Summary

RMgm-141
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0412900; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein (CTRP)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 18 February 2009, 14:24
  *RMgm-141
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) : 10562534
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/ResearcherDessens, J.T., Sinden, R.E.
Name Group/DepartmentInfection and Immunity Section, Department of Biology
Name InstituteImperial College of Science, Technology and Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-141
Principal nameCTRP KO (31); CTRP KO (45)
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 ookineteMutants developed gametocytes and formed ookinetes in vitro in similar numbers to, and morphologically indistinguishable from, wild type parasites in Giemsa-stained preparations.
Ookinetes do not develop into oocysts.
After removal of the blood meal from dissected, gametocyte-infected Anopheles gambiae midguts, wild type ookinetes were found in the epithelium. In sharp contrast, no mutant ookinetes were observed. When dissected, infected A.stephensi midguts were examined for the presence of ookinetes or young oocysts on the basal surface, only wild type and never mutant parasites were observed. When ookinete motility was assessed by recording their positions on glass surfaces at 5 min time intervals, displacement of wildtype was observed but not mutant ookinetes, indicating that CTRP is involved in ookinete locomotion. The KO ookinetes were occasionally seen to straighten and bend.
OocystNormal numbers of ookinetes are produced. Ookinetes do not develop into oocysts.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of a functional CTRP protein (circumsporozoite protein [CSP] and thrombospondin-related adhesive protein [TRAP]-related protein; CSP and TRAP-related protein (CTRP).
By Western analysis of proteins of ookinete preparations with antibodies against CTRP a smaller protein of Mr 190 000 was detected in mutants compared with that detected in wild type parasites. This protein must result from translation of truncated mRNA transcribed from the CTRP gene in the mutants. It is expected to be 266 amino acids smaller as a result of the TgDHFR/TS insertion

Protein (function)
CTRP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). CTRP has six tandemly arrayed A domains followed by seven TSP type 1-like domains. CTRP is located in the micronemes of ookinetes. This structure is similar to that of TRAP (PF13_0201; PB000374.03.0), a malaria sporozoite protein critical for sporozoite motility and invasion into host cells.
CTRP plays a role in the motility of ookinetes and invasion of midgut epithelial cells.

Phenotype
The phenotype analyses indicate a role in the transformation of the ookinete into the oocyst. This and other studies indicate a role in motility and invasion of midgut epithelial cells.

Additional information
Ookinetes lacking expression of CTRP that are directly injected into the hemocoel, thereby by-passing the midgut wall, develop into oocysts and these ookinetes can transform in vitro into oocysts, indicating that CTRP is not essential for transformation of ookinetes into oocysts (Nacer, A. et al., Malar J.  19;7:82).

A P. falciparum mutant lacking CTRP (PFC0640w) has been generated which show a comparable defect in the formation of oocysts (Templeton, T.J. 2000. Mol. Microbiol. 36, 1-9. 

A mutant has been generated (RMgm-150) that expresses a mutated form of TRAP (thrombospondin-related anonymous protein; PB000374.03.0; PF13_0201), in which the cytoplasmic tail domain (CTD) of TRAP is replaced with the CTD domain of CTRP. The CTD of CTRP can complement the function of the CTD of TRAP, albeit not as well as TRAP as shown by the reduced invasion of salivary glands and hepatocytes. 

Other mutants
RMgm-140:  An independent mutant lacking expression of CTRP.
RMgm-150: A mutant that expresses a mutated form of TRAP (thrombospondin-related anonymous protein; PB000374.03.0; PF13_0201), in which the cytoplasmic tail domain (CTD) of TRAP is replaced with the CTD domain of CTRP.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0412900
Gene Model P. falciparum ortholog PF3D7_0315200
Gene productcircumsporozoite- and TRAP-related protein
Gene product: Alternative nameCTRP
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 genePartial
Additional remarks partial/complete disruption Disruption was achieved by inserting a modified Toxoplasma gondii dihydrofolate reductase–thymidylate synthase gene (TgDHFR/TS) into the CTRP locus at nucleotide position 4955 by double cross-over homologous recombination. The partial, 1080 bp CTRP sequence was used to flank the TgDHFR/TS cassette and allow homologous recombination. By Western analysis of proteins of ookinete preparations with antibodies against CTRP a smaller protein of Mr 190 000 was detected in mutants compared with that detected in wild type parasites. This protein must result from translation of truncated mRNA transcribed from the CTRP gene in the mutants. It is expected to be 266 amino acids smaller as a result of the TgDHFR/TS insertion.
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationDisruption was achieved by inserting a modified Toxoplasma gondii dihydrofolate reductase–thymidylate synthase gene (TgDHFR/TS) into the CTRP locus at nucleotide position 4955 by double cross-over homologous recombination. The partial, 1080 bp CTRP sequence was used to flank the TgDHFR/TS cassette and allow homologous recombination. By Western analysis of proteins of ookinete preparations with antibodies against CTRP a smaller protein of Mr 190 000 was detected in mutants compared with that detected in wild type parasites. This protein must result from translation of truncated mRNA transcribed from the CTRP gene in the mutants. It is expected to be 266 amino acids smaller as a result of the TgDHFR/TS insertion.
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'-GGGGTACCTTGTTCTTCACAAGTTAAACCA-3'
Additional information primer 1CTRP-Kpn
Sequence Primer 25'-CCATCGATAAATATAGTTGGCTTTAATGAAG-3'
Additional information primer 2CTRP-Cla
Sequence Primer 35'-CGGGATCCGTAATAAAATGCCTTATTCTCAT-3'
Additional information primer 3CTRP-Bam
Sequence Primer 45'-CCCGGCCGACAATGAAATGTGCCAAACA-3'
Additional information primer 4CTRP-Eag
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6