RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5204
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1423100; Gene model (P.falciparum): PF3D7_0814400; Gene product: putative phospholipase, DDHD1 (PA-PLA1, Pla1)
Name tag: 3XHA-mCherry
Phenotype Asexual bloodstage; Liver stage;
Last modified: 26 May 2022, 16:37
  *RMgm-5204
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) : 35427579
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/ResearcherSrivastava PN, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-5204
Principal namePla1-3XHA-mCherry (Pla1mch)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagemCherry fluorescence was detected in the parasite’s blood stages indicating the presence of PbPla1 protein during the entire erythrocytic cycle
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNo fluorescence could be detected in oocyst or salivary gland sporozoites. Expression of Pla1 protein was maintained during the mixed blood stage (MBS) and early to mid-liver stages. Protein expression is maintained during late EEF development and is also detected in detached cells marking the end of the hepatic phase. Further analysis indicated that PbPla1 is not associated with either the PVM or the merozoite plasma membrane as is evident by its co-staining with anti-UIS4 and anti-MSP1.
Additional remarks phenotype

Mutant/mutation
The mutant (Pla1mch) expresses a C-terminal 3XHA-mCherry tagged version of of Pla1.

Protein (function)
A total of 22 genes have been identified thus far, having similarities to the representative members of at least one of the four groups of known phospholipases; PLA, PLB, PLC and PLD. Out of these, 15 have the α/β hydrolase fold, four have the patatin-like phospholipase (PLP) domain and the remaining three have lecithin:cholesterol acyltransferase (LCAT), exonuclease-endonuclease-phosphatase (EEP) and DDHD domains. An interesting class of enzymes is the intracellular PLA1 family. In most eukaryotes, there is only one enzyme of this family while there are three members in mammals. Early  characterization efforts identified a phosphatidic acid (PA) preferring phospholipase A1 (PAPLA1) a putative phospholipase, DDHD1 (PbPla1, PlasmoDB accession number: PBANKA_1423100), which is the only P. berghei homolog of PA-PLA1 (Pla1)

Phenotype
mCherry fluorescence was detected in the parasite’s blood stages indicating the presence of PbPla1 protein during the entire erythrocytic cycle. Protein expression of Pla1 representing the correct size of the fusion protein was confirmed by western blot. No fluorescence could be detected in oocyst or salivary gland sporozoites. Expression of Pla1 protein was maintained during the mixed blood stage (MBS) and early to mid-liver stages. Protein expression is maintained during late EEF development and is  also detected in detached cells marking the end of the hepatic phase. Further analysis indicated that PbPla1 is not associated with either the PVM or the merozoite plasma membrane as is evident by its co-staining with anti-UIS4 and anti-MSP1.

Additional information
See mutant RMgm-5203 lacking expression of Pla1, showing evidence for a role in liver stage merosome release

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1423100
Gene Model P. falciparum ortholog PF3D7_0814400
Gene productputative phospholipase, DDHD1
Gene product: Alternative namePA-PLA1, Pla1
Details of the genetic modification
Name of the tag3XHA-mCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid 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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor the C-terminal tagging of PbPla1 with 3XHA-mCherry, a transgenic construct was generated as described previously (Al-Nihmi et al., 2017). For this, a fragment was amplified from the 3’ end of Pla1 not containing its stop codon, using primer pair 1153/1154 (F3). F3 was cloned in the pBC-3XHA-mCherry-hDHFR vector at the XhoI/BglII site and the previously described F2 was cloned at the NotI/AscI site. The final construct was linearized by XhoI/AscI digestion.
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