RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4855

Malaria parasite	P. berghei
Genotype
Disrupted	Gene 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 modification	The 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
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 2.34
Other information parent line	P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/Researcher	Zheng W, Cao Y
Name Group/Department	Department of Immunology, College of Basic Medical Sciences
Name Institute	China Medical University
City	Shenyang
Country	China
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Name of the mutant parasite
RMgm number	RMgm-4855
Principal name	Δpbqsox
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	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.
Oocyst	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
Sporozoite	Not tested
Liver stage	Not 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

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1455400

Gene Model P. falciparum ortholog

PF3D7_1242000

Gene product

sulfhydryl oxidase, putative

Gene product: Alternative name

Quiescin sulfhydryl oxidase, QSOX

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

To 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

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