RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5516
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1241800; Gene model (P.falciparum): PF3D7_0523800; Gene product: food vacuole resident transporter 1 (FVRT1, PyDMT1 (Divalent metal transporter 1))
Name tag: HA
Phenotype Asexual bloodstage;
Last modified: 16 June 2024, 21:45
  *RMgm-5516
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) : 38049852
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherZhong M, Zhou B
Name Group/DepartmentFaculty of Synthetic Biology
Name InstituteShenzhen Institute of Advanced Technology, Chinese Academy of Sciences
CityShenzhen
CountryChina
Name of the mutant parasite
RMgm numberRMgm-5516
Principal namePyDMT1-HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageConfocal analysis after immunostaining with HA antibody showed that PyDMT-HA surrounded the hemozoin in the blood stage, implying that PyDMT1 is likely localized on the membrane of the food vacuole.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of DMT1

Protein (function)
Divalent metal transporter (DMT1) is a critical iron transporter with 12 transmembrane structures responsible for iron uptake in many organisms.
Through bioinformatics analysis, we found a homolog of human Dmt1 in the malaria parasite genome, which we named PyDMT1 in P. yoelii. Bioinformatic analysis shows that all Plasmodium genomes encode one gene product with homology to Saccharomyces cerevisiae (the yeast) Smf1-3, Homo sapiens Nramp1 and Dmt1 (Nramp2). PyDMT1 shares 29.53% amino-acid sequence identity with Homo sapiens Dmt1, and 24.94% with S. cerevisiae Smf3.

Phenotype
Confocal analysis after immunostaining with HA antibody showed that PyDMT-HA surrounded the hemozoin in the blood stage, implying that PyDMT1 is likely localized on the membrane of the food vacuole.

Additional information:
The unsuccessful attempts to disrupt DMT1 indicate an essential function during asexual blood stage growth/multiplication (see RMgm-5515)

To test whether PyDMT1 acts as an iron importer in P. yoelii, a mutant was generated with a partial loss of function allele of PyDMT1 by a single crossover to the regulatory region of Pydmt1. This mutation caused a partial loss of PyDMT1 expression but resulted in severe defects, rescued by iron supplementation (RMgm-5517).

From the paper: 'Plasmodium CRT (chloroquine-resistant transport) is known to localize on the vacuolar membrane, responsible for chloroquine resistance. To further confirm that PyDMT1 is localized on the food vacuole membrane, we fused a FLAG tag at the C-terminal of CRT by single crossover to label the food vacuole. Confocal imaging revealed that PyDMT1 indeed mainly colocalized with CRT at all blood stages, and slightly with the Golgi marker Rab6, but completely not with the endoplasmic reticulum marker PMV. During the schizont stage, a small amount of PyDMT1 signal is found in the merozoites, mainly in the hemozoin region. These data strongly suggest that PyDMT1 mainly localizes on the digestive vacuole membrane'.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1241800
Gene Model P. falciparum ortholog PF3D7_0523800
Gene productfood vacuole resident transporter 1
Gene product: Alternative nameFVRT1, PyDMT1 (Divalent metal transporter 1)
Details of the genetic modification
Name of the tagHA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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 modificationIn order to reveal the expression and subcellular localization of PyDMT1 protein, we fused a HA tag in frame with the endogenous PyDMT1 coding region at the carboxy terminus by CRISPR-Cas9 in P. yoelii 17X (using CRISPR-Cas9 vector pYCm), and obtained a recombinant PyDMT1-HA P. yoelii strain.
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