RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5548
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1241000; Gene model (P.falciparum): PF3D7_0523000; Gene product: multidrug resistance protein 1 | ABC transporter B family member 1 (MDR1; ABCB1; Pgh1)
Name tag: mCherry
Phenotype Asexual bloodstage;
Last modified: 23 September 2024, 11:27
  *RMgm-5548
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) : 38413566
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineNSS or NSR
Name parent line/clone Not applicable
Other information parent lineTwo isogenic clones NSR and NSS, derived from P. yoelii NSM, were established after MFQ selective pressure. NSS grew faster than NSR without drug pressure. The NSR and NSS are isogenic parasites with potential mutations in the NSR to confer high-level MFQ resistance
The mutant parasite was generated by
Name PI/ResearcherXu R, Li J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Li
Name InstituteXiamen University
CityXiamen
CountryChina
Name of the mutant parasite
RMgm numberRMgm-5548
Principal nameNSS(IP/mdr1::mCherry)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagemCherry fluorescence was detected specifically at digestive vacuole membrane (DVM) and parasite plasmalemma membrane (PPM) in NSS(IP). Next, we investigated the accumulation of a known PfMDR1 substrate Fluo-49 in the NSS(IP/mdr1::mCherry)
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutants express an N-terminal mCherry-tagged version of MDR1.
To investigate the substrate transport directions of the MDR1 in parasites carrying UBP1 IP and NT haplotypes, we engineered two parasite clones, NSS(IP/mdr1::mCherry)(RMgm-5548) and NSS(NT/mdr1::mCherry) that had the N-terminus of the MDR1 fused with mCherry protein

Protein (function)
MDR1: Drug resistance in Plasmodium has been associated with single nucleotide polymorphisms and an increased copy number of the multidrug resistance protein 1 (MDR1), an ATP-binding cassette (ABC) protein family member. It is a P-glycoprotein that localizes to the digestive vacuole membrane and import solutes, including some antimalarial drugs, into the digestive vacuole.

UBP1: Mutations of V2697F and V2728F in a de-ubiquitinase (or ubiquitin carboxyl-terminal hydrolase 1, UBP1; PF3D7_0104300) have been linked to Plasmodium chabaudi responses to artesunate (ATS), artemisinin (ART), and/or chloroquine (CQ). Introductions of equivalent mutation of PcUBP1 V2697F into P. falciparum and Plasmodium berghei increased ART resistant levels and the introduction of the PcUBP1 V2728F equivalent mutation caused ART and CQ-resistance in P. berghei, but not in P. falciparum. PfUBP1 variants were associated with ART-delayed parasite clearance in P. falciparum isolates from Kenya and Thailand. A PfUBP1 R3138H substitution was shown to confer ART resistance in a ring-stage survival assay in vitro.

Phenotype
mCherry fluorescence was detected specifically at digestive vacuole membrane (DVM) and parasite plasmalemma membrane (PPM) in NSS(IP) and NSS(NT), respectively. Next, we investigated the accumulation of a known PfMDR1 substrate Fluo-49 in the NSS(IP/mdr1::mCherry) and NSS(NT/mdr1::mCherry) parasites.

Additional information
Evidence is presented that:
- Mefloquine reistance is P. yoelii is associated with two mutated amino acids in UBP1: I1560N and P2874T located in the conserved central region (CCR) of UBP1. The CCR has some homology with the motifs III-IV of the glycosyltransferase (GTase) domain of bacterial penicillin-binding proteins (PBPs).
- UBP1 double mutations (NT) impair parasite growth, hemozoin formation, and de-ubiquitinase activity (RMgm-5544).
- UBP1 double mutations (NT) alters UBP1 localization pattern and aggregation.
NT substitutions can affect UBP1 aggregation and distribution possibly by destabilizing hydrophobic interaction after the introduction of the two hydrophilic residues.
To study the UBP1 protein localization and expression in different parasite stages, we tagged the P. yoelii UBP1 with a sextuple hemagglutinin (6HA) at N-terminus and a variable coding region between GDVKT and VQQSG residues in both NSS and NSR parasites using CRISPR/Cas9 method (see RMgm-5545). UBP1 was expressed as punctate structures in the cytoplasm of asexual blood-stage trophozoites and schizonts; it was also expressed in gametocytes, gametes, zygotes, and ookinetes, but not in oocysts and sporozoites of the NSS-IP. UBP1 in the NSR(NT) or NSS(NT) mutant parasites was distributed more widely in the cytoplasm than NSS(IP) and NSS(IT) in the intra-erythrocytic stages. We engineered two additional parasite clones, NSS(IP/ubp1::GFP), and NSS (NT/ubp1::GFP), with ubp1 being endogenously tagged with a GFP coding sequence at the N-terminus. The GFP-tagged parasites further verified that NSS(NT) had a more diffused expression of UBP1 than NSS(IP) in the asexual blood stages. These data show that IP → NT substitutions can affect UBP1 aggregation and distribution possibly by destabilizing hydrophobic interaction after the introduction of the two hydrophilic residues.
- Plasmodium UBP1 NT substitutions increase MDR1 ubiquitination.
To investigate the impact of the altered MDR1 ubiquitination on protein expression and trafficking, we tagged the endogenous MDR1 in the NSS(IP), NSR(NT), and NSS(NT) parasites with quadruple Myc epitope (4Myc) at the C-terminus to obtain mdr1::4Myc parasites (RMgm-5546). Because PfCRT and PfK13 contribute to parasite responses to CQ, DHA, and other drugs, we also tagged P. yoelii K13 with 4Myc at the N-terminus and CRT with 3HA at the C-terminus obtaining the k13::4Myc and crt::3HA. Immunoblotting analysis confirmed that NT substitutions in the UBP1 rendered higher levels of MDR1 ubiquitination.
- UBP1 NT substitutions translocate MDR1 from the DV membrane to the plasma membrane.
In the NSS(IP) trophozoites,MDR1 was expressed in hemozoin-containing DV scattered in the cytoplasm; in schizonts, most of the MDR1 co-localized with large hemozoin. Similar pattern of MDR1 localization was observed in the NSS(NP) and NSS(IT) parasites. The MDR1 was mostly (more than 95% of cells) localized at the parasite plasma membrane (PPM) of trophozoites and schizonts in both NSR(NT) and NSS(NT) parasites. To confirm the altered MDR1 protein localization, we genetically engineered three doubly tagged parasites (DTPs), mdr1::4Myc/ubp1::6HA (RMgm-5547), mdr1::4Myc/crt::3HA and mdr1::4Myc/sep1::3HA from the mdr1::4Myc parasite (RMgm-5546). Parasite proteins CRT and SEP1 are natively localized to DVM and parasitophorous vacuole membrane (PVM) that surrounds the parasite PPM, respectively. These results confirm the MDR1 localization on DVM in the NSS(IP) parasites and on PPM in the NSRNT and NSSNT parasites.
- Variants of UBP1 display reduced Fluo-4 and MFQ accumulation in the parasite cytoplasm.
To investigate the substrate transport directions of the MDR1 in parasites carrying UBP1 IP and NT haplotypes, we engineered two parasite clones, NSS(IP/mdr1::mCherry)(RMgm-5548) and NSS(NT/mdr1::mCherry) that had the N-terminus of the MDR1 fused with mCherry protein.  As expected, mCherry fluorescence was detected specifically at DVM and PPM in NSS(IP) and NSS(NT), respectively. Next, we investigated the accumulation of a known PfMDR1 substrate Fluo-49,37 in the NSS(IP/mdr1::mCherry) and NSS(NT/mdr1::mCherry) parasites.

We  performed allelic exchanges of the two UBP1 mutations (I1560N and P2874T) between NSS and NSR lines to investigate the roles of the mutations in modulating drug response and parasite fitness. Two DNA fragments covering the mutation sites were amplified from the parasites: Fragment R1 was a 644 bp DNA having I1560N substitution, and fragment R2 was a 667 bp DNA harboring P2874T substitution. Two plasmids each containing the R1 or R2 sequence from NSS or NSR were cross transfected sequentially to replace IP with NT in the NSS parasite and NT with IP in the NSR parasite. Self-replacements (IP replaced with IP in NSS and NT replaced with NT in NSR) were also performed to serve as transfection controls, generating eight allelic replacements with four haplotype combinations, namely NS(SIP-self), NSS(NP), NSS(IT), NSS(NT), NSR(NT-self), NSR(IT), NSR(NP), and NS(RIP). Single replacement clonal parasites of NSS(NP), NSS(IT), NSR(IT), and NS(RNP), double replacement NSR(IP), self-replacement NSS(IP-self), and NSR(NT-self) were obtained from one to three rounds of transfection and pyrimethamine (Pyr) selection, but not NSS(NT) parasites. NSS(NT) clones were finally obtained after additional selection with 20mg/kg MFQ for 4 days, suggesting a disadvantage in vivo growth and MFQ-resistant trait of the NSS(NT) parasites. This result is consistent with the finding that NSR parasite grew slower than the NSS parasite without drug selection.
In vivo, drug assays testing parasite responses to MFQ, LUM, QN, PPQ, CQ, and DHA of the allelic exchanged parasites with NSS background showed that introduction of a single mutation, NSS(NP) or NSS(IT), had a slight increase in IC90 to MFQ, LUM, and PPQ, but not IC50 values or no effect on parasite responses to high levels of MFQ, LUM, and PPQ. In contrast, NSS(NT) parasites with double substitutions dramatically increased parasite survival after MFQ, LUM, and PPQ treatments. 
As expected, in the set of parasites with NSR background, both NSR parent and NSR(NT-self) were resistant to MFQ, LUM, and PPQ, while single replacements (NSR-IT and NSR-NP) and double replacement (NSR-IP) were sensitive to these antimalarials. These findings indicate that double mutations (1560 N plus 2874 T) are required for simultaneous resistance to high levels of MFQ, LUM, and PPQ. The NT mutations in the UBP1 had no significant effect on parasite responses to CQ, QN, and DHA.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1241000
Gene Model P. falciparum ortholog PF3D7_0523000
Gene productmultidrug resistance protein 1 | ABC transporter B family member 1
Gene product: Alternative nameMDR1; ABCB1; Pgh1
Details of the genetic modification
Name of the tagmCherry
Details of taggingN-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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe CRISPR/Cas9 plasmid pYCm was utilized for gene editing. To construct plasmids for ubp1 allelic replacement at amino acid codon 1560 and 2874 between P. yoelii lines NSS and NSR, four DNA fragments from two regions (R1, from 4393 to 5036; and R2, from 8368 to 9034) were amplified and inserted into the NcoI/XhoI sites of the pYCm vector individually. Three silent nucleotide substitutions were introduced into the single guide RNA (sgRNA) binding sites in the donor templates using synthetic oligonucleotides and PCR amplification to yield the editing plasmids.

To construct plasmids for gene tagging or tag insertion, a fragment (450–800 bp) from the C- or N-terminal of coding regions as left or right homologous arm and a fragment (450–800 bp) from 3’UTR or 5’UTR regions as right or left homologous arm were amplified, respectively. DNA sequence encoding 6HA, 4Myc, mCherry, GFP, APEX2, or 3HA was inserted between the left and right arms in the frame and cloned into the pYCm vector.

To construct the plasmid to delete the coding region of the P. yoelii ubp1 gene, 5’- and 3’- genomic fragments (400–600 bp) were amplified as left and right arms and cloned them into restriction sites of HindIII/NcoI and XhoI/EcoRI in the pYCm vector, respectively. Synthesized sgRNAs were annealed and ligated into the restriction site of BsmBI in the vector.
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