Summary

RMgm-143
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1116000; Gene model (P.falciparum): PF3D7_0616500; Gene product: TRAP-like protein (TLP)
Phenotype Sporozoite; Liver stage;
Last modified: 24 July 2011, 12:18
  *RMgm-143
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) : 18346224
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherC.K. Moreira, T.J. Templeton, A. Coppi
Name Group/DepartmentDepartment of Microbiology and Immunology
Name InstituteWeill Cornell Medical College
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-143
Principal namePbTLP-
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
OocystNot different from wild type
SporozoiteSporozoites showed normal gliding motility.
Sporozoites showed a twofold decrease in infectivity compared with wild type after infection of mice by intravenous injection. The sporozoites showed a 20-fold decrease in infectivity when mice were infected by intradermal inoculation.
The level of HepG2 invasion in vitro of mutant sporozoites was similar to wild type sporozoites and invaded parasites developed efficiently into mature schizonts.
In vitro migration/cell traversal assays using Hepa 1–6 cells and mouse dermal fibroblasts (MDFs) showed a twofold reduction in the migratory activity of sporozoites compared with wild type. Treatment with the cysteine protease inhibitor E-64d, which prevents invasion and results in maximally migratory sporozoites, increased the cell traversal activity but not to the level that was observed with wild type sporozoites.
Liver stageSporozoites showed a twofold decrease in infectivity compared with wild type after infection of mice by intravenous injection. The sporozoites showed a 20-fold decrease in infectivity when mice were infected by intradermal inoculation.
The level of HepG2 invasion in vitro of mutant sporozoites was similar to wild type sporozoites and invaded parasites developed efficiently into mature schizonts.
In vitro migration/cell traversal assays using Hepa 1–6 cells and mouse dermal fibroblasts (MDFs) showed a twofold reduction in the migratory activity of sporozoites compared with wild type. Treatment with the cysteine protease inhibitor E-64d, which prevents invasion and results in maximally migratory sporozoites, increased the cell traversal activity but not to the level that was observed with wild type sporozoites.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of TLP (Trap-Like Protein; TRAP2).

Protein (function)
TLP belongs to the TRAP/MIC2 family of transmembrane proteins, members of which link extracellular adhesion to the intracellular actomyosin motor complex that plays a role in (gliding) motility and cell invasion.
Similar to the Plasmodium sporozoite protein, TRAP, and the ookinete protein, CTRP, TLP possesses an extracellular domain architecture that is comprised of von Willebrand factor A (vWA) and thrombospondin type 1 (TSP1) domains, plus a short cytoplasmic domain.

Phenotype
The phenotype analyses indicate that TLP is not involved in the typical gliding motility of sporozoites and invasion of hepatocytes but plays a role in sporozoite passage through cells. The decreased infectivity of mutant sporozoites is most likely the result of the reduced capacity of cell-traversal. However, the lack of a 'clear' phenotype indicates redundancy of the function of TLP.
An independent mutant RMgm-144 has been generated which lacks expression of TLP. This mutant shows  a gliding motility and infectivity which are both comparable to wild type. See also the paper of Lacroix and Menard ( 2008, Trends Parasitol, 24, 431-34) for a comparison of the phenotype of both mutants.

Additional information
In the same study a P. falciparum mutant has been generated that lacks expression of TLP. This mutant shows normal production of gametocytes.
A mutant has been generated (RMgm-149) 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 TLP. The CTD of TLP can complement the function of the CTD of TRAP, albeit not as well as TRAP as shown by the (strongly) reduced invasion of salivary glands and hepatocytes.

Other mutants
RMgm-144: An independant mutant lacking expression of TLP.
RMgm-149:  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 TLP.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1116000
Gene Model P. falciparum ortholog PF3D7_0616500
Gene productTRAP-like protein
Gene product: Alternative nameTLP
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 parasitehdhfr
Promoter of the selectable markereef1a
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 15'-CATAAATGGCAACCCAAGCC
Additional information primer 1PbTLP5S-HindIII
Sequence Primer 25'-GACAGAGTAACAACAAAAGGAGA
Additional information primer 2PbTLP5AS-PstI
Sequence Primer 35'-CTACAAAATTGTTCGGCTTTAAG
Additional information primer 3PbTLP3S-KpnI
Sequence Primer 45'-GATAATATCGATACAGACCC
Additional information primer 4PbTLP3AS-EcoRI
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6