Summary

RMgm-116
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1035200; Gene model (P.falciparum): PF3D7_1407000; Gene product: LCCL domain-containing protein | scavenger receptor-like protein (PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3)
Name tag: mCherry
TaggedGene model (rodent): PBANKA_1035200; Gene model (P.falciparum): PF3D7_1407000; Gene product: LCCL domain-containing protein | scavenger receptor-like protein (PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3)
Name tag: EGFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 9 December 2009, 17:00
  *RMgm-116
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 18452513
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherV. Carter; J.T. Dessens
Name Group/DepartmentDepartment of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene and Tropical Medicine
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-116
Principal namemCHERRY/PbSR/EGFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteExpression of the mCHERRY/PbSR/EGFP protein in female gametocytes (red and green fluorescence). No expression in blood stages and male gametocytes. Western blot analysis revealed that the C-terminal GFP tag was efficiently cleaved off the expressed PbSR protein (see also RMgm-117). mCherry (RFP) antibody detected full-length mCherry/PbSR protein (predicted to be 175 kDa in size).
Fertilization and ookinetePbSR protein expression was analysed by western blot with anti-mCherry (RFP) antibody. This analysis confirmed that PbSR is present in gametocytes and is also present, at lower levels, in ookinetes. However, PbSR was not detected in oocysts and sporozoites with this assay. Confocal microscopic examination of mCherry/PbSR/EGFP parasites revealed green and red fluorescence throughout the cytoplasm of female gametocytes/macrogametes. This localization was not obviously associated with the periphery of the parasites, or with their host erythrocytes. A similar fluorescence pattern was observed in zygotes and early retorts (i.e. immature ookinetes). In contrast, in mature ookinetes fluorescence had become condensed into one to two cytoplasmic focal spots. Similar spots were observed in young oocysts on the basal side of mosquito midguts for up to 2 days post mosquito infection. PbSR-fluorescence was not co-localized with mitochondrial nor to lysosomal organelles but co-localized with hemozoin clusters. Fluorescence microscopy and immunoelectronmicroscopy show that the protein is associated with crystalloids in the ookinetes. Plasmodium crystalloids are cytoplasmic aggregations of closely packed spherical particles 25–35 nm in diameter. In P. berghei, haemozoin-containing vacuoles accumulate around the edges of the crystalloid inclusion bodies.
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses an mCherry- and GFP-tagged version of  PbSR (PbSR (P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; LCCL domain containing protein CCp3). The endogenous pbsr gene is replaced with an mCherry-tagged (N-terminal) and gfp-tagged (C-terminal) version of pbsr (see also 'Additional information'). The mutant gave rise to large numbers of sporozoites in parasite-infected mosquitoes, and were efficiently transmitted to naïve mice by infected mosquito bites , demonstrating that the fluorescent protein tags did not affect the functionality of the PbSR protein.

Protein (function)
The pbsr gene is a member of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's. PbSR contains four LCCL domains; a LH2 (lipoxygenase homology 2) domain; two SR (scavenger receptor cysteine-rich) domains and a PTX/LamG (pentraxin/laminin-G) domain related to concanavalin A-like module.
Phenotype analyses of mutants lacking expression of PbSR (RMgm-113, RMgm-114) indicate a role of PbSR in the formation of sporozoites in the oocyst.

Phenotype
Analysis of the location of the tagged PbSR by fluorescence microscopy and immunoelectronmicroscopy shows that the protein is expressed in gametocytes and ookinetes and  is associated with crystalloids in the ookinetes. Crystalloids are transient organelles that form in developing ookinetes and disappear after ookinete-to-oocyst transition. Plasmodium crystalloids are cytoplasmic aggregations of closely packed spherical particles 25–35 nm in diameter. In P. berghei, haemozoin-containing vacuoles accumulate around the edges of the crystalloid inclusion bodies.

Additional information
Western blot analysis of gametocyte proteins revealed that the C-terminal GFP tag was efficiently cleaved off the expressed PbSR protein (see also RMgm-117). mCherry (RFP) antibody detected full-length mCherry/PbSR protein (predicted to be 175 kDa in size).
Under nonreducing conditions the RFP/PbSR protein migrated as a smear of high-molecular-weight bands, indicating that PbSR forms protein complexes through disulphide bond formation.

Other mutants
RMgm-113: A mutant lacking PbSR
RMgm-114: A mutant lacking PbSR
RMgm-115: A mutant containing a mutated form of PbSR
RMgm-117: A mutant expressing a GFP- tagged version of PbSR
 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1035200
Gene Model P. falciparum ortholog PF3D7_1407000
Gene productLCCL domain-containing protein | scavenger receptor-like protein
Gene product: Alternative namePbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3
Details of the genetic modification
Name of the tagmCherry
Details of taggingN-terminal
Additional remarks: taggingExpression of full length PbSR containing an N-terminal and C-terminal tag of red fluorescent protein (mCherry, RFP) and green fluorescent protein (GFP), respectively.
Commercial source of tag-antibodiesAnti mCherry (Abcam, ab34764)
Type of plasmid/constructPlasmid double 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 modificationThe endogenous pbsr gene was replaced with an n-terminal mcherry and c-terminal gfp-tagged version of pbsr.
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1035200
Gene Model P. falciparum ortholog PF3D7_1407000
Gene productLCCL domain-containing protein | scavenger receptor-like protein
Gene product: Alternative namePbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: taggingExpression of full length PbSR containing an N-terminal and C-terminal tag of red fluorescent protein (mCherry, RFP) and green fluorescent protein (GFP), respectively.
Commercial source of tag-antibodiesAnti GFP (Invitrogen 33-2600)
Type of plasmid/constructPlasmid double 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 modificationThe endogenous pbsr gene was replaced with an n-terminal mcherry and c-terminal gfp-tagged version of pbsr.
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