A Forward Genetic Screen Reveals that Calcium-dependent Protein Kinase 3 Regulates Egress in Toxoplasma

PLoS Pathogens

Tamila Garbuz¹  Moritz Treeck²  John Boothroyd²  Erin Garrison³  Emma Ehret³  Heidi Butz³  Gustavo Arrizabalaga³  Benji P. Oswald⁴  Matt Settles⁴ 

[1] Indiana University School of Medicine, Department of Pharmacology and Toxicology, Indianapolis, Indiana, United States of America;Stanford University School of Medicine, Department of Microbiology and Immunology, Stanford, California, United States of America;University of Idaho, Department of Biological Sciences, Moscow, Idaho, United States of America;University of Idaho, The Institute for Bioinformatics and Evolutionary Studies, Moscow, Idaho, United States of America

关键词: Toxoplasma gondii;    Polymerase chain reaction;    Vacuoles;    Phenotypes;    Ionophores;    Parasitic diseases;    Mutant strains;    Genetic loci;   

DOI  :  10.1371/journal.ppat.1003049

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.

【 授权许可】

CC BY