Biogenesis and secretion of micronemes in Toxoplasma gondii

One of the hallmarks of the parasitic phylum of Apicomplexa is the presence of highly specialised, apical secretory organelles, called the micronemes and rhoptries that play critical roles in ensuring survival and dissemination. Upon exocytosis, the micronemes release adhesin complexes, perforins, and proteases that are crucially implicated in egress from infected cells, gliding motility, migration across biological barriers, and host cell invasion. Recent studies on Toxoplasma gondii and Plasmodium species have shed more light on the signalling events and the machinery that trigger microneme secretion. Intracellular cyclic nucleotides, calcium level, and phosphatidic acid act as key mediators of microneme exocytosis, and several downstream effectors have been identified. Here, we review the key steps of microneme biogenesis and exocytosis, summarising the still fractal knowledge at the molecular level regarding the fusion event with the parasite plasma membrane.     

Role of ATG3 in the parasite Toxoplasma gondii

Toxoplasma gondii belongs to the phylum Apicomplexa, a diverse group of early branching unicellular eukaryotes related to dinoflagellates and ciliates. Like several other Apicomplexa such as Plasmodium (the causative agent of malaria), T. gondii is a human pathogen responsible for a potentially lethal disease called toxoplasmosis. Most Apicomplexa have complex life cycles, involving intermediate hosts and vectors, which include obligatory intracellular developmental stages. In the case of malaria and toxoplasmosis, it is that replicative process, leading to the ultimate lysis of the host cell, which is causing the symptoms of the disease. For Toxoplasma, the invasive and fast-replicating form of the parasite is called the tachyzoite. While autophagy has been a fast-growing field of research in recent years, not much was known about the relevance of this catabolic process in medically important apicomplexan parasites. Vesicles resembling autophagosomes had been described in drug-treated Plasmodium parasites in the early 1970s and a potential role for autophagy in organelle recycling during differentiation between Plasmodium life stages has also been recently described. Interestingly, recent database searches have identified putative orthologs of the core machinery responsible for the formation of autophagosomes in several protists, including Toxoplasma. In spite of an apparently reduced machinery (only about one-third of the yeast ATG genes appear to be conserved), T. gondii seemed thus able to perform macroautophagy, but the cellular functions of the pathway for this parasite remained to be demonstrated.     

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto-myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable propeptide. Although MIC propeptides have been implicated in forward targeting to micronemes, the specific amino acids involved have not been identified. It was also not known if the propeptide has a general function in MICs trafficking in T. gondii and other apicomplexans. Here we show that propeptide domains are extensively interchangeable between T. gondii MICs and also with that of Eimeria tenella MIC5 (EtMIC5), suggesting a common mechanism of function. We also performed N-terminal deletion and mutational analysis of M2AP and MIC5 propeptides to show that a valine at position +3 (relative to signal peptidase cleavage) of proM2AP and a leucine at position +1 of proMIC5 are crucial for targeting to micronemes. Valine and leucine are closely related amino acids with similar side chains, implying a similar mode of function, a notion that was confirmed by correct trafficking of TgM2AP-V/L and TgMIC5-L/V substitution mutants. Propeptides of AMA1, MIC3 and EtMIC5 have valine or leucine at or near the N-termini and mutagenesis of these conserved residues validated their role in microneme trafficking. Collectively, our findings suggest that discrete, aliphatic residues at the extreme N-termini of proMICs facilitate trafficking to the micronemes.     

Protein trafficking inside Toxoplasma gondii

The accurate targeting of proteins to their final destination is an essential process in all living cells. Apicomplexans are obligate intracellular protozoan parasites that possess a compartmental organization similar to that of free-living eukaryotes but can be viewed as professional secretory cells. Establishment of parasitism involves the sequential secretion from highly specialized secretory organelles, including micronemes, rhoptries and dense granules. Additionally, apicomplexans harbor a tubular mitochondrion, a nonphotosynthetic plastid organelle termed the apicoplast, acidocalcisomes and an elaborated inner membrane complex composed of flattened membrane cisternae that are derived from the secretory pathway. Given the multitude of destinations both inside and outside the parasite, the endoplasmic reticulum/Golgi of the apicomplexans constitutes one of the most busy roads intersections in eukaryotic traffic.     

Characterisation of hexokinase in Toxoplasma gondii tachyzoites

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.     

The immunobiology of the innate response to Toxoplasma gondii

Toxoplasma gondii is a unique intracellular parasite. It can infect a variety of cells in virtually all warm-blooded animals. It has a worldwide distribution and, overall, around one-third of people are seropositive for the parasite, with essentially the entire human population being at risk of infection. For most people, T. gondii causes asymptomatic infection but the parasite can cause serious disease in the immunocompromised and, if contracted for the first time during pregnancy, can cause spontaneous abortion or congenital defects, which have a substantial emotional, social and economic impact. Toxoplasma gondii provokes one of the most potent innate, pro-inflammatory responses of all infectious disease agents. It is also a supreme manipulator of the immune response so that innate immunity to T. gondii is a delicate balance between the parasite and its host involving a coordinated series of cellular interactions involving enterocytes, neutrophils, dendritic cells, macrophages and natural killer cells. Underpinning these interactions is the regulation of complex molecular reactions involving Toll-like receptors, activation of signalling pathways, cytokine production and activation of anti-microbial effector mechanisms including generation of reactive nitrogen and oxygen intermediates.     

Toxoplasma gondii: the model apicomplexan

Toxoplasma gondii is an obligate intracellular protozoan parasite which is a significant human and veterinary pathogen. Other members of the phylum Apicomplexa are also important pathogens including Plasmodium species (i.e. malaria), Eimeria species, Neospora, Babesia, Theileria and Cryptosporidium. Unlike most of these organisms, T. gondii is readily amenable to genetic manipulation in the laboratory. Cell biology studies are more readily performed in T. gondii due to the high efficiency of transient and stable transfection, the availability of many cell markers, and the relative ease with which the parasite can be studied using advanced microscopic techniques. Thus, for many experimental questions, T. gondii remains the best model system to study the biology of the Apicomplexa. Our understanding of the mechanisms of drug resistance, the biology of the apicoplast, and the process of host cell invasion has been advanced by studies in T. gondii. Heterologous expression of apicomplexan proteins in T. gondii has frequently facilitated further characterisation of proteins that could not be easily studied. Recent studies of Apicomplexa have been complemented by genome sequencing projects that have facilitated discovery of surprising differences in cell biology and metabolism between Apicomplexa. While results in T. gondii will not always be applicable to other Apicomplexa, T. gondii remains an important model system for understanding the biology of apicomplexan parasites.     

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii

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

Toxoplasma gondii uses unusual sorting mechanisms to deliver transmembrane proteins into the host-cell vacuole

A critical step in infection by the apicomplexan parasite Toxoplasma gondii is the formation of a membrane-bound compartment within which the parasite proliferates. This process relies on a set of secretory organelles that discharge their contents into the host cell upon invasion. Among these organelles, the dense granules are specialized in the export of transmembrane (TM) GRA proteins, which are major components of the mature parasitophorous vacuole (PV) membrane. How eukaryotic pathogens export and sort membrane-bound proteins destined for the host cell is still poorly understood at the mechanistic level. In this study, we show that soluble trafficking of the PV-targeted GRA5 TM protein is parasite specific: when expressed in mammalian cells, GRA5 is targeted to the plasma membrane and behaves as an integral membrane protein with a type I toplogy. We also demonstrate the dual role of the GRA5 N-terminal ectodomain, which is sufficient to prevent membrane integration within the parasite and is essential for both sorting and post-secretory membrane insertion into the vacuolar membrane. These results contrast with the general rule that states that information contained within the cytoplasmic tail and/or the TM domain of integral membrane proteins dictates their cellular localization. They also highlight the diversity of sorting mechanisms that leads to the specialization of secretory processes uniquely adapted to intracellular parasitism.     

Migration of Apicomplexa across biological barriers: the Toxoplasma and Plasmodium rides

The invasive stages of Apicomplexa parasites, called zoites, have been largely studied in in vitro systems, with a special emphasis on their unique gliding and host cell invasive capacities. In contrast, the means by which these parasites reach their destination in their hosts are still poorly understood. We summarize here our current understanding of the cellular basis of in vivo parasitism by two well-studied Apicomplexa zoites, the Toxoplasma tachyzoite and the Plasmodium sporozoite. Despite being close relatives, these two zoites use different strategies to reach their goal and establish infection.     

Protein kinases in Toxoplasma gondii

Toxoplasma gondii is an obligatory intracellular pathogen that causes life threatening illness in immunodeficient individuals, miscarriage in pregnant woman, and blindness in newborn children. Similar to any other eukaryotic cell, protein kinases play critical and essential roles in the Toxoplasma life cycle. Accordingly, many studies have focused on identifying and defining the mechanism of function of these signalling proteins with a long-term goal to develop anti-Toxoplasma therapeutics. In this review, we briefly discuss classification and key components of the catalytic domain which are critical for functioning of kinases, with a focus on domains, families, and groups of kinases within Toxoplasma. More importantly, this article provides a comprehensive, current overview of research on kinase groups in Toxoplasma including the established eukaryotic AGC, CAMK, CK1, CMGC, STE, TKL families and the apicomplexan-specific FIKK, ROPK and WNG family of kinases. This work provides an overview and discusses current knowledge on Toxoplasma kinases including their localization, function, signalling network and role in acute and chronic pathogenesis, with a view towards the future in probing kinases as viable drug targets.     

Host cell surface sialic acid residues are involved on the process of penetration of Toxoplasma gondii into mammalian cells

Tachyzoites of Toxoplasma gondii are able to infect several cell types tested (wild-type chinese hamster ovary (CHO) cells and glycosylation mutants, Vero and LLCMK2 cells). However, the extent of infection varied. Mutant cells which present few or no surface-exposed sialic acid residues were infected to a lower extent. Similar results were obtained if sialic acid residues were removed by previous neuraminidase treatment. Addition of sialic acid residues to surface-exposed glycoconjugates using fetuin as a sialic acid donor and the trans-sialidase of Trypanosoma cruzi rendered the cells more easily infected by Toxoplasma gondii. These observations indicate that surface-exposed carbohydrate residues of the host cell are involved on the process of Toxoplasma gondii-host cell recognition.     

The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii

SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.     

Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells

Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8⁺ T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.     

Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.     

Kinetic modeling of Toxoplasma gondii invasion

The phylum Apicomplexa includes parasites responsible for global scourges such as malaria, cryptosporidiosis, and toxoplasmosis. Parasites in this phylum reproduce inside the cells of their hosts, making invasion of host cells an essential step of their life cycle. Characterizing the stages of host-cell invasion, has traditionally involved tedious microscopic observations of individual parasites over time. As an alternative, we introduce the use of compartment models for interpreting data collected from snapshots of synchronized populations of invading parasites. Parameters of the model are estimated via a maximum negative log-likelihood principle. Estimated parameter values and their 95% confidence intervals (95% CI), are consistent with reported observations of individual parasites. For RH strain parasites, our model yields that: (1) penetration of the host-cell plasma membrane takes 26s (95% CI: 22-30s); (2) parasites that ultimately invade, remain attached three times longer than parasites that eventually detach from the host cells, and (3) 25% (95% CI: 19-33%) of parasites invade while 75% (95% CI: 67-81%) eventually detach from their host cells without progressing to invasion. A key feature of the model is the incorporation of invasion stages that cannot be directly observed. This allows us to characterize the phenomenon of parasite detachment from host cells. The properties of this phenomenon would be difficult to quantify without a mathematical model. We conclude that mathematical modeling provides a powerful new tool for characterizing the stages of host-cell invasion by intracellular parasites.     

Apicoplast targeting of a Toxoplasma gondii transmembrane protein requires a cytosolic tyrosine-based motif

Toxoplasma gondii, like most apicomplexan parasites, possesses an essential relict chloroplast, the apicoplast. Several apicoplast membrane proteins lack the bipartite targeting sequences of luminal proteins. Vesicles bearing these membrane proteins are detected during apicoplast enlargement, but the means of cargo selection remains obscure. We used a combination of deletion mutagenesis, point mutations and protein chimeras to identify a short motif prior to the first transmembrane domain of the T. gondii apicoplast phosphate transporter 1 (APT1) that is necessary for apicoplast trafficking. Tyrosine 16 was essential for proper localization; any substitution resulted in misdirection of APT1 to the Golgi body. Glycine 17 was also important, with significant Golgi body accumulation in the alanine mutant. Separation of at least eight amino acids from the transmembrane domain was required for full motif function. Similarly placed YG motifs are present in apicomplexan APT1 orthologs and the corresponding N‐terminal domain from Plasmodium vivax was able to route T. gondii APT1 to the apicoplast. Differential permeabilization showed that both the N‐ and C‐termini of APT1 are exposed to the cytosol. We propose that this YG motif facilitates APT1 trafficking via interactions that occur on the cytosolic face of nascent vesicles destined for the apicoplast.     

弓形虫RH株SAG1基因序列体外扩增及生物信息学分析

根据sAG1基因序列,自行设计一对寡核苷酸引物,利用PCR技术从弓形虫RH株基因组DNA中成功扩增出编码SAG1抗原的基因片段,扩增出的基因片段大小与预期长度（1006bp）相符,结果经测序验证,并利用生物信息学方法对SAG1蛋白理化性质、结构和功能进行了预测．     

Proteomic analysis of calcium-dependent secretion in Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite that invades a wide range of nucleated cells. In the course of intracellular parasitism, the parasite releases a large variety of proteins from three secretory organelles, namely, micronemes, rhoptries and dense granules. Elevation of intracellular Ca(2+) in the parasite causes microneme discharge, and microneme secretion is essential for the invasion. In this study, we performed a proteomic analysis of the Ca(2+)-dependent secretion to evaluate the protein repertoire. We found that Ca(2+)-mobilising agents, such as thapsigargin, NH(4)Cl, ethanol and a Ca(2+) ionophore, A23187, promoted the secretion of the parasite proteins. The proteins, artificially secreted by A23187, were used in a comparative proteomic analysis by 2-DE followed by PMF analysis and/or N-terminal sequencing. Major known microneme proteins (MICs), such as MIC2, MIC4, MIC6 and MIC10 and apical membrane antigen 1 (AMA1), were identified, indicating that the proteomic analysis worked accurately. Interestingly, new members of secretory proteins, namely rhoptry protein 9 (ROP9) and Toxoplasma SPATR (TgSPATR), which was a homologue of a Plasmodium secreted protein with an altered thrombospondin repeat (SPATR), were detected in Ca(2+)-dependent secretion. Thus, we succeeded in detecting Ca(2+)-dependent secretory proteins in T. gondii, which contained novel secretory proteins.