Summary

RMgm-115
Malaria parasiteP. berghei
Genotype
MutatedGene 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)
Details mutation: Two central SRCR (scavenger receptor) domains removed
Phenotype Oocyst; Sporozoite;
Last modified: 4 March 2010, 23:43
  *RMgm-115
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
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-115
Principal name∆SRCR/EGFP
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 numbers of oocysts are formed. Highly reduced formation of sporozoites within the oocysts. Only a few oocysts were observed that contained sporozoites.
SporozoiteHighly reduced formation of sporozoites within the oocysts. In samples of pooled oocyst-infected guts, gently homogenized to release sporozoites, no sporozoites were detected (compared with >10 000 sporozoites per gut in wild type with comparable oocyst numbers), although occasionally a sporulating oocyst was observed by light microscopy. These appeared normal and released sporozoites when subjected to gentle pressure. Moreover, these sporozoites displayed normal CS expression on their surface, and left CS reactive trails, indicating they possessed gliding motility. No sporozoites were detected in salivary glands. No transmission of parasites to naïve mice by infected mosquito bites.
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of PbSR (P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; LCCL domain containing protein CCp3). The mutated protein lacks the two central SRCR domains (see below) from the PbSR protein which is (c-terminal) tagged with EGFP.

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
Phenotype analyses of mutants lacking the complete PbSR protein (RMgm-113, RMgm-114) indicate a role of PbSR in the formation of sporozoites in the oocyst (see also Additional information). The phenotype analyses of the mutant that expresses the mutated form of PbSR indicate that the SRCR domains are essential for sporozoite formation.

Additional information
The protein is expressed in female gametocytes and in ookinetes. In the ookinetes PbSR is associated with crystalloids, transient organelles that form in developing ookinetes and disappear after ookinete-to-oocyst transition (Carter, V. et al., 2008, Mol. Microbiol. 68, 1560-1569; RMgm-116).

A P. falciparum mutant has been generated that lacks expression of CCp3 (PbSR). This mutant showed 'normal' sporozoite formation (sporulation) within oocysts in Anopheles freeborni. Howver, no hemocoel or salivary gland sporozoites were detected (Pradel G. et al., 2004, J. Exp. Med 199, 1533-1544).


Other mutants
RMgm-113: A mutant lacking PbSR
RMgm-114: A mutant lacking PbSR
RMgm-116: A mutant expressing a GFP- and mCherry-tagged version of PbSR
RMgm-117: A mutant expressing a GFP- tagged version of PbSR


  Mutated: Mutant parasite with a mutated 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
Short description of the mutationTwo central SRCR (scavenger receptor) domains removed
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
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 KpnI/SacII double digest
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 is replaced with a mutated Pbsr gene, lacking the two central SRCR (scavenger receptor cysteine-rich) domains. The mutated pbsr is tagged with EGFP (c-terminal).
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