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Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system.
- Source :
-
Parasitology research [Parasitol Res] 2016 Feb; Vol. 115 (2), pp. 697-702. - Publication Year :
- 2016
-
Abstract
- Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii.
- Subjects :
- Animals
Calcium metabolism
Drug Delivery Systems
Female
Gene Knockout Techniques
Genotype
Humans
Mice
Mice, Inbred BALB C
Phenotype
Plasmids
Protein Kinases genetics
Specific Pathogen-Free Organisms
Toxoplasma genetics
Toxoplasma pathogenicity
Virulence
CRISPR-Associated Proteins genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats genetics
Protein Kinases metabolism
Toxoplasma enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1432-1955
- Volume :
- 115
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Parasitology research
- Publication Type :
- Academic Journal
- Accession number :
- 26499803
- Full Text :
- https://doi.org/10.1007/s00436-015-4791-6