scholarly journals Serial Dissection of Parasite Gene Families

2016 ◽  
Vol 84 (5) ◽  
pp. 1252-1254 ◽  
Author(s):  
David J. Bzik
Keyword(s):  
Gene Families ◽  
Genetic System ◽  
Genetic Dissection ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Calcium Dependent ◽  
Large Gene ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases ◽  
Calcium Ion Signaling

Calcium ion signaling regulates central aspects of the biology controlling stage and life cycle transitions of apicomplexan parasites. In the current issue ofInfection and Immunity, Long and coworkers (S. Long, Q. Wang, and L. D. Sibley, Infect Immun 84:1262–1273, 2016,http://dx.doi.org/10.1128/IAI.01173-15) describe a powerful genetic system enabling reliable serial genetic dissection of a large gene family encoding novel calcium-dependent protein kinases (CDPKs) that provides new insights into the roles of CDPKs duringToxoplasma gondiiinfection.

scholarly journals Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

2016 ◽  
Vol 84 (5) ◽  
pp. 1262-1273 ◽  
Author(s):  
Shaojun Long ◽  
Qiuling Wang ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Kinases ◽  
Gene Deletion ◽  
Cyst Formation ◽  
Content Type ◽  
Calcium Dependent ◽  
Targeted Gene Deletion ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially inToxoplasma gondiiwhere 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division inT. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growthin vitroor infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes inT. gondii.

scholarly journals Calcium negatively regulates secretion from dense granules in Toxoplasma gondii

2018 ◽  
Author(s):  
Nicholas J Katris ◽  
Geoffrey I McFadden ◽  
Giel G. van Dooren ◽  
Ross F Waller
Keyword(s):  
Toxoplasma Gondii ◽  
Invasion Biology ◽  
Dense Granule ◽  
Cell Penetration ◽  
Parasite Invasion ◽  
Apicomplexan Parasites ◽  
Calcium Dependent ◽  
Dense Granules ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases

AbstractApicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite’s invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+, and we show that this Ca2+-mediated response is contingent on the function of calcium-dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.

scholarly journals Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Matthew A. Child ◽  
Megan Garland ◽  
Ian Foe ◽  
Peter Madzelan ◽  
Moritz Treeck ◽  
...  
Keyword(s):  
Host Cell ◽  
Kinase Activity ◽  
Effector Proteins ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Calcium Dependent ◽  
Intracellular Parasites ◽  
Dependent Protein ◽  
Protective Environment ◽  
Calcium Dependent Protein Kinases

ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles. IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

Calcium-Dependent Protein Kinases (CDPK) in Abiotic Stress Tolerance

2018 ◽  
Vol 34 (2) ◽  
pp. 259-265 ◽  
Author(s):  
Hemant B Kardile ◽  
◽  
Vikrant ◽  
Nirmal Kant Sharma ◽  
Ankita Sharma ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Stress Tolerance ◽  
Protein Kinases ◽  
Abiotic Stress Tolerance ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases

scholarly journals Genetic mapping and transcriptomic characterization of a new fuzzless-tufted cottonseed mutant

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian-Hao Zhu ◽  
Warwick Stiller ◽  
Philippe Moncuquet ◽  
Stuart Gordon ◽  
Yuman Yuan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Agronomic Traits ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Wild Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Flanking Region ◽  
Comparative Transcriptomic Analysis

Abstract Fiber mutants are unique and valuable resources for understanding the genetic and molecular mechanisms controlling initiation and development of cotton fibers that are extremely elongated single epidermal cells protruding from the seed coat of cottonseeds. In this study, we reported a new fuzzless-tufted cotton mutant (Gossypium hirsutum) and showed that fuzzless-tufted near-isogenic lines (NILs) had similar agronomic traits and a higher ginning efficiency compared to their recurrent parents with normal fuzzy seeds. Genetic analysis revealed that the mutant phenotype is determined by a single incomplete dominant locus, designated N5. The mutation was fine mapped to an approximately 250-kb interval containing 33 annotated genes using a combination of bulked segregant sequencing, SNP chip genotyping, and fine mapping. Comparative transcriptomic analysis using 0–6 days post-anthesis (dpa) ovules from NILs segregating for the phenotypes of fuzzless-tufted (mutant) and normal fuzzy cottonseeds (wild-type) uncovered candidate genes responsible for the mutant phenotype. It also revealed that the flanking region of the N5 locus is enriched with differentially expressed genes (DEGs) between the mutant and wild-type. Several of those DEGs are members of the gene families with demonstrated roles in cell initiation and elongation, such as calcium-dependent protein kinase and expansin. The transcriptome landscape of the mutant was significantly reprogrammed in the 6 dpa ovules and, to a less extent, in the 0 dpa ovules, but not in the 2 and 4 dpa ovules. At both 0 and 6 dpa, the reprogrammed mutant transcriptome was mainly associated with cell wall modifications and transmembrane transportation, while transcription factor activity was significantly altered in the 6 dpa mutant ovules. These results imply a similar molecular basis for initiation of lint and fuzz fibers despite certain differences.

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