Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance in Toxoplasma gondii.

The single mitochondrion of the obligate intracellular parasite Toxoplasma gondii is highly dynamic. Toxoplasma's mitochondrion changes morphology as the parasite moves from the intracellular to the extracellular environment and during division. Toxoplasma's mitochondrial dynamic is dependent on an outer mitochondrion membrane-associated protein LMF1 and its interaction with IMC10, a protein localized at the inner membrane complex (IMC). In the absence of either LMF1 or IMC10, parasites have defective mitochondrial morphology and inheritance defects. As little is known about mitochondrial inheritance in Toxoplasma, we have used the LMF1/IMC10 tethering complex as an entry point to dissect the machinery behind this process. Using a yeast two-hybrid screen, we previously identified Myosin A (MyoA) as a putative interactor of LMF1. Although MyoA is known to be located at the parasite's pellicle, we now show through ultrastructure expansion microscopy (U-ExM) that this protein accumulates around the mitochondrion in the late stages of parasite division. Parasites lacking MyoA show defective mitochondrial morphology and a delay in mitochondrion delivery to the daughter parasite buds during division, indicating that this protein is involved in organellar inheritance. Disruption of the parasite's actin network also affects mitochondrion morphology. We also show that parasite-extracted mitochondrion vesicles interact with actin filaments. Interestingly, mitochondrion vesicles extracted out of parasites lacking LMF1 pulled down less actin, showing that LMF1 might be important for mitochondrion and actin interaction. Accordingly, we are showing for the first time that actin and Myosin A are important for Toxoplasma mitochondrial morphology and inheritance.

IMC10 and LMF1 mediate mitochondrial morphology through mitochondrion-pellicle contact sites in Toxoplasma gondii.

The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular parasites. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion. Intracellular parasites lacking LMF1 fail to form the lasso-shaped mitochondrion. To identify other proteins that tether the mitochondrion of the parasite to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors localized in different cellular compartments, such as the apical end of the parasite, mitochondrial membrane and the inner membrane complex (IMC), including with the pellicle protein IMC10. Using protein-protein interaction assays, we confirmed the interaction of LMF1 with IMC10. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between the mitochondrion and the IMC in Toxoplasma. This article has an associated First Person interview with the first author of the paper.

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress.

Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.

TgDrpC, an atypical dynamin-related protein in Toxoplasma gondii, is associated with vesicular transport factors and parasite division.

Dynamin-related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP-2) complex subunit alpha-1 and a homolog of the ezrin-radixin-moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re-distribution of TgDrpC to the IMC during division is dependent on post-Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.

Targeting of tail-anchored membrane proteins to subcellular organelles in Toxoplasma gondii.

Proper protein localization is essential for critical cellular processes, including vesicle-mediated transport and protein translocation. Tail-anchored (TA) proteins are integrated into organellar membranes via the C-terminus, orienting the N-terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C-terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early-branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow-up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.

The serine/threonine phosphatases of apicomplexan parasites.

The balance between phosphorylation and de-phosphorylation, which is delicately regulated by protein kinases and phosphatases, is critical for nearly all biological processes. The Apicomplexa are a large phylum which contains various parasitic protists, including human pathogens, such as Plasmodium, Toxoplasma, Cryptosporidium and Babesia species. The diverse life cycles of these parasites are highly complex and, not surprisingly, many of their key steps are exquisitely regulated by phosphorylation. Interestingly, many of the kinases and phosphatases, as well as the substrates involved in these events are unique to the parasites and therefore phosphorylation constitutes a viable target for antiparasitic intervention. Most progress on this realm has come from studies in Toxoplasma and Plasmodium of their respective kinomes and phosphoproteomes. Nonetheless, given their likely importance, phosphatases have recently become the focus of research within the apicomplexan parasites. In this review, we concentrate on serine/threonine phosphatases in apicomplexan parasites, with the focus on comprehensively identifying and naming protein phosphatases in available apicomplexan genomes, and summarizing the progress of their functional analyses in recent years.

Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii.

Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.

A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.

The common parasite Toxoplasma gondii induces prostatic inflammation and microglandular hyperplasia in a mouse model.

BACKGROUND: Inflammation is the most prevalent and widespread histological finding in the human prostate, and associates with the development and progression of benign prostatic hyperplasia and prostate cancer. Several factors have been hypothesized to cause inflammation, yet the role each may play in the etiology of prostatic inflammation remains unclear. This study examined the possibility that the common protozoan parasite Toxoplasma gondii induces prostatic inflammation and reactive hyperplasia in a mouse model. METHODS: Male mice were infected systemically with T. gondii parasites and prostatic inflammation was scored based on severity and focality of infiltrating leukocytes and epithelial hyperplasia. We characterized inflammatory cells with flow cytometry and the resulting epithelial proliferation with bromodeoxyuridine (BrdU) incorporation. RESULTS: We found that T. gondii infects the mouse prostate within the first 14 days of infection and can establish parasite cysts that persist for at least 60 days. T. gondii infection induces a substantial and chronic inflammatory reaction in the mouse prostate characterized by monocytic and lymphocytic inflammatory infiltrate. T. gondii-induced inflammation results in reactive hyperplasia, involving basal and luminal epithelial proliferation, and the exhibition of proliferative inflammatory microglandular hyperplasia in inflamed mouse prostates. CONCLUSIONS: This study identifies the common parasite T. gondii as a new trigger of prostatic inflammation, which we used to develop a novel mouse model of prostatic inflammation. This is the first report that T. gondii chronically encysts and induces chronic inflammation within the prostate of any species. Furthermore, T. gondii-induced prostatic inflammation persists and progresses without genetic manipulation in mice, offering a powerful new mouse model for the study of chronic prostatic inflammation and microglandular hyperplasia.

Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.

Members of the family of calcium dependent protein kinases (CDPK's) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.

The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.

Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

Guanabenz repurposed as an antiparasitic with activity against acute and latent toxoplasmosis.

Toxoplasma gondii is a protozoan parasite that persists as a chronic infection. Toxoplasma evades immunity by forming tissue cysts, which reactivate to cause life-threatening disease during immune suppression. There is an urgent need to identify drugs capable of targeting these latent tissue cysts, which tend to form in the brain. We previously showed that translational control is critical during infections with both replicative and latent forms of Toxoplasma. Here we report that guanabenz, an FDA-approved drug that interferes with translational control, has antiparasitic activity against replicative stages of Toxoplasma and the related apicomplexan parasite Plasmodium falciparum (a malaria agent). We also found that inhibition of translational control interfered with tissue cyst biology in vitro. Toxoplasma bradyzoites present in these abnormal cysts were diminished and misconfigured, surrounded by empty space not seen in normal cysts. These findings prompted analysis of the efficacy of guanabenz in vivo by using established mouse models of acute and chronic toxoplasmosis. In addition to protecting mice from lethal doses of Toxoplasma, guanabenz has a remarkable ability to reduce the number of brain cysts in chronically infected mice. Our findings suggest that guanabenz can be repurposed into an effective antiparasitic with a unique ability to reduce tissue cysts in the brain.

Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs.

We have previously shown that genetic disruption of Toxoplasma gondii calcium-dependent protein kinase 3 (TgCDPK3) affects calcium ionophore-induced egress. We examined whether Plasmodium falciparum CDPK1 (PfCDPK1), the closest homolog of TgCDPK3 in the malaria parasite P. falciparum, could complement a TgCDPK3 mutant strain. PfCDPK1 is essential and plays critical roles in merozoite development, motility, and secretion. We show that expression of PfCDPK1 in the TgCDPK3 mutant strain rescues the egress defect. This phenotypic complementation requires the localization of PfCDPK1 to the plasma membrane and kinase activity. Interestingly, PfCDPK1-expressing Toxoplasma becomes more sensitive to egress inhibition by purfalcamine, a potent inhibitor of PfCDPK1 with low activity against TgCDPK3. Based on this result, we tested eight small molecules previously determined to inhibit the kinase activity of recombinant PfCDPK1 for their abilities to inhibit ionophore-induced egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we found that six drugs affected this process selectively in PfCDPK1-expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivities of dasatinib and PLX-4720 are regulated by the gatekeeper residue in the ATP binding site. Importantly, we have confirmed that the three most potent inhibitors of egress in the PfCDPK1-expressing strain effectively kill P. falciparum. Thus, we have established and validated a recombinant strain of Toxoplasma that can be used as a surrogate for the discovery and analysis of PfCDPK1-specific inhibitors that can be developed as antimalarials.

A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma.

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.

Myosin A and F-Actin play a critical role in mitochondrial dynamics and inheritance in Toxoplasma gondii.

The single mitochondrion of the obligate intracellular parasite Toxoplasma gondii is highly dynamic. Toxoplasma's mitochondrion changes morphology as the parasite moves from the intracellular to the extracellular environment and during division. Toxoplasma's mitochondrial dynamic is dependent on an outer mitochondrion membrane-associated protein LMF1 and its interaction with IMC10, a protein localized at the inner membrane complex (IMC). In the absence of either LMF1 or IMC10, parasites have defective mitochondrial morphology and inheritance defects. As little is known about mitochondrial inheritance in Toxoplasma, we have used the LMF1/IMC10 tethering complex as an entry point to dissect the machinery behind this process. Using a yeast two-hybrid screen, we previously identified Myosin A (MyoA) as a putative interactor of LMF1. Although MyoA is known to be located at the parasite's pellicle, we now show through ultrastructure expansion microscopy (U-ExM) that this protein accumulates around the mitochondrion in the late stages of parasite division. Parasites lacking MyoA show defective mitochondrial morphology and a delay in mitochondrion delivery to the daughter parasite buds during division, indicating that this protein is involved in organellar inheritance. Disruption of the parasite's actin network also affects mitochondrion morphology. We also show that parasite-extracted mitochondrion vesicles interact with actin filaments. Interestingly, mitochondrion vesicles extracted out of parasites lacking LMF1 pulled down less actin, showing that LMF1 might be important for mitochondrion and actin interaction. Accordingly, we are showing for the first time that actin and Myosin A are important for Toxoplasma mitochondrial morphology and inheritance.

The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii.

Intracellular replication is crucial for the success of apicomplexan parasites, including Toxoplasma gondii. Therefore, essential players in parasite replication present potential targets for drug development. In this study, we have characterized TgGSK, a glycogen synthase kinase homolog that plays an important role in Toxoplasma endodyogeny. We have shown that TgGSK has a dynamic localization that is concurrent with the cell cycle. In non-dividing parasites, this kinase is highly concentrated in the nucleus. However, during division, TgGSK displays a cytosolic localization, with concentration foci at the centrosomes, a key organelle involved in parasite division, and the basal end. Conditional knockdown of TgGSK determined that it is essential for the completion of the lytic cycle and proper parasite division. Parasites lacking endogenous protein levels of TgGSK exhibited defects in division synchronicity and the segregation of the nucleus and apicoplast into forming daughter cells. These phenotypes are associated with defects in centrosome duplication and fission. Global phosphoproteomic analysis determined TgGSK-dependent phosphorylation of RNA-processing, basal end, and centrosome proteins. Consistent with the putative regulation of RNA-processing proteins, global transcriptomic analysis suggests that TgGSK is needed for proper splicing. Finally, we show that TgGSK interacts with GCN5b, a well-characterized acetyltransferase with roles in transcriptional control. Conversely, GCN5b chemical inhibition results in specific degradation of TgGSK. Thus, these studies reveal the involvement of TgGSK in various crucial processes, including endodyogeny and splicing, and identify acetylation as a possible mechanism by which this essential kinase is regulated.

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.

Apicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the stability of cortical microtubules. Both co-immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 causes parasites to exhibit division defects with predominantly asynchronous divisions. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated protein SPM1 in PPKL-depleted parasites, suggesting that PPKL regulates cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway. IMPORTANCE Toxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma, like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase PPKL appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.

Apicomplexan parasites, including Toxoplasma gondii, encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of the nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the rigidity and arrangement of the cortical microtubules. Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 phenocopies lack of PPKL, strongly suggesting a functional relationship between these two signaling proteins. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated proteins SPM1 in PPKL-depleted parasites, suggesting PPKL regulates the cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway.

A Toxoplasma gondii protein with homology to intracellular type Na⁺/H⁺ exchangers is important for osmoregulation and invasion.

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na(+)/H(+) exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca(2+) concentration [Ca(2+)](i), and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings.

BACKGROUND: The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. METHODS: We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. RESULTS: Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. CONCLUSION: We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of osteogenic extracellular matrix. The coated nanosprings enhance normal human osteoblasts cellular behaviors needed for improving osseointegration of orthopedic materials. Thus, metal-coated nanosprings represent a novel biomaterial that could be exploited for improving success rates of orthopedic implant procedures.