The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii.

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Checkpoints of apicomplexan cell division identified in Toxoplasma gondii.

The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication, and are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.

A novel bipartite centrosome coordinates the apicomplexan cell cycle.

Apicomplexan parasites can change fundamental features of cell division during their life cycles, suspending cytokinesis when needed and changing proliferative scale in different hosts and tissues. The structural and molecular basis for this remarkable cell cycle flexibility is not fully understood, although the centrosome serves a key role in determining when and how much replication will occur. Here we describe the discovery of multiple replicating core complexes with distinct protein composition and function in the centrosome of Toxoplasma gondii. An outer core complex distal from the nucleus contains the TgCentrin1/TgSfi1 protein pair, along with the cartwheel protein TgSas-6 and a novel Aurora-related kinase, while an inner core closely aligned with the unique spindle pole (centrocone) holds distant orthologs of the CEP250/C-Nap protein family. This outer/inner spatial relationship of centrosome cores is maintained throughout the cell cycle. When in metaphase, the duplicated cores align to opposite sides of the kinetochores in a linear array. As parasites transition into S phase, the cores sequentially duplicate, outer core first and inner core second, ensuring that each daughter parasite inherits one copy of each type of centrosome core. A key serine/threonine kinase distantly related to the MAPK family is localized to the centrosome, where it restricts core duplication to once per cycle and ensures the proper formation of new daughter parasites. Genetic analysis of the outer core in a temperature-sensitive mutant demonstrated this core functions primarily in cytokinesis. An inhibition of ts-TgSfi1 function at high temperature caused the loss of outer cores and a severe block to budding, while at the same time the inner core amplified along with the unique spindle pole, indicating the inner core and spindle pole are independent and co-regulated. The discovery of a novel bipartite organization in the parasite centrosome that segregates the functions of karyokinesis and cytokinesis provides an explanation for how cell cycle flexibility is achieved in apicomplexan life cycles.

Discovery of a splicing regulator required for cell cycle progression.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

A nucleolar AAA-NTPase is required for parasite division.

Apicomplexa division involves several distinct phases shared with other eukaryote cell cycles including a gap period (G1) prior to chromosome synthesis, although how progression through the parasite cell cycle is controlled is not understood. Here we describe a cell cycle mutant that reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA-ATPase/CDC48 family member called TgNoAP1. TgNoAP1 is tightly regulated and expressed in the nucleolus during the G1/S phases. A tyrosine to a cysteine change upstream of the second AAA+ domain in the temperature sensitive TgNoAP1 allele leads to conditional protein instability, which is responsible for rapid cell cycle arrest and a primary defect in 28S rRNA processing as confirmed by knock-in of the mutation back into the parent genome. The interaction of TgNoAP1 with factors of the snoRNP and R2TP complexes indicates this protein has a role in pre-rRNA processing. This is a novel role for a cdc48-related chaperone protein and indicates that TgNoAP1 may be part of a dynamic mechanism that senses the health of the parasite protein machinery at the initial steps of ribosome biogenesis and conveys that information to the parasite cell cycle checkpoint controls.

Essential role of the conserved oligomeric Golgi complex in Toxoplasma gondii.

IMPORTANCE: The Golgi is an essential eukaryotic organelle and a major place for protein sorting and glycosylation. Among apicomplexan parasites, Toxoplasma gondii retains the most developed Golgi structure and produces many glycosylated factors necessary for parasite survival. Despite its importance, Golgi function received little attention in the past. In the current study, we identified and characterized the conserved oligomeric Golgi complex and its novel partners critical for protein transport in T. gondii tachyzoites. Our results suggest that T. gondii broadened the role of the conserved elements and reinvented the missing components of the trafficking machinery to accommodate the specific needs of the opportunistic parasite T. gondii.

Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration.

Cells require ribonucleotide reductase (RNR) activity for DNA replication. In bacteria, electrons can flow from NADPH to RNR by either a thioredoxin-reductase- or a glutathione-reductase-dependent route. Yeast and plants artificially lacking thioredoxin reductases exhibit a slow-growth phenotype, suggesting glutathione-reductase-dependent routes are poor at supporting DNA replication in these organisms. We have studied proliferation of thioredoxin-reductase-1 (Txnrd1)-deficient hepatocytes in mice. During development and regeneration, normal mice and mice having Txnrd1-deficient hepatocytes exhibited similar liver growth rates. Proportions of hepatocytes that immunostained for PCNA, phosphohistone H3 or incorporated BrdU were also similar, indicating livers of either genotype had similar levels of proliferative, S and M phase hepatocytes, respectively. Replication was blocked by hydroxyurea, confirming that RNR activity was required by Txnrd1-deficient hepatocytes. Regenerative thymidine incorporation was similar in normal and Txnrd1-deficient livers, further indicating that DNA synthesis was unaffected. Using genetic chimeras in which a fluorescently marked subset of hepatocytes was Txnrd1-deficient while others were not, we found that the multigenerational contributions of both hepatocyte types to development and to liver regeneration were indistinguishable. We conclude that, in mouse hepatocytes, a Txnrd1-independent route for the supply of electrons to RNR can fully support DNA replication and normal proliferative growth.

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.

Agonist-dependent phosphorylation of the formyl peptide receptor is regulated by the membrane proximal region of the cytoplasmic tail.

Formyl peptide receptor (FPR) is a chemoattractant G protein-coupled receptor (GPCR) involved in the innate immune response against bacteria. Receptor activation is terminated by receptor phosphorylation of two serine- and threonine-rich regions located in the distal half of the cytoplasmic tail. In this study we show that introduction of an amino acid with a bulky side chain (leucine or glutamine) adjacent to a single leucine, L320, in the membrane-proximal half of the cytoplasmic tail, significantly enhanced receptor phosphorylation, beta-arrestin1/2 translocation, and receptor endocytosis, without affecting G(i)-mediated ERK1/2 activation and release of intracellular calcium. In addition, the point mutations resulted in diminished susceptibility to trypsin, suggesting a conformation different from that of wild type FPR. Alignment of the FPR sequence with the rhodopsin sequence showed that L320 resides immediately C-terminal of an amphipathic region that in rhodopsin forms helix 8. Deletion of seven amino acids (Delta309-315) from the predicted helix 8 of FPR (G307-S319) caused reduced cell signaling as well as defects in receptor phosphorylation, beta-arrestin1/2 translocation and endocytosis. Thus, the amino acid content in the N-terminal half of the cytoplasmic tail influences the structure and desensitization of FPR.

The RESTRICTION checkpoint: a window of opportunity governing developmental transitions in Toxoplasma gondii.

The life cycle of Toxoplasma gondii is characterized by active replication alternating with periods of rest. Encysted dormant sporozoites and bradyzoites initiate active replication as tachyzoites and merozoites. Here we explore the role of the cell cycle with a focus on the canonical G1 RESTRICTION checkpoint (R-point) as the integrator governing developmental decisions in T. gondii. This surveillance mechanism, which licenses replication, creates a window of opportunity in G1 for cellular reorganization in the execution of developmental transitions. We also explore the unique status of the bradyzoite, the only life cycle stage executing both a forward (entry into the sexual cycle) and reverse (recrudescence) developmental transitions as a multipotent cell. These opposing decisions are executed through the common machinery of the RESTRICTION checkpoint.

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies.

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte-specific recombination of loxP-flanked ("floxed") alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two-color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre-dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development.

Role of the carboxyl terminal di-leucine in phosphorylation and internalization of C5a receptor.

The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane-proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK. Normal agonist-induced activation of ERK1/2 and intracellular calcium release suggested that the L318A mutation did not affect receptor signaling. Binding of GRK2 and PKCbetaII to intracellular loop 3 of C5aR in vitro indicated that mutagenesis of L318 did not affect kinase binding. Limited proteolysis with trypsin revealed a conformational difference between wild type and mutant receptor. Our studies support a model in which the L318/L319 stabilizes an amphipathic helix (Q305-R320) in the membrane-proximal region of C5aR.

The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins.

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.

Apicomplexa Cell Cycles: Something Old, Borrowed, Lost, and New.

Increased parasite burden is linked to the severity of clinical disease caused by Apicomplexa parasites such as Toxoplasma gondii, Plasmodium spp, and Cryptosporidium. Pathogenesis of apicomplexan infections is greatly affected by the growth rate of the parasite asexual stages. This review discusses recent advances in deciphering the mitotic structures and cell cycle regulatory factors required by Apicomplexa parasites to replicate. As the molecular details become clearer, it is evident that the highly unconventional cell cycles of these parasites is a blending of many ancient and borrowed elements, which were then adapted to enable apicomplexan proliferation in a wide variety of different animal hosts.

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors.

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, Tgondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.IMPORTANCE Parasites of the apicomplexan family are important causes of human disease, including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance, the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole-genome sequencing data because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of well-studied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model. An example of this approach is described here with the discovery of a putative E3 ligase/protein kinase mechanism involved in regulating chromosome replication and mitotic processes of asexual stage parasites.

Toxoplasma gondii Arginine Methyltransferase 1 (PRMT1) Is Necessary for Centrosome Dynamics during Tachyzoite Cell Division.

UNLABELLED: The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T. gondii mutants lacking PRMT1 (Δprmt1) by deletion of the PRMT1 gene. Δprmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the Δprmt::PRMT1mRFP complemented strain. Tagged PRMT1 localizes primarily in the cytoplasm with enrichment at the pericentriolar material, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, Δprmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole-genome expression profiling demonstrated differences in expression of cell-cycle-regulated genes in the Δprmt1 strain relative to the complemented Δprmt1::PRMT1mRFP and parental wild-type strains, but these changes do not correlate with a specific block in cell cycle. Although PRMT1's primary biological function was previously proposed to be methylation of histones, our studies suggest that PRMT1 plays an important role within the centrosome to ensure the proper replication of the parasite. IMPORTANCE: Apicomplexan parasites include several important pathogens, including Toxoplasma gondii, a major cause of opportunistic infections and congenital birth defects. These parasites divide using a unique form of cell division called endodyogeny that is different from those of most eukaryotes. PRMT1 is a conserved arginine methyltransferase that was thought to regulate gene expression of T. gondii by modifying histone methylation. Using genetic techniques, we show that disruption of PRMT1 affects the parasite's ability to perform accurate cell division. Our studies reveal an unexpected role for arginine methylation in centrosome biology and regulation of parasite replication.

Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development.

UNLABELLED: Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals. IMPORTANCE: Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation.

Transcript maturation in apicomplexan parasites.

The complex life cycles of apicomplexan parasites are associated with dynamic changes of protein repertoire. In Toxoplasma gondii, global analysis of gene expression demonstrates that dynamic changes in mRNA levels unfold in a serial cascade during asexual replication and up to 50% of encoded genes are unequally expressed in development. Recent studies indicate transcription and mRNA processing have important roles in fulfilling the 'just-in-time' delivery of proteins to parasite growth and development. The prominence of post-transcriptional mechanisms in the Apicomplexa was demonstrated by mechanistic studies of the critical RNA-binding proteins and regulatory kinases. However, it is still early in our understanding of how transcription and post-transcriptional mechanisms are balanced to produce adequate numbers of specialized forms that is required to complete the parasite life cycle.