RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4855
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1455400; Gene model (P.falciparum): PF3D7_1242000; Gene product: sulfhydryl oxidase, putative (Quiescin sulfhydryl oxidase, QSOX)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 25 August 2020, 14:36
  *RMgm-4855
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) : 32670896
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherZheng W, Cao Y
Name Group/DepartmentDepartment of Immunology, College of Basic Medical Sciences
Name InstituteChina Medical University
CityShenyang
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4855
Principal nameΔpbqsox
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 ookineteNormal numbers of gametocytes are produced. Whereas, pbqsox deletion did not affect the formation of macrogametes, it reduced the number of exflagellation centers by 28.0%. Ookinetes in the Δpbqsox line retained normal morphology, but the proportion of mature ookinetes (stage IV-VI) was reduced by 43.1%.
Consistent with the in vitro ookinete conversion result, the number of ookinetes formed in the blood bolus of mosquitoes feeding on Δpbqsox-infected mice was 60.2–64.6% lower than that with the WT parasites.
OocystA 61.8–62.8% reduction in oocyst number/midgut in mosquitoes after feeding on Δpbqsox-infected mice as compared to those fed on WT parasite-infected mice. The gross morphology of oocysts appeared normal in the Δpbqsox parasites compared to the WT
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of QSOX

Protein (function)
To adapt to  environmental changes in the blood meal inside the mosquito midgut, the malaria parasite produces antioxidant proteins such as thioredoxin-1, peroxiredoxin-1 and 1-Cys peroxiredoxin-1, to ensure survival and escape of the ookinete. However, it is not known whether other antioxidant proteins are involved in this process or how the ookinete deals with the oxidative damage of its surface proteins.
Quiescin sulfhydryl oxidase (QSOX) family enzymes, found in eukaryotes except fungi, specifically catalyze the direct and facile introduction of disulfide bonds into unfolded, reduced proteins with the reduction of a molecular oxygen, generating hydrogen peroxide.
This putative sulfhydryl oxidase protein in P. berghei (PBANKA_145540) encodes 516 amino acids, with a predicted molecular weight of 61.5 kDa. Analysis of the domain structures by SMART showed that the predicted protein contains a signal peptide, a highly conserved N-terminal Trx1 domain and C-terminal Erv/ALR domain, which is typical of QSOX enzymes. From the sequence alignment and predicted domains, Plasmodium QSOX-like proteins are different from the metazoan homologs in that it lacks the Trx2 domain completely. This feature closely resembles QSOX proteins in plants and protozoan parasites. The Plasmodium QSOX-like proteins have all three conserved CXXC motifs. Within the Trx1 domain, the most prevalent redox active Trx-CXXC motif is CGHC, whereas the corresponding PbQSOX sequence is CPAC, which is the same as in Arabidopsis. The Erv/ALR domain Erv-CXXC motif serves to communicate with the Trx-CXXC and interacts with the flavin adenine dinucleotide (FAD) co-factor, and this motif in PbQSOX is CRNC. In addition, a C-terminal CXXC motif (CT-CXXC) is also highly retained in QSOX family enzymes. Thus, the putative sulfhydryl oxidase protein in P. berghei has all these conserved features of the QSOX family, and is thus designated as the P. berghei quiescin sulfhydryl oxidase (PbQSOX)

Phenotype
Normal numbers of gametocytes are produced. Whereas, pbqsox deletion did not affect the formation of macrogametes, it reduced the number of exflagellation centers by 28.0%. Ookinetes in the Δpbqsox line retained normal morphology, but the proportion of mature ookinetes (stage IV-VI) was reduced by 43.1%.
Consistent with the in vitro ookinete conversion result, the number of ookinetes formed in the blood bolus of mosquitoes feeding on Δpbqsox-infected mice was 60.2–64.6% lower than that with the WT parasites. A 61.8–62.8% reduction in oocyst number/midgut in mosquitoes after feeding on Δpbqsox-infected mice as compared to those fed on WT parasite-infected mice. The gross morphology of oocysts appeared normal in the Δpbqsox parasites compared to the WT.

Additional information
Evidence is presented that:
-  Recombinant PbQSOX possesses thiol oxidase activity
- PbQSOX Is Expressed Primarily in Sexual Stages (gametocytes/ookinetes with higher 
- PbQSOX Is Secreted and Associated With Plasma Membranes of ookinetes (in gametocytes in cytoplasm). Also expression in sporozoites
- PbQSOX Is Required for Disulfide Bridges of Surface Proteins in Ookinetes

Other mutants

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1455400
Gene Model P. falciparum ortholog PF3D7_1242000
Gene productsulfhydryl oxidase, putative
Gene product: Alternative nameQuiescin sulfhydryl oxidase, QSOX
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo knock out pbqsox, an 824-bp upstream fragment containing the 5′ UTR was amplified from P. berghei genomic DNA using primers 5UTR-F and 5UTR-R, and cloned in the plasmid PL0034 at the HindIII and PstI sites upstream of the hdhfr cassette. Similarly, an 805-bp downstream fragment containing the 3′ UTR was amplified using primers 3UTR-F and 3UTR-R, and inserted at the XhoI and EcoRI sites downstream of the hdhfr cassette. This would replace the pbqsox protein-coding region with the hdhfr expression cassette which confers resistance to pyrimethamine. The plasmid was linearized by HindIII/EcoRI 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