Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii.

Human toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T. gondii cell cycle mechanism, the G1 restriction checkpoint (R-point), that operates the switch between parasite growth and differentiation. Apicomplexans lack conventional R-point regulators, suggesting adaptation of alternative factors. We showed that Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2, and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. Examination of cyclins verified the correlation of cyclin expression with growth dependence and development capacity of RH and ME49 strains. We demonstrated that rapidly dividing RH tachyzoites were dependent on TgCycP1 expression, which interfered with bradyzoite differentiation. Using the conditional knockdown model, we established that TgCycP2 regulated G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally incompetent RH tachyzoites. We tested the functions of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models. Based on functional and global gene expression analyses, we determined that TgCycP2 also regulated bradyzoite replication, while signal-induced TgCyc5 was critical for efficient tissue cyst maturation. In conclusion, we identified the central machinery of the T. gondii restriction checkpoint comprised of TgCrk2 kinase and three atypical T. gondii cyclins and demonstrated the independent roles of TgCycP1, TgCycP2, and TgCyc5 in parasite growth and development. IMPORTANCE Toxoplasma gondii is a virulent and abundant human pathogen that puts millions of silently infected people at risk of reactivation of the chronic disease. Encysted bradyzoites formed during the chronic stage are resistant to current therapies. Therefore, insights into the mechanism of tissue cyst formation and reactivation are major areas of investigation. The fact that rapidly dividing parasites differentiate poorly strongly suggests that there is a threshold of replication rate that must be crossed to be considered for differentiation. We discovered a cell cycle mechanism that controls the T. gondii growth-rest switch involved in the conversion of dividing tachyzoites into largely quiescent bradyzoites. This switch operates the T. gondii restriction checkpoint using a set of atypical and parasite-specific regulators. Importantly, the novel T. gondii R-point network was not present in the parasite's human and animal hosts, offering a wealth of new and parasite-specific drug targets to explore in the future.

Novel CRK-Cyclin Complex Controls Spindle Assembly Checkpoint in Toxoplasma Endodyogeny.

Opportunistic parasites of the Apicomplexa phylum use a variety of division modes built on two types of cell cycles that incorporate two distinctive mechanisms of mitosis: uncoupled from and coupled to parasite budding. Parasites have evolved novel factors to regulate such unique replication mechanisms that are poorly understood. Here, we have combined genetics, quantitative fluorescence microscopy, and global proteomics approaches to examine endodyogeny in Toxoplasma gondii dividing by mitosis coupled to cytokinesis. In the current study, we focus on the steps controlled by the recently described atypical Cdk-related kinase T. gondii Crk6 (TgCrk6). While inspecting protein complexes, we found that this previously orphaned TgCrk6 kinase interacts with a parasite-specific atypical cyclin, TgCyc1. We built conditional expression models and examined primary cell cycle defects caused by the lack of TgCrk6 or TgCyc1. Quantitative microscopy assays revealed that tachyzoites deficient in either TgCrk6 or the cyclin partner TgCyc1 exhibit identical mitotic defects, suggesting cooperative action of the complex components. Further examination of the mitotic structures indicated that the TgCrk6/TgCyc1 complex regulates metaphase. This novel finding confirms a functional spindle assembly checkpoint (SAC) in T. gondii. Measuring global changes in protein expression and phosphorylation, we found evidence that canonical activities of the Toxoplasma SAC are intertwined with parasite-specific tasks. Analysis of phosphorylation motifs suggests that Toxoplasma metaphase is regulated by CDK, mitogen-activated kinase (MAPK), and Aurora kinases, while the TgCrk6/TgCyc1 complex specifically controls the centromere-associated network. IMPORTANCE The rate of Toxoplasma tachyzoite division directly correlates with the severity of the disease, toxoplasmosis, which affects humans and animals. Thus, a better understanding of the tachyzoite cell cycle would offer much-needed efficient tools to control the acute stage of infection. Although tachyzoites divide by binary division, the cell cycle architecture and regulation differ significantly from the conventional binary fission of their host cells. Unlike the unidirectional conventional cell cycle, the Toxoplasma budding cycle is braided and is regulated by multiple essential Cdk-related kinases (Crks) that emerged in the place of missing conventional cell cycle regulators. How these novel Crks control apicomplexan cell cycles is largely unknown. Here, we have discovered a novel parasite-specific complex, TgCrk6/TgCyc1, that orchestrates a major mitotic event, the spindle assembly checkpoint. We demonstrated that tachyzoites incorporated parasite-specific tasks in the canonical checkpoint functions.

CCR4-associated factor 1 coordinates the expression of Plasmodium falciparum egress and invasion proteins.

Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.

Activation of HydA(DeltaEFG) requires a preformed [4Fe-4S] cluster.

The H-cluster is a complex bridged metal assembly at the active site of [FeFe]-hydrogenases that consists of a [4Fe-4S] subcluster bridged to a 2Fe-containing subcluster with unique nonprotein ligands, including carbon monoxide, cyanide, and a dithiolate ligand of unknown composition. Specific biosynthetic gene products (HydE, HydF, and HydG) responsible for the biosynthesis of the H-cluster and the maturation of active [FeFe]-hydrogenase have previously been identified and shown to be required for the heterologous expression of active [FeFe]-hydrogenase [Posewitz, M. C., et al. (2004) J. Biol. Chem. 279, 25711-25720]. The precise roles of the maturation proteins are unknown; the most likely possibility is that they are directed at the synthesis of the entire 6Fe-containing H-cluster, the 2Fe subcluster, or only the unique ligands of the 2Fe subcluster. The spectroscopic and biochemical characterization of HydA(DeltaEFG) (the [FeFe]-hydrogenase structural protein expressed in the absence of the maturation machinery) reported here indicates that a [4Fe-4S] cluster is incorporated into the H-cluster site. The purified protein in a representative preparation contains Fe (3.1 +/- 0.5 Fe atoms per HydA(DeltaEFG)) and S(2-) (1.8 +/- 0.5 S(2-) atoms per HydA(DeltaEFG)) and exhibits UV-visible spectroscopic features characteristic of iron-sulfur clusters, including a bleaching of the visible chromophore upon addition of dithionite. The reduced protein gave rise to an axial S = (1)/(2) EPR signal (g = 2.04 and 1.91) characteristic of a reduced [4Fe-4S](+) cluster. Mossbauer spectroscopic characterization of (57)Fe-enriched HydA(DeltaEFG) provided further evidence of the presence of a redox active [4Fe-4S](2+/+) cluster. Iron K-edge EXAFS data provided yet further support for the presence of a [4Fe-4S] cluster in HydA(DeltaEFG). These spectroscopic studies were combined with in vitro activation studies that demonstrate that HydA(DeltaEFG) can be activated by the specific maturases only when a [4Fe-4S] cluster is present in the protein. In sum, this work supports a model in which the role of the maturation machinery is to synthesize and insert the 2Fe subcluster and/or its ligands and not the entire 6Fe-containing H-cluster bridged assembly.

HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis.

In an effort to determine the specific protein component(s) responsible for in vitro activation of the [FeFe] hydrogenase (HydA), the individual maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum were purified from heterologous expressions in Escherichia coli. Our results demonstrate that HydF isolated from a strain expressing all three maturation proteins is sufficient to confer hydrogenase activity to purified inactive heterologously expressed HydA (expressed in the absence of HydE, HydF, and HydG). These results represent the first in vitro maturation of [FeFe] hydrogenase with purified proteins, and suggest that HydF functions as a scaffold upon which an H-cluster intermediate is synthesized.