RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5459
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0515350; Gene model (P.falciparum): PF3D7_1031400; Gene product: OTU-like cysteine protease (Otu)
DisruptedGene model (rodent): PBANKA_1025400; Gene model (P.falciparum): PF3D7_1417300; Gene product: cysteine protease ATG4, putative (ATG4)
PhenotypeNo phenotype has been described
Last modified: 25 April 2024, 12:40
  *RMgm-5459
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption, Gene disruption
Number of attempts to introduce the genetic modification 2
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37910326
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
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherMishra A, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0515350
Gene Model P. falciparum ortholog PF3D7_1031400
Gene productOTU-like cysteine protease
Gene product: Alternative nameOtu
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate/select mutants with both genes simultaneously disrupted indicate that asexual blood stages require that at least one of the genes is active for growth/multiplication of asexual blood stages. See alos below for the information of single KO mutants of Atg4 and Otu.

For the generation of Atg4/Otu double knockout parasites, the linearized targeting cassette of Atg4 with GFP reporter and hDHFR selection marker was transfected into the purified schizonts of Otu KO parasites with mCherry reporter and TgDHFR selection marker. The transfected parasites were selected by the subcutaneous injection of WR drug (Sigma-Aldrich, SML2976) for 3 consecutive days.
In the second approach, Otu–mCherry–TgDHFR and Atg4–GFP–hDHFR linearized cassettes were cotransfected into P. berghei ANKA schizonts. The transfected parasites were selected by the oral administration of pyrimethamine.

Additional information on mutants generated in this study:

For the generation of Otu (PBANKA_0515350) knockout parasites, two fragments, F1 (591 bp) and F2 (546 bp), were amplified using primer sets 1964/1965 and 1966/1967 from the 5′ and 3′ UTRs, respectively, and sequentially cloned into the pBC–mCherry–TgDHFR vector at KpnI/ClaI and NotI/AscI, respectively. The plasmid was linearized using KpnI/AscI and transfected into P. berghei ANKA schizonts.

For the endogenous tagging of Atg8 (PBANKA_0504100) to generate Atg8-3XHAmCherry transgenic parasites, two fragments, F1 (630 bp) and F2 (547 bp), were amplified using primers 1560/1561 and 1562/1563, respectively, and cloned sequentially into the pBC–3XHA–mCherry–hDHFR vector at XhoI/BglII and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts.

To generate Atg4 (PBANKA_1025400) knockout parasites, double-crossover homologous recombination was used. For this, two fragments, F1 (967 bp) and F2 (556 bp), were amplified using primer sets 1655/1656 and 1333/1355. from the 5′ and 3′ UTRs of the gene, respectively, and were sequentially cloned into the pBC–GFP–hDHFR vector at XhoI/ClaI and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts.

From the paper:
'We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 (RMgm-5457) and Otu (RMgm-5458) showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling'.
Attempts to generate and select Atg4/Otu double knockout parasites were unsuccessful.
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

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1025400
Gene Model P. falciparum ortholog PF3D7_1417300
Gene productcysteine protease ATG4, putative
Gene product: Alternative nameATG4
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct 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 modificationFor the generation of Atg4/Otu double knockout parasites, the linearized targeting cassette of Atg4 with GFP reporter and hDHFR selection marker was transfected into the purified schizonts of Otu KO parasites with mCherry reporter and TgDHFR selection marker. The transfected parasites were selected by the subcutaneous injection of WR drug (Sigma-Aldrich, SML2976) for 3 consecutive days.
In the second approach, Otu–mCherry–TgDHFR and Atg4–GFP–hDHFR linearized cassettes were cotransfected into P. berghei ANKA schizonts. The transfected parasites were selected by the oral administration of pyrimethamine.

Additional information on mutants generated in this study:

For the generation of Otu (PBANKA_0515350) knockout parasites, two fragments, F1 (591 bp) and F2 (546 bp), were amplified using primer sets 1964/1965 and 1966/1967 from the 5′ and 3′ UTRs, respectively, and sequentially cloned into the pBC–mCherry–TgDHFR vector at KpnI/ClaI and NotI/AscI, respectively. The plasmid was linearized using KpnI/AscI and transfected into P. berghei ANKA schizonts.

For the endogenous tagging of Atg8 (PBANKA_0504100) to generate Atg8-3XHAmCherry transgenic parasites, two fragments, F1 (630 bp) and F2 (547 bp), were amplified using primers 1560/1561 and 1562/1563, respectively, and cloned sequentially into the pBC–3XHA–mCherry–hDHFR vector at XhoI/BglII and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts.

To generate Atg4 (PBANKA_1025400) knockout parasites, double-crossover homologous recombination was used. For this, two fragments, F1 (967 bp) and F2 (556 bp), were amplified using primer sets 1655/1656 and 1333/1355. from the 5′ and 3′ UTRs of the gene, respectively, and were sequentially cloned into the pBC–GFP–hDHFR vector at XhoI/ClaI and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts.

From the paper:
'We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 (RMgm-5457) and Otu (RMgm-5458) showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling'.
Attempts to generate and select Atg4/Otu double knockout parasites were unsuccessful.
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