
IFAs on limp::gfp sporozoites recovered from salivary glands, fluorescence was found in a dusted, speckled distribution along the entire length of the parasite.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of LIMP.

Protein (function)
The gene comprises six exons and five introns (this organisation is conserved throughout the genus) and encodes a protein of 110 amino acids (aa) with a 22 aa long signal peptide and a molecular weight of 13 kDa. Ab initio protein structure predictions  indicate that LIMP (I23 to G110) consists of three beta sheets opposed to two a-helices. LIMP is highly conserved among the various Plasmodium species; similarly short proteins are present in related api-complexan parasites, where the homology is focused on a 22 amino acid proline-rich region adjacent to the signal peptide.

Phenotype
Analyses of mutants lacking expression of LIPM (RMgm-4165) indicate a role for LIMP during mosquito midgut colonisation by the ookinete, and a crucial function for the protein during salivary gland invasion, but not for development; neither ookinete nor sporozoite formation were affected by the absence of LIMP. Δlimp sporozoites are impaired in hepatocyte transmigration, adhesion and invasion and do not glide.

The analyses a GFP-tagged version of LIMP (RMgm-4166) showed the following:
'No protein expression in asexual stage parasites or gametocytes; limp::gfp is translated in the ookinete stage and its expression is visible in crystalloids (transient and putative storage organelles of the ookinete) and the surface; the protein produced a faint ‘dusting’ on the surface of midgut and salivary gland sporozoites. In agreement with previously published results limp mRNA was not detected in asexual blood stages; mRNA is however present in gametocytes (while protein is not) and in ookinetes (where protein is present); this is consistent with limp being translationally repressed and maternally provided to the developing ookinete. Western blot analysis of ookinete material confirmed expression of a GFP fusion protein of the expected size. Parasites expressing LIMP::GFP showed no defects in liver stage development in vitro nor during transmission to the rodent host by mosquito bite'.

Evidence is presented for:
- limp::gfp sporozoites  move significantly slower  despite limp::gfp salivary gland sporozoites producing normal numbers of CSP trails
- No evidence for shedding of LIMP during sporozoite migration
- LIMP::GFP parasites glide with a ‘limp’

GFP tagging of LIMP caused a limping defect during movement with reduced speed and transient curvature changes of the parasite.

The aberrant gliding motility was not observed in sporozoites expressing N-terminal mCherry-tagged LIMP (RMgm-4167) or C-terminal myc-tagged LIMP (RMgm-4168)

Additional information
Using an immuno-electron microscopy (immuno-EM) approach, we corroborated LIMP::GFP surface localisation in sporozoites in three independent staining experiments with three different parasite samples (using anti-GFP antibodies). Seventy-six percent of gold particles (from a total of 281) were associated with the parasite PM, 11% outside of parasites; 13% were intracellular although not clearly associated with any specific organelle.

Other mutants
See PF3D7_1207300
 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0605800
Gene Model P. falciparum ortholog PF3D7_1207300
Gene productLIMP protein, putative
Gene product: Alternative nameLIMP
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
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 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