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Calcium-Dependent Signaling and Kinases in Apicomplexan Parasites
Abstract
Calcium controls many critical events in the complex life cycles of apicomplexan parasites including protein secretion, motility, and development. Calcium levels are normally tightly regulated and rapid release of calcium into the cytosol activates a family of calcium-dependent protein kinases (CDPKs), which are normally characteristic of plants. CDPKs present in apicomplexans have acquired a number of unique domain structures likely reflecting their diverse functions. Calcium regulation in parasites is closely linked to signaling by cyclic nucleotides and their associated kinases. This Review summarizes the pivotal roles that calcium- and cyclic nucleotide-dependent kinases play in unique aspects of parasite biology.
... 接区域, 而CaMLD结构域有4个EF手型区供钙离子结 合 [16] . CPK的激酶结构域和类钙调素结构域通过结合结 构域连接, AI-JD结构域与激酶结构域可以发生物理互 作, 从而作为假底物(pseudo-substrate)抑制其激酶活 性 [11] . ...
... CPK的N端具有较高比例的脯氨酸、谷氨酰胺、 丝氨酸和苏氨酸(PEST)序列, 可以进行快速的蛋白降 [16] 解 [22] . GA合成及信号通路 [25] . ...
... 左侧为N端, 右侧为C端. 共包 括4个结构域: (1) N端可变结构域(VNTD); (2) Ser/Thr蛋白激酶域 (PKD), 如图示具有一活性位点(active site); (3) 自抑制-连接域(AI-JD); (4) 类钙调素调控结构域(CaMLD), 为EF手型区[16] ...
... While cyclic nucleotide signaling enzymes are expressed in every life stage of Toxo plasma, the bulk of knowledge concerning cGMP and cAMP signaling comes from tachyzoite research. Cyclic GMP activation of TgPKG is required for tachyzoite motility to permit extracellular migration, host cell invasion, and host cell egress (24,31,(68)(69)(70)(71)(72). TgPKG regulates motility by controlling microneme secretion. ...
... This involves converting GTP to cGMP by TgGC at the apical plasma membrane (78,79) while inactivating PDEs that degrade cGMP such as TgPDE1. Activation of TgPKG likely induces the phosphory lation of a network of proteins for Ca 2+ influx or mobilization, microneme secretion, and glideosome activation (24,31,(68)(69)(70)(71)(72). Thus, cGMP signaling is required for host cell egress, extracellular migration, and host cell invasion. ...
Apicomplexa encompasses a large number of intracellular parasites infecting a wide range of animals. Cyclic nucleotide signaling is crucial for a variety of apicomplexan life stages and cellular processes. The cyclases and kinases that synthesize and respond to cyclic nucleotides (i.e., 3′,5′-cyclic guanosine monophosphate and 3′,5′-cyclic adenosine monophosphate) are highly conserved and essential throughout the parasite phylum. Growing evidence indicates that phosphodiesterases (PDEs) are also critical for regulating cyclic nucleotide signaling via cyclic nucleotide hydrolysis. Here, we discuss recent advances in apicomplexan PDE biology and opportunities for therapeutic interventions, with special emphasis on the major human apicomplexan parasite genera Plasmodium , Toxoplasma , Cryptosporidium , and Babesia . In particular, we show a highly flexible repertoire of apicomplexan PDEs associated with a wide range of cellular requirements across parasites and lifecycle stages. Despite this phylogenetic diversity, cellular requirements of apicomplexan PDEs for motility, host cell egress, or invasion are conserved. However, the molecular wiring of associated PDEs is extremely malleable suggesting that PDE diversity and redundancy are key for the optimization of cyclic nucleotide turnover to respond to the various environments encountered by each parasite and life stage. Understanding how apicomplexan PDEs are regulated and integrating multiple signaling systems into a unified response represent an untapped avenue for future exploration.
... Despite their similarity to Ca 2+ /CaM-dependent protein kinases, CDPKs are absent from mammals (42). Canonical CDPKs have four C-terminal calmodulin-like EF hands, linked by an autoinhibitory domain to the kinase domain (42,43). In plants, CDPKs control diverse stress responses, starch accumulation, cell morphology, and viability (44). ...
... CDPK2A has a long N-terminal extension, which is absent from CDPK1 and CDPK3 (42). We examined whether the N-terminal extension is required for CDPK2A function. ...
In apicomplexan parasites, the transition between replication and dissemination is regulated by fluctuations in cytosolic calcium concentrations, transduced in part by calcium-dependent protein kinases (CDPKs). We examined the role of CDPK2A in the lytic cycle of Toxoplasma , analyzing its role in the regulation of cellular processes associated with parasite motility. We used chemical-genetic approaches and conditional depletion to determine that CDPK2A contributes to the initiation of parasite motility through microneme discharge. We demonstrate that the N-terminal extension of CDPK2A is necessary for the protein’s function. Conditional depletion revealed an epistatic interaction between CDPK2A and CDPK1, suggesting that the two kinases work together to mediate motility in response to certain stimuli. This signaling module appears distinct from that of CDPK3 and protein kinase G, which also control egress. CDPK2A is revealed as an important regulator of the Toxoplasma kinetic phase, linked to other kinases that govern this critical transition. Our work uncovers extensive interconnectedness between the signaling pathways that regulate parasite motility.
IMPORTANCE
This work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium-dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating that it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive kinase alleles and conditionally depleted alleles uncovered epistasis between CDPK2A and CDPK1, implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting kinases for anti-parasitic therapies.
... The lytic cycle is essential for T. gondii's survival and encompasses five steps which include: attachment (adhesion), invasion of the host cell, parasitophorous vacuole (PV) formation, replication of the parasite within the parasitophorous vacuole, egress, and emergence of new daughter parasites after the destruction of the invaded host cell ( Figure 12). (Billker et al., 2009). Tachyzoites actively invade host cells by means of gliding motility which is based on actin-myosin motility allowing for active invasion of the host cell. ...
... The peak in calcium concentration is involved in signaling through the activation of specific kinases and microneme secretion coordination (Lourido & Moreno, 2015). The presence of several calcium-dependent kinases (CDPKs) which are usually found in plants have been demonstrated to be crucial for microneme secretion (Billker et al., 2009). TgCDPK1 has an important role in microneme exocytosis and is an essential kinase for microneme protein secretion (Lourido et al., 2010). ...
During its complex life cycle, the T. gondii parasite differentiates into distinct developmental stages. The transition between these life stages is associated with changes in the parasite's transcriptome. In Toxoplasma, it has been demonstrated that specific transcription factors of the ApiAP2 family play a role in regulating gene expression during these developmental transitions. ApiAP2 transcription factors are characterized by the presence of an AP2 domain. These AP2 TFs possess an Apetala2/ERF integrase DNA binding domain similar to those of plants. While certain ApiAP2 TFs have been demonstrated to play a role in controlling the tachyzoite to bradyzoite developmental transition, several remain unstudied. Thus, for the first part of this thesis, the role of two constitutively expressed ApiAP2 TFs, TgAP2X-10 and TgAP2III-1 was studied. The effect of TgAP2X-10 and TgAP2III-1 on bradyzoite differentiation was determined using two in vitro models. We also study the effect of TgAP2X-10 and TgAP2III-1 loss in vivo in mice and demonstrate that these proteins might be potential regulators of bradyzoite differentiation. In an attempt to continue with the characterization of ApiAP2 TFs, a third ApiAP2 TF, TgAP2IX-5 was studied. The study of TgAP2IX-5 represents the second part of this PhD project. TgAP2IX-5 was demonstrated to have a role in asexual cell cycle division of the T. gondii tachyzoite. The conditional depletion of TgAP2IX-5 blocks the progression of the cell cycle at a precise time-point when the plastid is elongated before its segregation. By using RNA-seq, we determined that TgAP2IX-5 differentially regulates hundreds of genes, a majority of which are targeted to the Inner Membrane Complex (IMC) and apical complex. ChIP-seq allowed to identify the promoters of hundreds of genes targeted by TgAP2IX-5 which are necessary for the progression of the budding cycle. In addition, TgAP2IX-5 was demonstrated to activate known bradyzoite repressors. Strikingly, we demonstrate that the re-expression of TgAP2IX-5 re-initiates cell cycle division, yet the parasite switches its mode of division from endodyogeny to endopolygeny. Further studies regarding the elucidation of the different regions of the TgAP2IX-5 protein were carried out and a novel domain was identified and shown to be essential for the function of TgAP2IX-5. In the third and final part of this PhD study, we demonstrated the crucial role of the TgPP1 phosphatase in the cell cycle. The production of the daughter cell IMC and the nuclear cycle is affected in absence of this protein. Phospho-proteomics analysis revealed that the depletion of TgPP1 results in the differential phosphorylation of several IMC proteins. Overall, this study suggests that TgPP1 is important for controlling phosphorylation events within the tachyzoite's cell cycle.
... Under stress, plants can trigger the expression of genes that are involved in multiple signal transduction pathways and further activate downstream regulatory pathways that are associated with physiological adaptation. Ca 2+ , as an important second messenger in plants, which is changed by nearly all signals about developmental, hormonal, and stresses [44,45], was activated in T. mongolicum in response to the leaf loss due to grazing. In our results, we found several genes relating to Ca 2+ -related proteins. ...
... Both CBLs and CIPKs, as the key step of Ca 2+ signal transduction, belonged to the group of serine/threonine protein kinases, that interact and regulate plant responses to various environmental stress [46,47]. After being stimulated by biotic and abiotic stress, plants form specific Ca 2+ signals in the cells that directly binds to the EF-hand domain to change the conformation of CDPKs with kinase active sites that are exposed and kinase activity is activated, which play a key role in decoding Ca 2+ signatures and transducing signals [44]. CDPKs also can interact with different kinds of pathways to control plant growth and development, hormone signal transduction, and adaptation to stress [45]. ...
Grazing, as an important land use method in grassland, has a significant impact on the morphological and physiological traits of plants. However, little is known about how the molecular mechanism of plant responds to different grazing intensities. Here, we investigated the response of Taraxacum mongolicum to light grazing and heavy grazing intensities in comparison with a non-grazing control. Using de novo transcriptome assembly, T. mongolicum leaves were compared for the expression of the different genes under different grazing intensities in natural grassland. In total, 194,253 transcripts were de novo assembled and comprised in nine leaf tissues. Among them, 11,134 and 9058 genes were differentially expressed in light grazing and heavy grazing grassland separately, with 5867 genes that were identified as co-expression genes in two grazing treatments. The Nr, SwissProt, String, GO, KEGG, and COG analyses by BLASTx searches were performed to determine and further understand the biological functions of those differentially expressed genes (DEGs). Analysis of the expression patterns of 10 DEGs by quantitative real-time RT-PCR (qRT-PCR) confirmed the accuracy of the RNA-Seq results. Based on a comparative transcriptome analysis, the most significant transcriptomic changes that were observed under grazing intensity were related to plant hormone and signal transduction pathways, carbohydrate and secondary metabolism, and photosynthesis. In addition, heavy grazing resulted in a stronger transcriptomic response compared with light grazing through increasing the of the secondary metabolism- and photosynthesis-related genes. These changes in key pathways and related genes suggest that they may synergistically respond to grazing to increase the resilience and stress tolerance of T. mongolicum. Our findings provide important clues for improving grassland use and protection and understanding the molecular mechanisms of plant response to grazing.
... Genomic searches for canonical Ca 2+ -binding domains in apicomplexans have identified several individual proteins involved in transducing and effectuating Ca 2+ signals (Farrell et al., 2012;Huet et al., 2018;McCoy et al., 2017), like kinases, phosphatases, and transporters (Hortua Triana et al., 2018;Lourido et al., 2012;Lourido et al., 2010;Luo et al., 2005;Márquez-Nogueras et al., 2021). However, many of the key signaling elements involved in fundamental Ca 2+ responses-including the channels responsible for its stimulated release-are either missing from apicomplexan genomes or have diverged beyond recognition, suggesting that eukaryotic pathogens evolved novel pathways for Ca 2+ mobilization and transduction (Billker et al., 2009;Lourido and Moreno, 2015). ...
... In apicomplexans, Ca 2+ -dependent protein kinases (CDPKs) have garnered the most attention as the only known Ca 2+ -regulated kinases in the phylum (Billker et al., 2009). These kinases possess intrinsic Ca 2+ -binding sites and do not rely on CaM-like mammalian CaMKs. ...
Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca ²⁺ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca ²⁺ -responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca ²⁺ -responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca ²⁺ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca ²⁺ -responsive proteins, such as parasite Ca ²⁺ -dependent protein kinases. Our approach also identified numerous Ca ²⁺ -responsive proteins that are not predicted to bind Ca ²⁺ , yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca ²⁺ -responsive enzyme that relocalized to the parasite apex upon Ca ²⁺ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca ²⁺ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca ²⁺ -regulated serine/threonine phosphatase activity in apicomplexan parasites.
... Signal-transducing components downstream of Ca 2+ release are largely unknown yet likely 26 essential for apicomplexan viability and virulence (Lourido and Moreno, 2015;Nagamune and Sibley, 2006). fundamental Ca 2+ responses-including the channels responsible for its stimulated release-are either missing 37 from apicomplexan genomes or have diverged beyond recognition, suggesting that eukaryotic pathogens 38 evolved novel pathways for Ca 2+ mobilization and transduction (Billker et al., 2009;Lourido and Moreno, 2015). 39 ...
... In apicomplexans, Ca 2+ -dependent protein kinases (CDPKs) have garnered the most attention as the only 40 known Ca 2+ -regulated kinases in the phylum (Billker et al., 2009). These kinases possess intrinsic Ca 2+ parasite Ca 2+ signaling field, dephosphorylation has garnered comparatively little attention (Yang and 44 Arrizabalaga, 2017). ...
Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca ²⁺ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca ²⁺ -responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca ²⁺ -responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca ²⁺ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca ²⁺ -responsive proteins, such as parasite Ca ²⁺ -dependent protein kinases. Our approach also identified numerous Ca ²⁺ -responsive proteins that are not predicted to bind Ca ²⁺ , yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP 1 ) as a Ca ²⁺ -responsive enzyme that relocalized to the parasite apex upon Ca ²⁺ store release. Conditional depletion of PP 1 revealed that the phosphatase regulates Ca ²⁺ uptake to promote parasite motility. PP 1 may thus be partly responsible for Ca ²⁺ -regulated serine/threonine phosphatase activity in apicomplexan parasites.
... CDPKs, a family of kinases that are restricted to plants and Alveolates (the taxon that includes Ciliates and Apicomplexa), are major effectors of calcium signaling in Plasmodium as well as the related apicomplexan parasite Toxoplasma gondii, and collectively regulate crucial processes, including host cell invasion, egress, growth, and sexual differentiation (Billker et al, 2004(Billker et al, , 2009Dvorin et al, 2010;Lourido et al, 2010;Sebastian et al, 2012;Gaji et al, 2014;Kumar et al, 2014Kumar et al, , 2017Morlon-Guyot et al, 2014;Treeck et al, 2014). Classical CDPKs contain a protein kinase domain and C-terminal calmodulin (CaM)-like domain, which are connected by a regulatory Junction domain (Billker et al, 2009;Ahmed et al, 2012). ...
... CDPKs, a family of kinases that are restricted to plants and Alveolates (the taxon that includes Ciliates and Apicomplexa), are major effectors of calcium signaling in Plasmodium as well as the related apicomplexan parasite Toxoplasma gondii, and collectively regulate crucial processes, including host cell invasion, egress, growth, and sexual differentiation (Billker et al, 2004(Billker et al, , 2009Dvorin et al, 2010;Lourido et al, 2010;Sebastian et al, 2012;Gaji et al, 2014;Kumar et al, 2014Kumar et al, , 2017Morlon-Guyot et al, 2014;Treeck et al, 2014). Classical CDPKs contain a protein kinase domain and C-terminal calmodulin (CaM)-like domain, which are connected by a regulatory Junction domain (Billker et al, 2009;Ahmed et al, 2012). The domain architecture of PfCDPK7 diverges from that of other members of the CDPK family. ...
PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P. falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.
... Microneme discharge plays an essential role in the egress, gliding motility, and cell invasion of apicomplexan parasites and is regulated by a set of intracellular signaling enzymes, including calcium-dependent protein kinases (32,33), phosphoinositide phospholipase C (PI-PLC) (34), and diacylglycerol (DAG) kinase (35). We evaluated whether these enzymes are also involved in the gliding motility of P. falciparum merozoites. ...
... After merozoite egress from the erythrocyte, merozoite adhesin(s) are secreted from micronemes (green) via a signaling pathway involving PI-PLC and DAG kinase (DGK) and bind to environmental substrates including the erythrocyte membrane. A pathway involving PI-PLC and Ca 2+ activates calcium-dependent protein kinases (CDPKs) and phosphorylates the components of the glideosome machinery(32)(33)(34)(35)64). Gray, nucleus; blue, rhoptries. ...
Significance
Plasmodium malaria parasites use a unique substrate-dependent locomotion, termed gliding motility, to migrate through tissues and invade cells. Previously, it was thought that the small labile invasive stages that invade erythrocytes, merozoites, use this motility solely to penetrate target erythrocytes. Here we reveal that merozoites use gliding motility for translocation across host cells prior to invasion. This forms an important preinvasion step that is powered by a conserved actomyosin motor and is regulated by a complex signaling pathway. This work broadens our understanding of the role of gliding motility and invasion in the blood and will have a significant impact on our understanding of blood stage host–pathogen interactions and parasite biology, with implications for interventions targeting erythrocyte invasion.
... Secondary messengers like Ca 2+ regulate a multitude of cellular events in apicomplexan protozoa and serve as important intermediaries during their life cycle stages. In fact, changes in Ca 2+ concentration play a pivotal role in protein secretion, motility, invasion, differentiation, and egress from infected cells [139]. Calcium-dependent protein kinases (CDPKs) are major effector molecules involved in calcium signaling pathways [140], thereby affecting abovementioned physiological processes. ...
Despite tremendous advances in the prevention and treatment of infectious diseases, only few antiparasitic drugs have been developed to date. Protozoan infections such as malaria, leishmaniasis, and trypanosomiasis continue to exact an enormous toll on public health worldwide, underscoring the need to discover novel antiprotozoan drugs. Recently, there has been an explosion of research into the antiprotozoan properties of quercetin, one of the most abundant flavonoids in the human diet. In this review, we tried to consolidate the current knowledge on the antiprotozoal effects of quercetin and to provide the most fruitful avenues for future research. Quercetin exerts potent antiprotozoan activity against a broad spectrum of pathogens such as Leishmania spp., Trypanosoma spp., Plasmodium spp., Cryptosporidium spp., Trichomonas spp., and Toxoplasma gondii. In addition to its immunomodulatory roles, quercetin disrupts mitochondrial function, induces apoptotic/necrotic cell death, impairs iron uptake, inhibits multiple enzymes involved in fatty acid synthesis and the glycolytic pathways, suppresses the activity of DNA topoisomerases, and downregulates the expression of various heat shock proteins in these pathogens. In vivo studies also show that quercetin is effective in reducing parasitic loads, histopathological damage, and mortality in animals. Future research should focus on designing effective drug delivery systems to increase the oral bioavailability of quercetin. Incorporating quercetin into various nanocarrier systems would be a promising approach to manage localized cutaneous infections. Nevertheless, clinical trials are needed to validate the efficacy of quercetin in treating various protozoan infections.
... Ca 2+ signals in apicomplexans are primarily transduced by Ca 2+ -dependent protein kinases (CDPKs) (Billker et al., 2009 ;Farrell et al., 2012 ;Garrison et al., 2012 ;Kumar et al., 2017 ;Lourido et al., 2012Lourido et al., , 2010Lourido and Moreno, 2015 ;Luo et al., 2005 ;Márquez-Nogueras et al., 2021 ;McCoy et al., 2012 ;Sebastian et al., 2012 ). CDPKs are serine/threonine protein kinases that are unique to apicomplexans and plants. ...
Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
... The key proteins associated with RBC parasite invasion include various proteins located on the merozoite surface, in micronemes, rhoptries, and dense granules [27][28][29]. When a merozoite invades RBCs, the parasite's surface proteins bind to RBC surface receptors, increasing calcium concentration in the cytoplasm of the parasite; this in turn stimulates the secretion of microneme proteins, such as TRAP, which plays an important role in the gliding motility and invasion, leading to a series of invasion steps [30][31][32][33][34]. Therefore, adhesion is the first step for Plasmodium to invade RBCs, and proteins located on the surface of merozoites play pivotal roles in the process of invasion. ...
Background
Malaria caused by Plasmodium species is a prominent public health concern worldwide, and the infection of a malarial parasite is transmitted to humans through the saliva of female Anopheles mosquitoes. Plasmodium invasion is a rapid and complex process. A critical step in the blood-stage infection of malarial parasites is the adhesion of merozoites to red blood cells (RBCs), which involves interactions between parasite ligands and receptors. The present study aimed to investigate a previously uncharacterized protein, PbMAP1 (encoded by PBANKA_1425900), which facilitates Plasmodium berghei ANKA (PbANKA) merozoite attachment and invasion via the heparan sulfate receptor.
Methods
PbMAP1 protein expression was investigated at the asexual blood stage, and its specific binding activity to both heparan sulfate and RBCs was analyzed using western blotting, immunofluorescence, and flow cytometry. Furthermore, a PbMAP1-knockout parasitic strain was established using the double-crossover method to investigate its pathogenicity in mice.
Results
The PbMAP1 protein, primarily localized to the P. berghei membrane at the merozoite stage, is involved in binding to heparan sulfate-like receptor on RBC surface of during merozoite invasion. Furthermore, mice immunized with the PbMAP1 protein or passively immunized with sera from PbMAP1-immunized mice exhibited increased immunity against lethal challenge. The PbMAP1-knockout parasite exhibited reduced pathogenicity.
Conclusions
PbMAP1 is involved in the binding of P. berghei to heparan sulfate-like receptors on RBC surface during merozoite invasion.
... Therefore, it constitutes a rich source of potential targets for drug development as demonstrated for calcium-dependent protein kinases containing calmodulin-like domains (calmodulin-like domain protein kinase; CDPK). These CDPKs are homologous to kinases commonly found in plants [80] and are essential for protein secretion, invasion, and differentiation [81]. The genome of T. gondii ME49 comprises 16 proteins annotated as CDPKs (toxoDB.org; ...
Introduction:
Toxoplasmosis constitutes a challenge for public health, animal production and welfare. So far, only a limited panel of drugs has been marketed for clinical applications. Besides classical screening, the investigation of unique targets of the parasite may lead to the identification of novel drugs.
Areas covered:
Herein, the authors describe the methodology to identify novel drug targets in Toxoplasma gondii and review the literature with a focus on the last two decades.
Expert opinion:
During the last two decades, the investigation of essential proteins of T. gondii as potential drug targets has fostered the hope to identify novel compounds for the treatment of toxoplasmosis. Despite good efficacies in vitro, only a few classes of these compounds are effective in suitable rodent models, and none has cleared the hurdle to applications in humans. This shows that target-based drug discovery is in no way better than classical screening approaches. In both cases, off-target effects and adverse side effects in hosts must be considered. Proteomics-driven analyses of parasite- and host-derived proteins that physically bind drug candidates may constitute a suitable tool to characterize drug targets, irrespectively of the drug discovery methods.
... and ΔTgPDE1/TgPDE2-mAID-3HA strains cultured without or with auxin ( Figure 22F-G). As envisaged, A23187, an ionophore activating calcium signaling downstream of cyclic nucleotides [152][153][154][155] , triggered a complete egress of the three strains irrespective of the IAA treatment ( Figure 22F). Equally, BIPPO induced almost total lysis of all samples but the auxin-treated double mutant, which responded by only 20-25% egress ( Figure 22G). ...
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in human and warm-blood organisms. Cyclic nucleotide signaling is crucial for the successful intracellular survival and replication of the parasites. Here, we dissected the physiological and biochemical importance of the essential phosphodiesterases (PDEs) in Toxoplasma gondii tachyzoite. By C-terminal tagging of 18 PDEs, we detected the expression of 11 PDEs. Immunogold staining revealed that TgPDE1, TgPDE2 and TgPDE9 are distributed throughout the parasite body, including the inner membrane complex, the apical pole, the plasma membrane, the cytosol, dense granules, and rhoptry, suggesting the spatial control of signaling within tachyzoites. Subsequently, we identified that most enzymes are notorious dual-specific phosphodiesterases, and TgPDE2 is cAMP specific differently, whilst T.gondii lacks of cGMP specific phosphodiesterase. Our enzyme kinetic data demonstrated that the highest affinity to its substrate belongs to TgPDE2, while the dual PDEs (TgPDE1, TgPDE7 and TgPDE9) have higher affinity with cGMP than cAMP. Inhibition screening of commonly-used PDE inhibitors on TgPDEs, signifying TgPDE1 as the target of BIPPO and zaprinast. Furthermore, the biological significance revealed TgPDE1 and TgPDE2 are individually necessary for parasite growth, and their loss associatively results in parasite death, implying their functional redundancy. In addition, we identified kinases and phosphatases within the TgPDE1 and TgPDE2 interactomes, which may operate the enzymatic activity via protein-protein interactions or posttranslational modifications. Collectively, our findings on subcellular localization, catalytic function, drug inhibition, and physiological relevance of major phosphodiesterases highlight the unforeseeable plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii. The data set of cAMP-binding interactors, which we disclosed in another aspect of this study, will provide valuable insight into the pervasive nature of cAMP-mediated signaling in T. gondii tachyzoites.
... Among the CDPKs that facilitate microneme protein (MIC) secretion are CDPK1 that was reported to induce secretion of MICs for invasion and egress [1,4], and CDPK3 which was reported to induce egress by inducing MIC secretion, permeabilization of the parasitophorous vacuole membrane (PVM), parasite conversion to latent stage and phosphorylation of aldolase 1 to facilitate gliding motility [5,6]. On the other hand, the CDPK7 functions to ensure the integrity of the centromeres required for the intracellular replication during cell division and growth [2]. ...
Infection with Toxoplasma gondii remains widespread among humans and animals as water, soil and food continue to serve as the major carriers of the sporulated oocyst. The infection is poorly controlled due to lack of a potent vaccine against the parasite, and the current medication presents severe side effects on the host, less efficacy on the parasite and is accompanied by the potential development of resistance by the parasite. The aim of this study was to evaluate the in vitro activities of ethanolic extract of Tinospora crispa (EETC) on protein kinases involved in the lytic cycle of T. gondii infection in Vero cell line. The EETC was obtained through the maceration of dried stem powder. Vero cells infected with the RH strain of T. gondii were used to evaluate the inhibitory effect of EETC against T. gondii calcium dependent protein kinase (CDPK) genes and microneme proteins (MIC) that are essential for host cell invasion and intracellular replication of the tachyzoite. Gene expression profiling of CDPK genes was determined through quantitative real-time PCR (qPCR) after 24 h of treatment. The expression of microneme protein was determined through western blot technique. The RT-qPCR revealed downregulation of most protein kinase (PK) genes after treatment with EETC. The expression of CDPK1, PKG, CDPK3, CDPK6, CDPK7 genes that participate in the lytic cycle of T. gondii infection was downregulated. The expression of the TgMIC1 and TgMIC2 proteins were observed to have decreased in both 4 and 24 h post-infection treatment models. This study showed that EETC contains promising drug candidates effective against T. gondii that can target the protein kinase genes involved in the lytic cycle of the parasite to prevent disease progression. HIGHLIGHTS This study explored the possible mechanism of action of phytochemicals on Toxoplasma gondii parasite-host cell invasion and intracellular replication The study evaluated the effects of ethanolic extract of Tinospora crispa Miers on protein kinase genes that are involved in host cell invasion The target protein kinase genes were downregulated which prevents the secretion of microneme proteins Inhibition of microneme secretion prevents host cell invasion and intracellular replication by Toxoplasma gondi GRAPHICAL ABSTRACT
... While the exocytosis of micronemes and rhoptries is known to be critical for parasite motility, the mechanisms linking Ca 2+ signaling to their trafficking and fusion to the plasma membrane are still unclear. Ca 2+ signals in apicomplexans are primarily transduced by Ca 2+ -dependent protein kinases (CDPKs) (Billker et al., 2009;Farrell et al., 2012;Garrison et al., 2012;Kumar et al., 2017;Lourido et al., 2012;Lourido and Moreno, 2015;Luo et al., 2005;Márquez-Nogueras et al., 2021;McCoy et al., 2012;Sebastian et al., 2012). CDPKs are serine/threonine protein kinases that are unique to apicomplexans and plants. ...
Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
... can shed lights on the discovery of novel targets for the development of drugs and vaccines against cryptosporidiosis. Calcium-associated signals have been reported to control protein secretion, parasite motility, host cell invasion, and differentiation in several apicomplexan parasites such as Toxoplasma gondii and Plasmodium (Billker et al. 2009;Moreno et al. 2011). Notably, calmodulin (CaM) is a universal regulator in almost all eukaryotic cells. ...
Cryptosporidium parvum is an important apicomplexan parasite causing severe diarrhea in both humans and animals. Calmodulin (CaM), a multifunctional and universal calcium-binding protein, contributes to the growth and development of apicomplexan parasites, but the role of CaM in C. parvum remains unknown. In this study, the CaM of C. parvum encoded by the cgd2_810 gene was expressed in Escherichia coli, and the biological functions of CpCaM were preliminarily investigated. The transcriptional level of the cgd2_810 gene peaked at 36 h post infection (pi), and the CpCaM protein was mainly located around the nucleus of the whole oocysts, in the middle of sporozoites and around the nucleus of merozoites. Anti-CpCaM antibody reduced the invasion of C. parvum sporozoites by 30.69%. The present study indicates that CpCaM is potentially involved in the growth of C. parvum. Results of the study expand our knowledge on the interaction between host and Cryptosporidium.
... Calcium-mediated signaling is used by Apicomplexans parasites to control a range of vital events, such as the secretion of proteins and the movement of cells, and this signaling process is carried out through a variety of specialized systems. These systems are noted to differ from their hosts (22). A comparative analysis of the genome of three of the most important parasites of Apicomplexans (Toxoplasma gondii, Plasmodium spp. ...
Calcium signaling and lipid metabolism are crucial in the infection processes of Apicomplexans parasites. Thus, enzymes involved in these processes can be drug targets against Apicomplexans. For example, in malaria infection, in-depth research into lipid metabolic pathways is crucial in understanding the parasite's infection cycle, particularly during its erythrocytic infection cycle,
which has been demonstrated to be a critical stage during the disease progression. Most enzymes that play critical roles in lipid synthesis and calcium signaling have been extensively studied; nonetheless, a vast knowledge gap still exists, especially on specific enzymes and their roles in the transmission and progression of the Apicomplexan parasites. Many types of infections caused by Apicomplexans are life-threatening and hard to treat. These intracellular parasites proliferate within parasitophorous vacuoles in their host cells. As the parasites multiply, they need to meet their high demand for nutrients such as amino acids and lipids. They can acquire nutrients through scavenging and biosynthesis. This review summarizes a few interesting, unique pathways in selected Apicomplexa and how such unique pathways can be targets for drugs.
... Calcium-binding proteins (CBP) include calmodulin (CAM), calmodulin neuropilin B-like proteins (CBL), and calcium-dependent protein kinases (CDPK), all have highly conserved EF-chiral structural domains (Moreno et al. 2011). At least twelve CDPKs (CDPK1, CDPK2, CDPK2A, CDPK2B, CDPK3, CDPK4, CDPK4A, CDPK5, CDPK6, CDPK7, CDPK8, and CDPK9) are expressed in T. gondii (Billker et al. 2009), and several CDPKs are involved in the parasites propagation. For example, CDPK1 plays a role in the motility, host-cell invasion, and egress of T. gondii (Lourido et al. 2010), while deletion of CDPK2 causes the accumulation of starch granules in the bradyzoite stage, leading to morphological defects and inhibition of cyst formation (Uboldi et al. 2015;Wang et al. 2018). ...
Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress from the host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated.
... It has been hypothesised that inositol triphosphate (IP 3 ), generated by phosphoinositide phospholipase C (PI-PLC)-mediated cleavage of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), opens an (as yet unidentified) IP 3 -sensitive channel to release Ca 2+ from organelles that otherwise sequester Ca 2+ during immotile replicative stages [10,11]. Once released, Ca 2+ activates a range of effectors, including a group of 'plant-like' Ca 2+ -dependent protein kinases (CDPKs) [12] and proteins involved in vesicle exocytosis [13]. PI-PLC-mediated cleavage of PIP 2 also leads to the production of diacylglycerol (DAG), which can be converted to PA by the apicomplexan-specific DAG-kinase 1 (DGK1). ...
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 Ca ²⁺ 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 Ca ²⁺ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca ²⁺ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.
... CDPK homologs were previously thought to be plant-specific, but they have now been discovered in ciliates and apicomplexan parasites (Zhang and Choi. 2001;Billker et al., 2009). The first CDPK gene appeared prior to the basal split between green plants and alveolate protists (Zhang and Choi. ...
The second messenger calcium (Ca²⁺) is a ubiquitous intracellular signaling molecule found in eukaryotic cells. In plants, the multigene family of calcium-dependent protein kinases (CDPKs) plays an important role in regulating plant growth, development, and stress tolerance. CDPKs sense changes in intracellular Ca²⁺ concentration and translate them into phosphorylation events that initiate downstream signaling processes. Several functional and expression studies on different CDPKs and their encoding genes have confirmed their multifunctional role in stress. Here, we provide an overview of the signal transduction mechanisms and functional roles of CDPKs. This review includes details on the regulation of secondary metabolites, nutrient uptake, regulation of flower development, hormonal regulation, and biotic and abiotic stress responses.
... Thus CAMKs are essentially calcium signaling regulators (Junho et al., 2020). CDPK is one of the most important protein kinase groups in the CAMK family due to its role in protein secretion, motility, invasion and egress (Billker et al., 2009). Theileria annulata proteome contains 6 CDPKs [TA16570 (TaCDPK3), TA14625 (TaCDPK4), TA16180 (TaCDPK4), TA12965 (TaCDPK5), TA08785 (TaCDPK6) and TA08840 (TaCDPK7)]. ...
Tropical theileriosis is one of the major parasitic diseases of ruminants. It is a tick-borne disease caused by an apicomplexan parasite, Theileria annulata. In the infected cells, these parasites induce phenotypes similar to cancerous cells. Among the most critical changes induced by the parasite are immortalization, hyperproliferation, and dissemination. The proliferative signal in the T. annulata transformed cells are provided by different kinases such as mitogen-activated protein kinases, SRC family kinases, casein kinase-2, and phosphatidylinositide 3-kinase. Deregulation of protein kinases in cancer is also well known. Targeting protein kinases in a cancerous cell is one of the most common methods in cancer therapy. Here, we revisited the kinome of T. annulata and studied its evolutionary relationship with other piroplasms. This analysis revealed that T. annulata kinome encodes 54 protein kinases. Based on our analysis, 12 of these 54 kinases were identified for the first time in the T. annulata proteome. Three protein kinases, TA16570, TA09820, and TA07000, had <40% identity with Bos taurus and >40% identity with the previously identified potential drug targets present in the Therapeutic Target Database (TTD). These 3 proteins were predicted to be essential for the survival of T. annulata and were selected as drug targets. Screening these drug targets in the Protein Kinase Inhibitor Database (PKID) led to shortlisting of 5 drugs. Only Dabrafenib, out of these 5 drugs, could bind to the ATP binding site (in silico) of the Calcium Dependent Protein Kinase 3 of both T. annulata and Theileria parva. Further, dabrafenib could inhibit the proliferation of T. annulata infected bovine leucocytes in 6 days proliferation assay with the IC50 value of 0.66 µM. Also, this drug did not have a cytotoxic effect on bovine peripheral blood mononuclear cells. In summary, the analysis of T. annulata kinome led to the identification of dabrafenib as a potential drug for treating theileriosis.
... We tested their impact on egress of the parental, TgPDE2-mAID-3HA and DTgPDE1/TgPDE2-mAID-3HA strains cultured without or with auxin ( Fig. 6F-G). As envisaged, A23187, an ionophore activating calcium signaling downstream of cyclic nucleotides [21][22][23][24], triggered a complete egress of the three strains irrespective of the IAA treatment (Fig. 6F). Equally, BIPPO induced almost total lysis of all samples but the auxin-treated double mutant, which responded by only 20-25 % egress (Fig. 6G). ...
Toxoplasma gondii is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten enzymes are promiscuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP. TgPDE1 and TgPDE9, with a Km of 18 μM and 31 μM, respectively, are primed to hydrolyze cGMP, whereas TgPDE2 is highly specific to cAMP (Km, 14 μM). Immuno-electron microscopy revealed various subcellular distributions of TgPDE1, 2, and 9, including in the inner membrane complex, apical pole, plasma membrane, cytosol, dense granule, and rhoptry, indicating spatial control of signaling within tachyzoites. Notably, despite shared apical location and dual-catalysis, TgPDE8 and TgPDE9 are dispensable for the lytic cycle and show no functional redundancy. In contrast, TgPDE1 and TgPDE2 are individually required for optimal growth, and their collective loss is lethal to the parasite. In vitro phenotyping of these mutants revealed a role of TgPDE2 in intracellular proliferation and of TgPDE1 in gliding motility, invasion and egress events. Moreover, our enzyme inhibition assays in conjunction with chemogenetic phenotyping underpin TgPDE1 as a target of commonly-used PDE inhibitors, BIPPO and zaprinast. Finally, we identified a retinue of TgPDE1 and TgPDE2-interacting kinases and phosphatases, possibly regulating the enzymatic activity. In conclusion, our datasets on the catalytic function, physiological relevance, subcellular localization and drug inhibition of key phosphodiesterases highlight the previously-unanticipated plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii.
... T. gondii possesses 14 genes that code for Ca 2+ -dependent protein kinases (CDPKs), which are involved in motility, invasion, replication, and egress [26][27][28] . TgCDPK3 is essential for the rapid induction of parasite egress and the establishment of chronic infection in mice, and it is the key to the virulence of the parasite in vivo 29,30 . ...
Toxoplasmosis, a common parasitic disease, is caused by Toxoplasma gondii, which infects approximately 30% of the world’s population. This obligate intracellular protozoan causes significant economic losses and poses serious public health challenges worldwide. However, the development of an effective toxoplasmosis vaccine in humans remains a challenge to date. In this study, we observed that the knockout of calcium-dependent protein kinase 3 (CDPK3) in the type II ME49 strain greatly attenuated virulence in mice and significantly reduced cyst formation. Hence, we evaluated the protective immunity of ME49Δcdpk3 as a live attenuated vaccine against toxoplasmosis. Our results showed that ME49Δcdpk3 vaccination triggered a strong immune response marked by significantly elevated proinflammatory cytokine levels, such as IFN-γ, IL-12, and TNF-α, and increased the percentage of CD4+ and CD8+ T-lymphocytes. The high level of Toxoplasma-specific IgG was maintained, with mixed IgG1/IgG2a levels. Mice vaccinated with ME49Δcdpk3 were efficiently protected against the tachyzoites of a variety of wild-type strains, including type I RH, type II ME49, Chinese 1 WH3 and Chinese 1 WH6, as well as the cysts of wild-type strains ME49 and WH6. These data demonstrated that ME49Δcdpk3 inoculation induced effective cellular and humoral immune responses against acute and chronic Toxoplasma infections with various strains and was a potential candidate to develop a vaccine against toxoplasmosis.
... CDPK-related kinases (CRKs), on the other hand, can have a degenerated Calmodulin domain with non-functional EF hands (Yuan et al. 2018). CDPK6 from the apicomplexan species Toxoplasma gondii possesses extra N-terminal EF hands, in contrast to CDPKs from plants (Billker et al. 2009). Calcineurin B-like proteins (CBLs) belong to the calmodulin superfamily and are tiny Ca 2+ -binding proteins. ...
Despite its known phytotoxicity, cadmium (Cd) is being added in most arable soils via different agricultural activities. Additionally, information is meager on the Cd-phytotoxicity mitigation strategies employing sustainable approaches. Essential plant nutrient calcium (Ca 2+) and signaling molecule nitric oxide (NO) are known as the key regulators of plant biochemical and physiological processes under stress conditions. Though there exists a close functional synergism between Ca 2+ and NO in plant stress tolerance, the role of either Ca 2+ and/or NO has been explored in plant stress tolerance in isolated reports. Given this, in addition to briefly overviewing the availability, uptake, transport and toxicity (including impact on morpho-physiological traits, photosynthesis, oxidative stress, and genotoxicity), this paper aimed to highlight the general roles, transport, and signaling of Ca 2+ and/or NO in plants, and appraises the literature available on their significance in improvements of plant growth, photosynthesis and control of oxidative stress under Cd stress. Effort was also made to present a crosstalk on Ca 2+ and NO signaling, and the significance of its outcome in plant Cd-tolerance.
... During disease development, Plasmodium growth and responses to the host environment are mainly regulated by Ca +2 dependent signaling pathways [122]. Interestingly, Plasmodium is revealed to have both conserved and evolutionarily unique Ca +2 effectors modulating protein phosphorylation, known as Ca +2 dependent protein kinases (CDPKs) [123,124]. ...
Introduction:
Malaria and tuberculosis are highly infectious diseases declared a global health emergency by the World Health Organization, and together they account for more than 1.5 million deaths worldwide each year. In the case of both malaria and tuberculosis, emergence of multidrug resistance towards frontline drugs has been reported in the recent past. Therefore, an urgent need exists for the discovery and development of novel drugs or therapies to fight these diseases.
Areas covered:
We provide a detailed overview of major infection strategies, commonly used by both the parasite Plasmodium and by Mycobacterium tubercolosis (Mtb) during disease development. We also describe selected host-directed drugs which can be repurposed to treat both malaria and tuberculosis, and co-infections.
Expert opinion:
Investigation of common infection strategies used by both Plasmodium and Mtb, during the development of disease in humans, suggests that they are potential host targets for which to develop host-directed therapies. By taking advantage of these common infection strategies, there is a chance that a number of available drugs can be repurposed to fight both malaria and tuberculosis, and their co-infections.
... Canonical CDPK proteins, such as the 70-kDa CpCDPK1, have four EF hands at the C-terminus. Therefore, the presence of additional N-terminal EF domains in CpCDPK9, predicted to be 130 kDa, is unusual [28]. The expression of these two CDPKs in native proteins appears to be different. ...
As the invasion, egress, and growth of Cryptosporidium spp. are regulated by the calcium ion, calcium-dependent protein kinases (CDPKs) are considered potential drug targets against these pathogens. In this study, we expressed CpCDPK1 of Cryptosporidium parvum encoded by the cgd3_920 gene and CpCDPK9 encoded by the the cgd7_1260 gene in Escherichia coli, and we conducted some comparative studies with quantitative PCR, immunofluorescence staining, and in vitro neutralization assays. By immunofluorescence microscopy, CpCDPK1 was expressed over the entirety of the sporozoites, while CpCDPK9 was mainly expressed in the apical region. The expression of the cgd3_920 gene was the highest at 12 h of the in vitro culture, whereas the expression of the cgd7_1260 gene peaked between 2 h and 6 h. Polyclonal antibodies against these two CpCDPK proteins had similar neutralization efficiency on C. parvum growth, reaching approximately 40%. Of the 50 candidate compounds from the molecular docking of CpCDPK1, 10 had significant in vitro anti-cryptosporidial effects, but only one inhibited enzyme activity. For CpCDPK9, five of the forty-five candidate compounds showed significant in vitro anti-cryptosporidial effects. Results obtained from this study suggest that CpCDPK1 and CpCDPK9 might function differently in C. parvum infection.
... The parasite is apt to accomplish these activities using a multitude of parasite effectors ( Figure 18). (Billker et al., 2009). The extracellular tachyzoite uses gliding motility to move on the cell surface and adhere to the membrane of the host cell after MIC protein secretion. ...
Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into the host cell. These factors are contained in specific secretory organelles, the rhoptries (ROP), micronemes (MIC) and dense granules (DG) that release their content upon host adhesion and active invasion. DG proteins (GRA) are also secreted in a so called « constitutive manner » during parasite replication and play a crucial role in modulating host responses, ensuring parasite survival and dissemination. While the molecular mechanisms regulating ROP and MIC protein release during parasite invasion have been well studied, constitutive secretion of DG remains a fully unexplored aspect of T. gondii vesicular trafficking. During this thesis, we first investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. We demonstrated that during parasite replication, TgRab11A regulates actin-dependent DG motion and stimulates the final step of their exocytosis at the parasite plasma membrane and therefore GRA protein release in the vacuolar space and host cytosol. Moreover, we demonstrated a novel function for TgRab11A in the early steps of parasite adhesion to host cells and parasite motility, and thus host cell invasion. In agreement with these findings, the secretion of the MIC2 adhesin was severely perturbed in extracellular TgRab11A-defective parasites. Strikingly, extracellular adhering and invading parasites exhibited an apically polarized and focalized accumulation of TgRab11A-positive vesicles, suggesting a role for TgRab11A in early secretory events triggered during parasite invasion. Collectively, our data revealed TgRab11A as a crucial regulator of the constitutive secretory pathway in T. gondii. In a second part of this thesis, we functionally characterized a novel TgRab11A-binding partner, containing a unique HOOK-domain, that we called TgHOOK. We found that this protein forms a stable complex with a homologue of the Fused Toes (FTS) protein and a newly identified HOOK Interacting Protein (HIP) specific to coccidian parasites. HOOK and FTS are two conserved endosomal trafficking regulators known to promote vesicle trafficking and/or fusion in other eukaryotes. In T. gondii, we found that the TgHOOK-TgFTS-HIP complex accumulates at the apical tip of parasites and promotes microneme secretion, thereby contributing to parasite invasion.
... CDPKs are key mediators of Ca 21 signaling in the malaria parasite and function at various life cycle stages (22). The unique architecture of Plasmodium CDPKs that differentiates them from mammalian calmodulin-dependent protein kinases makes CDPKs attractive drug targets (10). PfCDPK1 and PfCDPK5 are involved in invasion and egress processes, respectively, during asexual blood stage infection (16,23). ...
Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence.
... CDPKs are not available in animals and fungi, but can be found in plants and in Apicomplexa (protists) e.g. Plasmodium falciparum (Billker et al., 2009;Valmonte et al., 2014). ...
Plants have a sessile lifestyle and cannot run away when attacked. They have therefore developed various defense systems to deal with potential predators or bacteria and fungi. But heat, drought or high concentrations of salt in the water also mean stress for the plant. Calcium plays an important role as a signaling molecule in the transmission of the stress stimulus within the plant. Plants of the species Arabidopsis thaliana can be genetically modified so that their calcium concentration is visible under the microscope. These plants were stimulated by my cooperation partners with various biotic and abiotic stress stimuli and recorded with the camera. As a response, the time-lapse recordings show a locally limited increase in the intracellular calcium concentration close to the root tip, which runs in waves through the plant. The aim of my work was to analyze the wave qualitatively and quantitatively and to investigate the connection between the type of stimulus and the spatio-temporal pattern of the wave, the calcium signature. By means of modeling, I investigated how this wave can propagate at high speed across cell boundaries and how different responses to a calcium signal can be triggered at the protein level. The course of the calcium wave can be displayed as a kymograph, a space-time diagram. Using the kymograph, I managed to quantify the wave and to determine characteristic parameters such as start time, start position, and speed. The image series of the individual experiments show a high variance with respect to intensity and shape of the calcium wave. To the purpose of convenient evaluation, I designed an analysis script that quantifies the calcium wave automatically. Firstly, the root is detected in an image series. Secondly, a kymograph is created from the mean calcium concentration along the root. Finally, the calcium wave is plotted as a thin, sharply outlined line. This so-called crestline plot highlights the characteristic properties of the individual wave and allows an easy approximation of aforementioned wave parameters. The analysis of the experiments revealed that stimulation with salt leads to an immediate, short-term increase in the calcium concentration, while bacteria or fungi trigger a delayed calcium wave that propagates at a speed of a few µm/s. It is known from the literature that after stimulation of the root with elevated levels of salt, the wave moves through the plant at a high speed of around 400 µm/s. A combined signal transmission of intracellular calcium and extracellular reactive oxygen species (ROS) is suggested as a possible explanation. Together with my cooperation partner, I designed a corresponding mathematical model and adapted it to the different cell sizes in the root tip. We were able to show that wave propagation based only on intracellular calcium is sufficient for the much slower calcium wave after stimulation with bacteria or fungi. However, the calcium wave after stimulation with salt requires additional components. Based on a simulation, I was able to demonstrate that the plasmodesmata, the narrow tubes between adjacent cells, slow down the expansion of the wave considerably and should not be ignored. The plant can use calcium-dependent protein kinases (CPKs) to decode the calcium signal and translate it into protein phosphorylations as a starting point for further reactions. For example, the closing process of the stomata is based on the calcium-regulated activation of CPKs. Based on experimental data, I developed a CPK protein model for different CPKs. In a computer simulation, I coupled calcium time series from a stimulation experiment of guard cells and epidermal cells to my protein model and examined the activity of the CPK proteins. I was able to show that by varying the calcium signal, different CPK proteins can be addressed and the stress response of the plant can be adapted to the type of stimulation.
... Herein, we extend the number of genes upregulated during B. bovis sexual stage development to include cdpk4, ttl, and mt. CDPK4, a serine/threonine kinase, is an enzyme that plays an important role in intracellular calcium signaling in plants, green algae, ciliates, and apicomplexan parasites [31]. In malaria parasites, CDPK4 is known to be involved in stage-specific cellular responses to calcium signaling transduction pathways, cell cycle regulation, and life cycle progression [32]. ...
Background
Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies.
Results
In this study, we induced B . bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B . bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B . bovis calcium-dependent protein kinase 4 ( cdpk4 ), tubulin-tyrosine ligase ( ttl ), and methyltransferase ( mt ) genes by in vitro induced sexual stages and during parasite development within tick midguts.
Conclusions
Similar to other apicomplexan parasites, it is likely that B . bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4 , ttl , and mt genes by both B . bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis.
Graphical abstract
... These signaling molecules require secondary messengers such as phosphatidylinositol phospholipase C (PI-PLC) (Bullen and Soldati-Favre, 2016), cADPR cyclase and hydrolase to mediate Ca 2+ release from the ER, however, there is no data on the existence of secondary messenger receptors (Nagamune et al., 2008;Jones et al., 2009;Singh et al., 2010). It has also been found that calcium-dependent protein kinase 1 (TgCDPK1) (Lourido et al., 2010(Lourido et al., , 2012Lourido and Moreno, 2015;Brochet and Billker, 2016) and cyclic GMP (cGMP) activated protein kinase G (PKG) (Brown et al., 2017) which play a role in the downstream pathway of Ca 2+ , specifically regulating the secretion of MICs until the end of invasion, and after that within the host cell, T. gondii ER uptakes Ca 2+ to store via SERCA-type Ca 2+ -ATPases, in order to use in the process of egress and the next invasion (Figure 2; Billker et al., 2009;Lourido and Moreno, 2015). Ca 2+ -mobilizing agents such as calcium ionophores, thapsigargin, ethanol have been shown to stimulate the increase of intracellular calcium levels in T. gondii FIGURE 2 | Ca 2+ -dependent secretory regulation pathway of TgMIC1/4/6. ...
Toxoplasma gondii microneme is a specialized secretory organelle that discharges its contents at the apical tip of this apicomplexan parasite in a sequential and regulated manner. Increasing number of studies on microneme proteins (MICs) have shown them as a predominant and important role in host cell attachment, invasion, motility and pathogenesis. In this review, we summarize the research advances in one of the most important MICs complexes, TgMIC1/4/6, which will contribute to improve the understanding of the molecular mechanism of T. gondii infection and provide a theoretical basis for the effective control against T. gondii.
Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum . We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte- a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which plays an important role in parasite invasion.
Apicomplexan parasites use Ca ²⁺ -regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca ²⁺ -dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii , consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
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.
Signalling pathways in malaria parasite remain poorly defined and major reason for this is the lack of understanding of the function of majority of parasite protein kinases and phosphatases in parasite signalling and its biology. In the present study, we have elucidated the function of Protein Kinase 2 (PfPK2), which is known to be indispensable for the survival of human malaria parasite Plasmodium falciparum . We demonstrate that it is involved in the invasion of host erythrocytes, which is critical for establishing infection. In addition, PfPK2 may also be involved in the maturation of the parasite post-invasion. PfPK2 regulates the release of microneme proteins like Apical Membrane Antigen 1 (AMA1), which facilitates the formation of Tight Junction between the merozoite and host erythrocyte-a key step in the process of invasion. Comparative phosphoproteomics studies revealed that PfPK2 may be involved in regulation of several key proteins involved in invasion and signalling. Furthermore, PfPK2 regulates the generation of cGMP and the release of calcium in the parasite, which are key second messengers for the process of invasion. These and other studies have shed light on a novel signalling pathway in which PfPK2 acts as an upstream regulator of important cGMP-calcium signalling, which is plays an important role in parasite invasion.
Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here we developed a genetically encoded FRET (Förster resonance energy transfer)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.
Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.
Cryptosporidium species has been identified as an important pediatric diarrheal pathogen in resource-limited countries, particularly in very young children (0-24 months). However, the only available drug (nita-zoxanide) has limited efficacy and can only be prescribed in a medical setting to children older than one year. Many drug development projects have started to investigate new therapeutic avenues. Cryptosporidium's unique biology is challenging for the traditional drug discovery pipeline and requires novel drug screening approaches. Notably, in recent years, new methods of oocyst generation, in vitro processing, and continuous three-dimensional cultivation capacities have been developed. This has enabled more physiologically pertinent research assays for inhibitor discovery. In a short time, many great strides have been made in the development of anti-Cryptosporidium drugs. These are expected to eventually turn into clinical candidates for cryptosporidiosis treatment in the future. This review describes the latest development in Cryptosporidium biology , genomics, transcriptomics of the parasite, assay development, and new drug discovery.
Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modeling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri (C. tyzzeri) infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23(recombinant P23), GST6P-1(expressed by pGEX-6P-1 transfected E. coli) , GST4T-1(expressed by pGEX-4T-1 transfected E. coli) , glutathione (GSH), adjuvant, and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similart to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.
Apicomplexan parasites require motility to actively infect host cells and cause disease. Cyclic nucleotide signaling governs apicomplexan motility, but it is unclear how cyclic nucleotide levels are maintained in these parasites.
Plasmodium spp. infection remains as a major global health problem, being annually responsible for a large number of deaths. Disease eradication remains a major challenge, although improvement on malaria elimination net has been seen over the past decade. Understanding parasite cell biology and its interactions with the human host, nevertheless, is essential to improve diagnostics and treatment. In this regard, existing knowledge of different aspects of malaria infection, replication, survival and transmission has provided key information to define the current strategies applied to treat different forms of malaria. In addition to its importance in the context of global health, Plasmodium spp. represent interesting cell biology models, being used as excellent tools to understand host-parasite interaction. Such interaction is based on specific balances between the parasite and different host tissues as well as on sophisticated mechanisms of evasion from the host immune system. Malaria parasites manage to migrate through different environments, develop inside highly differentiated host cells and nevertheless are successful on establishing the infection, in some cases leading the host organism to a severe disease. Understanding this complex system represents a bottleneck on malaria control and eradication. In this chapter we will explore different aspects of parasite development inside vertebrates, more specifically the human host. The basic concepts and main advances that molded our current understanding of the structure, biochemistry, proteomics and transcriptomics of the parasite as well as its interaction with the host will be here discussed. More than answers, the idea is to point to some of the many open questions regarding Plasmodium spp. development and discuss how multidisciplinary approaches and new tools have helped to understand the biology of this peculiar parasite.
Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these are connected is largely not known. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of ΔCDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates while the other PDEs are cGMP specific; two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role of PDE1 and PDE2 for growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that potentiates signalling during egress and links several signalling pathways together.
Recent studies suggest that immune-induced alternative splice variants of the Arabidopsis thaliana Ca2+-dependent protein kinase (CDPK) AtCPK28 may result in signal attenuation. We put forward the hypothesis that expression of alternative truncated variants may be a broadly conserved regulatory mechanism of CDPKs throughout the green lineage.
An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP–aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-Å and 2.7-Å structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix–loop–helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite.
• actin
• Apicomplexa
• cell invasion machinery
• gliding motility
• induced fit
A small, heat stable chromophore extracted from mosquitoes has recently been implicated as the signal that induces mating
of Plasmodium, the malaria parasite. We have used high resolution electrospray mass spectrometry to determine that this gamete activation
factor (GAF) has a m/z = 205.0450, suggesting a molecular species composition of C10H7NO4. Xanthurenic acid (XA), a product of tryptophan catabolism, was determined to have an elemental composition, ultraviolet
absorbance maxima, and mass spectrum consistent with those characteristics of GAF. XA activated gametogenesis of Plasmodium gallinaceum and P. falciparum in vitro at concentrations lower than 0.5 μm in saline buffered to pH 7.4. A structural analog of XA, kynurenic acid (C10H6NO3), also activated gametogenesis but only at higher concentrations and with less effect. We propose that XA is GAF. This is
the first evidence that XA has induction activity.
Recent studies have demonstrated that calcium-dependent protein kinases (CDPKs) are used by calcium to regulate a variety
of biological processes in the malaria parasite Plasmodium. CDPK4 has emerged as an important enzyme for parasite development, because its gene disruption in rodent parasite Plasmodium berghei causes major defects in sexual differentiation of the parasite (
Billker, O., Dechamps, S., Tewari, R., Wenig, G., Franke-Fayard, B., and Brinkmann, V. (2004) Cell 117, 503-514
). Despite these findings, it is not very clear how PfCDPK4 or any other PfCDPK is regulated by calcium at the molecular level.
We report the biochemical characterization and elucidation of molecular mechanisms involved in the regulation of PfCDPK4.
PfCDPK4 was detected on gametocyte periphery, and its activity in the parasite was regulated by phospholipase C. Even though
the Junction Domain (JD) of PfCDPK4 shares moderate sequence homology with that of the plant CDPKs, it plays a pivotal role
in PfCDPK4 regulation as previously reported for some plant CDPKs. The regions of the J-domain involved in interaction with
both the kinase domain and the calmodulin-like domain were mapped. We propose a model for PfCDPK regulation by calcium, which
may also prove useful for design of inhibitors against PfCDPK4 and other members of the PfCDPK family.
Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.
Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe Toxoplasma perforin-like protein 1 (TgPLP1), a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore formation. After intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to rapidly permeabilize the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture but resulted in a reduction greater than five orders of magnitude of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.
Toxoplasma gondii motility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light
chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble
complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all
proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear.
We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated
on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser163 and Ser167. The importance of these phosphorylation events was determined by mutation of Ser163 and Ser167 to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser163 and Ser167 to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA
and MLC1. Mutation of Ser163 and Ser167 to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser163 and Ser167 to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that
phosphorylation of Ser163 and Ser167 in GAP45 controls the final step in assembly of the myosin XIV motor complex.
Calcium-dependent protein kinases (CDPKs) of Apicomplexan parasites are crucial for the survival of the parasite throughout its life cycle. CDPK1 is expressed in the asexual blood stages of the parasite, particularly late stage schizonts. We have identified two substrates of Plasmodium falciparum CDPK1: myosin A tail domain-interacting protein (MTIP) and glideosome-associated protein 45 (GAP45), both of which are components of the motor complex that generates the force required by the parasite to actively invade host cells. Indirect immunofluorescence shows that CDPK1 localizes to the periphery of P. falciparum merozoites and is therefore suitably located to act on MTIP and GAP45 at the inner membrane complex. A proportion of both GAP45 and MTIP is phosphorylated in schizonts, and we demonstrate that both proteins can be efficiently phosphorylated by CDPK1 in vitro. A primary phosphorylation of MTIP occurs at serine 47, whereas GAP45 is phosphorylated at two sites, one of which could also be detected in phosphopeptides purified from parasite lysates. Both CDPK1 activity and host cell invasion can be inhibited by the kinase inhibitor K252a, suggesting that CDPK1 is a suitable target for antimalarial drug development.
An unusual protein kinase gene, termed PfCPK, was isolated from Plasmodium falciparum. The gene, which contains five exons and four introns, encodes a product with a predicted length of 524 amino acids. The amino-terminal segment of the predicted protein contains all of the conserved sequences characteristic of a protein kinase catalytic domain and has a high homology to several protein serine-threonine kinase subfamilies (30-41% amino acid identities). These subfamilies include calcium/calmodulin-dependent protein kinases, calcium-dependent protein kinase, ribosomal S6 protein kinase, cyclic nucleotide-dependent protein kinases, protein kinase C, and the yeast SNF1 subfamily. All of these protein kinases are relatively close in the phylogeny tree and within the kinase catalytic domains have about 35% amino acid identities to each other, suggesting that PfCPK is also in this region of the phylogeny tree. An unusual feature of PfCPK is that its carboxyl-terminal segment displays homology to the EF hand calcium-binding proteins, for example 34% amino acid identity to chicken fast skeletal muscle troponin C and 35% amino acid identity to human calmodulin. Like troponin Cs and calmodulins, PfCPK also contains four EF hand calcium-binding motifs. Furthermore, the four introns in the PfCPK gene are all located in the carboxyl-terminal putative EF hand calcium-binding region (EF hand calcium-binding proteins from higher eukaryotes generally contain multiple introns). This combination of a protein kinase and an EF hand calcium-binding protein in a single polypeptide implies that PfCPK may be directly activated by calcium. Constructs containing the full-length PfCPK cDNA have been expressed in Escherichia coli at a high level to generate a 60-kDa recombinant protein. Compared with similar fractions from control cells, the fraction containing PfCPK recombinant protein exhibited an elevated protein kinase activity which was Ca(2+)-dependent.
During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.
A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi. These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme. Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity. We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms.
Many protozoans of the phylum Apicomplexa are invasive parasites that exhibit a substrate-dependent gliding motility. Plasmodium (malaria) sporozoites, the stage of the parasite that invades the salivary glands of the mosquito vector and the liver of the vertebrate host, express a surface protein called thrombospondin-related anonymous protein (TRAP) that has homologs in other Apicomplexa. By gene targeting in a rodent Plasmodium, we demonstrate that TRAP is critical for sporozoite infection of the mosquito salivary glands and the rat liver, and is essential for sporozoite gliding motility in vitro. This suggests that in Plasmodium sporozoites, and likely in other Apicomplexa, gliding locomotion and cell invasion have a common molecular basis.
Developmentally arrested malarial gametocytes undergo gamete formation in the mosquito midgut immediately after ingestion of the infected bloodmeal. In the rodent malaria parasite Plasmodium berghei male gametogenesis (exflagellation) can be induced in vitro by a temperature decrease (from 39 degrees C in the vertebrate host to 20 degrees C) and a concomitant pH increase (from 7.3 in mouse blood to 8.0). We report the presence of additional Gametocyte Activating Factor(s) (GAF) present in Anopheles stephensi tissue extracts, which induce both male and female gametogenesis at the otherwise nonpermissive pH of 7.3 in vitro but are unable to overcome the low temperature requirement. All constituent cellular events of microgametogeneis studied here are induced by the same triggers in vitro. A temperature decrease is also required for exflagellation in the mosquito midgut. The possible role of GAF as a second obligatory natural trigger of gametogenesis is discussed.
Abscisic acid (ABA) is the primary hormone that mediates plant responses to stresses such as cold, drought, and salinity.
Single-cell microinjection experiments in tomato were used to identify possible intermediates involved in ABA signal transduction.
Cyclic ADP–ribose (cADPR) was identified as a signaling molecule in the ABA response and was shown to exert its effects by
way of calcium. Bioassay experiments showed that the amounts of cADPR in Arabidopsis thalianaplants increased in response to ABA treatment and before ABA-induced gene expression.
Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.
The role of calcium-dependent protein kinases in the invasion of Toxoplasma gondii into its animal host cells was analyzed. KT5926, an inhibitor of calcium-dependent protein kinases in other systems, is known
to block the motility of Toxoplasma tachyzoites and their attachment to host cells. In vivo, KT5926 blocks the phosphorylation of only three parasite proteins, and in parasite extracts only a single KT5926-sensitive
protein kinase activity was detected. This activity was calcium-dependent but did not require calmodulin. In a search for
calcium-dependent protein kinases in Toxoplasma, two members of the class of calmodulin-like domain protein kinases (CDPKs) were detected. TgCDPK2 was only expressed at
the mRNA level in tachyzoites, but no protein was detected. TgCDPK1 protein was expressed in Toxoplasmatachyzoites and cofractionated precisely with the peak of KT5926-sensitive protein kinase activity. TgCDPK1 kinase activity
was calcium-dependent but did not require calmodulin or phospholipids. TgCDPK1 was found to be inhibited effectively by KT5926
at concentrations that block parasite attachment to host cells.In vitro, TgCDPK1 phosphorylated three parasite proteins that migrated identical to the three KT5926-sensitive phosphoproteins detected
in vivo. Based on these observations, a central role is suggested for TgCDPK1 in regulatingToxoplasma motility and host cell invasion.
The ability of intracellular parasites to monitor the viability of their host cells is essential for their survival. The protozoan
parasite Toxoplasma gondii actively invades nucleated animal cells and replicates in their cytoplasm. Two to 3 days after infection, the parasite-filled
host cell breaks down and the parasites leave to initiate infection of a new cell. Parasite egress from the host cell is triggered
by rupture of the host plasma membrane and the ensuing reduction in the concentration of cytoplasmic potassium. The many other
changes in host cell composition do not appear be used as triggers. The reduction in the host cell [K+] appears to activate a phospholipase C activity inToxoplasma that, in turn, causes an increase in cytoplasmic [Ca2+] in the parasite. The latter appears to be necessary and sufficient for inducing egress, as buffering of cytoplasmic Ca2+ blocks egress and calcium ionophores circumvent the need for a reduction of host cell [K+] and parasite phospholipase C activation. The increase in [Ca2+]C brings about egress by the activation of at least two signaling pathways: the protein kinase TgCDPK1 and the calmodulin-dependent
protein phosphatase calcineurin.
The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1) inhibits the growth of Eimeria spp. bothin vitro and in vivo. The molecular target of Compound 1 was identified as cGMP-dependent protein kinase (PKG) using a tritiated analogue to purify
a ∼120-kDa protein from lysates of Eimeria tenella. This represents the first example of a protozoal PKG. Cloning of PKG from several Apicomplexan parasites has identified a
parasite signature sequence of nearly 300 amino acids that is not found in mammalian or DrosophilaPKG and which contains an additional, third cGMP-binding site. Nucleotide cofactor regulation of parasite PKG is remarkably
different from mammalian enzymes. The activity of both native and recombinantE. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from
a single copy gene. NH2-terminal sequence of the soluble isoform of PKG is consistent with alternative translation initiation within the open reading
frame of the enzyme. A larger, membrane-associated isoform corresponds to the deduced full-length protein sequence. Compound
1 is a potent inhibitor of both soluble and membrane-associated isoforms of native PKG, as well as recombinant enzyme, with
an IC50 of <1 nm.
The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elements in these parasites.
Calcium-mediated microneme secretion in Toxoplasma gondii is stimulated by contact with host cells, resulting in the discharge of adhesins that mediate attachment. The intracellular source of calcium and the signaling pathway(s) triggering release have not been characterized, prompting our search for mediators of calcium signaling and microneme secretion in T. gondii. We identified two stimuli of microneme secretion, ryanodine and caffeine, which enhanced release of calcium from parasite intracellular stores. Ethanol, a previously characterized trigger of microneme secretion, stimulated an increase in parasite inositol 1,4,5-triphosphate, implying that this second messenger may mediate intracellular calcium release. Consistent with this observation, xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, inhibited microneme secretion and blocked parasite attachment and invasion of host cells. Collectively, these results suggest that T. gondii possess an intracellular calcium release channel with properties of the inositol 1,4,5-triphosphate/ryanodine receptor superfamily. Intracellular calcium channels, previously studied almost exclusively in multicellular animals, appear to also be critical to the control of parasite calcium during the initial steps of host cell entry.
Obligate intracellular apicomplexan parasites rely on gliding motion powered by their actomyosin system to disperse throughout tissues and to penetrate host cells. Toxoplasma gondii myosin A has been implicated in this process, but direct proof has been lacking. We designed a genetic screen to generate a tetracycline-inducible transactivator system in T. gondii. The MyoA gene was disrupted in the presence of a second regulatable copy of MyoA. Conditional removal of this myosin caused severe impairment in host cell invasion and parasite spreading in cultured cells, and unambiguously established the pathogenic function of this motor in an animal model.
The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo
activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1.
The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this
class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic
efficacy of compound 1.
We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.
Most cultivated and characterized eukaryotes can be confidently assigned to one of eight major groups. After a few false starts, we are beginning to resolve relationships among these major groups as well. However, recent developments are radically revising this picture again, particularly (i) the discovery of the likely antiquity and taxonomic diversity of ultrasmall eukaryotes, and (ii) a fundamental rethinking of the position of the root. Together these data suggest major gaps in our understanding simply of what eukaryotes are or, when it comes to the tree, even which end is up.
Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.
Gliding motility and host cell invasion by apicomplexan parasites are empowered by an acto-myosin motor located underneath the parasite plasma membrane. The motor is connected to host cell receptors through trans-membrane invasins belonging to the thrombospondin-related anonymous protein (TRAP) family. A recent study indicates that aldolase bridges the cytoplasmic tail of MIC2, the homologous TRAP protein in Toxoplasma, and actin. Here, we confirm these unexpected findings in Plasmodium sporozoites and identify conserved features of the TRAP family cytoplasmic tail required to bind aldolase: a subterminal tryptophan residue and two noncontiguous stretches of negatively charged amino acids. The aldolase substrate and other compounds that bind to the active site inhibit its interaction with TRAP and with F-actin, suggesting that the function of the motor is metabolically regulated. Ultrastructural studies in salivary gland sporozoites localize aldolase to the periphery of the secretory micronemes containing TRAP. Thus, the interaction between aldolase and the TRAP tail takes place during or preceding the biogenesis of the micronemes. The release of their contents in the anterior pole of the parasite upon contact with the target cells should bring simultaneously aldolase, TRAP and perhaps F-actin to the proper subcellular location where the motor is engaged.
More than 20% of the human genome encodes proteins involved in transmembrane and intracellular signaling pathways. The cAMP-protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells and is involved in regulation of cellular functions in almost all tissues in mammals. Various extracellular signals converge on this signal pathway through ligand binding to G protein-coupled receptors, and the cAMP-PKA pathway is therefore tightly regulated at several levels to maintain specificity in the multitude of signal inputs. Ligand-induced changes in cAMP concentration vary in duration, amplitude, and extension into the cell, and cAMP microdomains are shaped by adenylyl cyclases that form cAMP as well as phosphodiesterases that degrade cAMP. Different PKA isozymes with distinct biochemical properties and cell-specific expression contribute to cell and organ specificity. A kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP-PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein signaling complexes that serve as crossroads for different paths of cell signaling. Targeting of PKA and integration of a wide repertoire of proteins involved in signal transduction into complex signal networks further increase the specificity required for the precise regulation of numerous cellular and physiological processes.
Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
Numerous stimuli can alter the Ca2+concentration in the cytoplasm, a factor common to many physiological responses in plant and animal cells. Calcium-binding proteins decode information contained in the temporal and spatial patterns of these Ca2+ signals and bring about changes in metabolism and gene expression. In addition to calmodulin, a calcium-binding protein found in all eukaryotes, plants contain a large family of calcium-binding regulatory protein kinases. Evidence is accumulating that these protein kinases participate in numerous aspects of plant growth and development.
SYNOPSIS
Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4 C for several days. P. berghei, P. cynomolgi , and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry‐microneme complex may play a role in sporozoite motility.
Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii.
The induction mechanism of gamete formation (gametogenesis) in a rodent malaria parasite, Plasmodium berghei, was investigated using Ca2+ antagonists, protein kinase inhibitors and amiloride, an inhibitor of monovalent cation/H+ exchange. Treatment with 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, a Ca2+ release inhibitor) and W-7/W-66 (calmodulin inhibitors) blocked formation of male gametes by inhibiting DNA synthesis from 1.5C to 8C level. In contrast, inhibitors of cAMP/cGMP-dependent protein kinases such as H-8, H-87, H-89 and staurosporine also ceased the development of gametocytes, but DNA synthesis in male gametocytes occurred as in the controls. Electron microscopy revealed that male gametocytes treated with TMB-8 and W-7 failed to enlarge nuclei and to form axonemes in the cytoplasm. In female gametocytes, treatment with both Ca2+ antagonists resulted in a dramatic morphological change in the endoplasmic reticulum (ER), which is thought to be a Ca2+ store. The ER network condensed near nuclei and was laminated by the abnormal attachment of ribosomes between two ER membranes. On the other hand, male gametocytes treated with protein kinase inhibitors or amiloride had enlarged nuclei and axonemes, but failed to develop further. The ER network in female gametocytes treated with these inhibitors was similar to that in the controls.
Malaria is transmitted from vertebrate host to mosquito vector by mature sexual blood-living stages called gametocytes. Within seconds of ingestion into the mosquito bloodmeal, gametocytes undergo gametogenesis. Induction requires the simultaneous exposure to at least two stimuli in vitro: a drop in bloodmeal temperature to 5 degrees C below that of the vertebrate host, and a rise in pH from 7.4 to 8.0-8.2. In vivo the mosquito bloodmeal has a pH of between 7.5 and 7.6. It is thought that in vivo the second inducer is an unknown mosquito-derived gametocyte-activating factor. Here we show that this factor is xanthurenic acid. We also show that low concentrations of xanthurenic acid can act together with pH to induce gametogenesis in vitro. Structurally related compounds are at least ninefold less effective at inducing gametogenesis in vitro. In Drosophila mutants with lesions in the kynurenine pathway of tryptophan metabolism (of which xanthurenic acid is a side product), no alternative active compound was detected in crude insect homogenates. These data could form the basis of the rational development of new methods of interrupting the transmission of malaria using drugs or new refractory mosquito genotypes to block parasite gametogenesis.
We describe here an efficient method for conditional gene inactivation in malaria parasites that uses the Flp/FRT site-specific recombination system of yeast. The method, developed in Plasmodium berghei, consists of inserting FRT sites in the chromosomal locus of interest in a parasite clone expressing the Flp recombinase via a developmental stage-specific promoter. Using promoters active in mosquito midgut sporozoites or salivary gland sporozoites to drive expression of Flp or its thermolabile variant, FlpL, we show that excision of the DNA flanked by FRT sites occurs efficiently at the stage of interest and at undetectable levels in prior stages. We applied this technique to conditionally silence MSP1, a gene essential for merozoite invasion of erythrocytes. Silencing MSP1 in sporozoites impaired subsequent merozoite formation in the liver. Therefore, MSP1 plays a dual role in the parasite life cycle, acting both in liver and erythrocytic parasite stages.
Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Prior in vitro studies implicated aldolase, a tetrameric glycolytic enzyme, in coupling actin filaments to the parasite's surface adhesin microneme protein 2 (MIC2). Here, we test the essentiality of this interaction in host cell invasion. Based on in vitro studies and homology modeling, we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity. We tested these mutants by complementing a conditional knockout of TgALD1. Mutations that affected glycolysis also reduced motility. Mutants only affecting binding to MIC2t had no motility phenotype, but were decreased in their efficiency of host cell invasion. Our studies demonstrate that aldolase is not only required for energy production but is also essential for efficient host cell invasion, based on its ability to bridge adhesin-cytoskeleton interactions in the parasite.
The pre-erythrocytic (PE) phase of malaria infection, which extends from injection of sporozoites into the skin to the release of the first generation of merozoites, has traditionally been the 'black box' of the Plasmodium life cycle. However, since the advent of parasite transfection technology 13 years ago, our understanding of the PE phase in cellular and molecular terms has dramatically improved. Here, we review and comment on the major developments in the field in the past five years. Progress has been made in many diverse areas, including identifying and characterizing new proteins of interest, imaging parasites in vivo, understanding better the cell biology of hepatocyte infection and developing new vaccines against PE stages of the parasite.
A sustained elevation of free Ca(2+) is observed on the rupture and release of merozoites of Plasmodium falciparum from the erythrocytes. The immunoelectron micrographs demonstrate that calmodulin is localized in merozoites. To elucidate the Ca(2+) signal of P. falciparum invasion, we attempted to characterize P. falciparum protein kinase 2 (PfPK2), which is homologous to human calcium calmodulin-dependent protein kinase (CaMK). PfPK2 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP; this labeling was then eliminated by phosphatase. This phosphorylation was eliminated when the putative catalytic lysine residue of PfPK2 was replaced with alanine. PfPK2 phosphorylated histone II(AS) as a representative substrate in a Ca(2+)- and calmodulin-dependent manner. Calmodulin antagonists inhibited the phosphorylation of PfPK2 in vitro and markedly decreased the parasitemia of ring forms in an invasion assay, whereas CaMKII-specific inhibitors had no effect. PfPK2 was localized in the merozoites in the culture of P. falciparum. Thus, purified PfPK2 possesses protein kinase activity in a Ca(2+)- and calmodulin-dependent manner and the catalytic lysine of this protein was determined. These data suggest that PfPK2 is the Plasmodium protein kinase expressed in the merozoites during the invasion stage.
Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4°C for several days. P. berghei, P. cynomolgi and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry microneme complex may play a role in sporozoite motility.
The Ca2+ ionophore A23187 consistently induced the exit of Toxoplasma gondii trophozoites from cultured macrophages which they had recently infected. Following exit of toxoplasmas, the host macrophages underwent degeneration. A23187 was active at concentrations higher than 0.25 μM and the activity reached a plateau at the concentration of 1.0 μM. Noninfected macrophages or those engulfing heat-killed toxoplasmas, or some other particles, were not affected by treatment with A23187. The toxoplasmas exiting host cells were capable of infecting and proliferating in normal macrophages. The A23187-mediated exit of toxoplasmas proceeded despite external Ca2+ and was enhanced by the addition of ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) in the reaction mixture. On the other hand, the A23187-mediated exit of toxoplasmas was inhibited significantly by exogenous Mg2+.
Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.
Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. We demonstrate that invasion is critically dependent on actin filaments in the parasite, but not the host cell. Invasion into cytochalasin D (CD)-resistant host cells was blocked by CD, while parasite mutants invaded wild-type host cells in the presence of drug. CD resistance in Toxoplasma was mediated by a point mutation in the single-copy actin gene ACT1. Transfection of the mutant act1 allele into wild-type Toxoplasma conferred motility and invasion in the presence of CD. We conclude that host cell invasion by Toxoplasma, and likely by related Apicomplexans, is actively powered by an actin-based contractile system in the parasite.
Invasion of vertebrate cells by the protozoan Toxoplasma gondii is accompanied by regulated protein secretion from three distinct parasite organelles called micronemes, rhoptries, and dense granules. We have compared the kinetics of secretion from these different compartments during host cell invasion using immunofluorescence, immunoelectron microscopy, and quantitative immunoassays. Binding to the host cell triggered apical release of the micronemal protein MIC2 at the tight attachment zone that forms between the parasite and the host cell. In a second step, invagination of the host cell plasma membrane was initiated by discharge of the rhoptry protein ROP1 to form a nascent parasitophorous vacuole (PV). ROP1 was fully discharged into the vacuole by the time invasion was complete. In contrast to these very rapid early events, release of the dense granule markers GRA1 and NTPase was delayed until after the parasite was fully within the PV, eventually peaking at 20 min post-invasion. The sequential triggering of secretion from different organelles implies that their release is governed by separate signals and that their contents mediate distinct phases of intracellular parasitism.
Apicomplexan parasites, including Toxoplasma gondii, apically attach to their host cells before invasion. Recent studies have implicated the contents of micronemes, which are small secretory organelles confined to the apical region of the parasite, in the process of host cell attachment. Here, we demonstrate that microneme discharge is regulated by parasite cytoplasmic free Ca2+ and that the micronemal contents, including the MIC2 adhesin, are released through the extreme apical tip of the parasite. Microneme secretion was triggered by Ca2+ ionophores in both the presence and the absence of external Ca2+, while chelation of intracellular Ca2+ prevented release. Mobilization of intracellular calcium with thapsagargin or NH4Cl also triggered microneme secretion, indicating that intracellular calcium stores are sufficient to stimulate release. Following activation of secretion by the Ca2+ ionophore A23187, MIC2 initially occupied the apical surface of the parasite, but was then rapidly treadmilled to the posterior end and released into the culture supernatant. This capping and release of MIC2 by ionophore-stimulated tachyzoites mimics the redistribution of MIC2 that occurs during attachment and penetration of host cells, and both events are dependent on the actin-myosin cytoskeleton of the parasite. These studies indicate that microneme release is a stimulus-coupled secretion system responsible for releasing adhesins involved in cell attachment.
Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of its pervasive cell invasion is not completely understood. Here, we demonstrate, using several independent assays, that Toxoplasma invasion of host cells is tightly coupled to the release of proteins stored within apical secretory granules called micronemes. Both microneme secretion and cell invasion were highly temperature dependent, and partial depletion of microneme resulted in a transient loss of infectivity. Chelation of parasite intracellular calcium strongly inhibited both microneme release and invasion of host cells, and this effect was partially reversed by raising intracellular calcium using the ionophore A23187. We also provide evidence that a staurosporine-sensitive kinase activity regulates microneme discharge and is required for parasite invasion of host cells. Additionally, we demonstrate that, during apical attachment to the host cell, the micronemal protein MIC2 is released at the junction between the parasite and the host cell. During invasion, MIC2 is successively translocated towards the posterior end of the parasite and is shed before entry of the parasite into the vacuole. Furthermore, we show that the full-length cellular form of MIC2, but not the proteolytically modified secreted form of MIC2, binds specifically to host cells. Collectively, these observations strongly imply that micronemal proteins play a role in Toxoplasma invasion of host cells.
Calmodulin (CaM) is an essential protein that serves as a ubiquitous intracellular receptor for Ca(2+). The Ca(2+)/CaM complex initiates a plethora of signaling cascades that culminate in alteration of cellular functions. Among the many Ca(2+)/CaM-binding proteins to be discovered, the multifunctional protein kinases CaMKI, II, and IV play pivotal roles. Our review focuses on this class of CaM kinases to illustrate the structural and biochemical basis for Ca(2+)/CaM interaction with and regulation of its target enzymes. Gene transcription has been chosen as the functional endpoint to illustrate the recent advances in Ca(2+)/CaM-mediated signal transduction mechanisms.
Sex is an obligate step in the life cycle of the malaria parasite and occurs in the midgut of the mosquito vector. With both Plasmodium falciparum and Plasmodium berghei, the tryptophan metabolite xanthurenic acid induces the release of motile male gametes from red blood cells (exflagellation), a prerequisite for fertilization. The addition of cGMP or phosphodiesterase inhibitors to cultures of mature gametocytes has also been shown to stimulate exflagellation. Here, we demonstrate that there is a guanylyl cyclase activity associated with mature P. falciparum gametocyte membrane preparations, which is dependent on the presence of Mg(2+)/Mn(2+) but is inhibited by Ca(2+). Significantly, this activity is increased on addition of xanthurenic acid. In contrast, a xanthurenic acid precursor (3-hydroxykynurenine), which is not an inducer of exflagellation, does not induce this guanylyl cyclase activity. These results therefore suggest that xanthurenic acid-induced exflagellation may be mediated by activation of the parasite cGMP signalling pathway.
Host cell invasion by apicomplexan parasites requires coordinated interactions between cell surface adhesins and the parasite cytoskeleton. We have identified a complex of parasite proteins, including the actin binding protein aldolase, which specifically interacts with the C-terminal domains of several parasite adhesins belonging to the thrombospondin-related anonymous protein (TRAP) family. Binding of aldolase to the adhesin was disrupted by mutation of a critical tryptophan in the C domain, a residue that was previously shown to be essential for parasite motility. Our findings reveal a potential role for aldolase in connecting TRAP family adhesins with the cytoskeleton, and provide a model linking adhesion with motility in apicomplexan parasites.
Invasion of host cells by Toxoplasma gondii is accompanied by secretion of parasite proteins that occurs coincident with increases in intracellular calcium. The source of calcium mobilized by the parasite and the signals that promote calcium increase remain largely undefined. We demonstrate here that intracellular stores of calcium in the parasite were both necessary and sufficient to support microneme secretion, motility and invasion of host cells. In contrast, host cell calcium was largely unaltered during parasite entry and not essential for this process. During parasite motility, cytosolic calcium levels underwent dramatic and rapid fluxes as imaged using the calcium indicator fluo-4 and time-lapse microscopy. Surprisingly, intracellular calcium in the parasite cytosol was rapidly quenched during the initial stages of host cell invasion, suggesting that while it is needed to initiate motility, it is not required to complete entry. These studies indicate that intracellular calcium stores govern secretion and motility by T. gondii and that the essential role of calcium in these events explains its requirement for cell entry.
The calcium ion (Ca(2+)) is used as a major signaling molecule in a diverse range of eukaryotic cells including several human parasitic protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, Leishmania spp, Plasmodium spp, Toxoplasma gondii, Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis. Ca(2+) is critical for invasion of intracellular parasites, and its cytosolic concentration is regulated by the concerted operation of several transporters present in the plasma membrane, endoplasmic reticulum, mitochondria and acidocalcisomes. Recent findings have shed light on the function of these transporters, the roles that they play in cellular metabolism and their potential use for targeting them for new therapies.