ABSTRACT Despite their critical role in chronic toxoplasmosis, the biology of Toxoplasma gondii bradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replication in vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cysts in vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage. IMPORTANCE The protozoan Toxoplasma gondii establishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cysts in vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease.

The neoGRID is under development in Quarry, a virtual hosting environment, for working with Taverna-based workflow utilizing grid computing. Taverna is a graphical workbench often used for biomedical informatics [1, and the references therein]. neoGRID is designed to offer a HPC-supported collaborative environment for the researchers from multidisciplinary scientific fields to gather data, integrate and analyze using teragrid resources. A significant number of resources including bioinformatics tools [2] has been deployed in TeraGrid, an NSF funded project. Besides, the myGrid team produced a suite of tools that are made available for analysis of proteins. In addition, their myExperiment site makes it easy to find, use and share scientific workflows and building scientific communities with common interests. This Cyberinfrastructure (CI)-supported neoGRID can be utilized for protein motifs analysis, particularly in the glycosyltransferase protein family and will be available to the Glyco-Community [3]. Workflows specifically designed for such analysis will be available in neoGRID. Moreover, a significant number of workflows are available in KEGG that are useful for protein analysis and drug discovery research. These and modification of these workflows in a collaborative environment will also be available for drug discovery research using members of glycosyltransferase enzyme family as target protein(s) [4]. Here, we demonstrate the usage of neoGRID for analysis of sialylmotifs of sialyltransferases. The presence of sialylmotifs is the cardinal feature of mammalian sialyltransferases [5], a group of enzymes that transfers sialic acid from CMP-NeuAc to the terminal carbohydrates group of various glycoproteins and glycolipids [6]. Sialic acid has been recognized as the key determinant of a diverse oligosaccharide structures involved in a large variety of biological events as diverse as animal cell-cell interaction to oncogenic transformation [7]. These conserved protein domains, sialylmotifs, have been shown to be involved in binding either the donor or acceptor substrates or both [8, 9], and a disulfide linkage between these two motifs has been shown to be essential for catalytic activity [10, 11]. Protein sequence [12] and structural analysis [11, 13] showed that mammalian sialyltransferase has no similarities with the bacterial enzymes, although a His residue serves as a catalytic center for both [11]. This provides a unique opportunity for discovery research on potential drug development. A thorough analysis of these motifs is now under study for drug discovery research using sialyltransferase as a target protein. Such analysis demanding high-performance computing power is available in neoGRID. The genome of protozoan parasite Toxoplasma gondii has been used here for bioinformatics analysis. T. gondii, which chronically infects roughly 30% of the world's population is typically asymptomatic but can cause life threatening disease in immune compromised individuals and birth defects of acquired during pregnancy [14]. Sequencing of this parasite genome of about 63 Mb in size with 14 chromosomes, has recently been completed [15, 16]. The availability of this sequence information from multiple isolates in a specially designed database (www.toxodb.org) that also include transcriptomic and proteomic data sets [16], has made this parasite an ideal candidate for in-depth bioinformatic interrogation. At this time, little is known about the parasite glycome or the encoded capacity for glycosylation. This deficit is in spite of the fact that the parasite form associated with chronic infection is heavily glycosylated [17]. Our initial studies have identified a broad spectrum of lectin reactivities [18]. Lectins recognize their specific glycan targets with high specificity and affinity [19]. We were particularly surprised to find evidence for sialylation of the parasite tissue cyst forms given that the genome does not appear to encode recognizable sialyltransferases based on the 'sialylmotif' [5]. This finding suggests that Toxoplasma may hijack the sialyltrasferase activities of the infected host cell or alternatively possess activities with entirely novel functional signatures. The integration of state of the art computational biology and bioinformatics with experimental validation provides a unique opportunity toward new discovery. By conducting a systematic survey of glycosyltransferase activities in silico we can establish both the presence and absence of specific activities in the Toxoplasma genome. Given the evolutionary antiquity of the parasitic protozoa we expect the combination of in silico analysis and experimental validation to offer new insights into the biology of these parasites. The availability of sequenced genomes of several related parasites housed at www.eupathdb.org [20] provides an ideal resource for expanding these studies to other parasites including Plasmodium species, the agent of malaria. In silico identification of potentially unique enzymatic activities could open doors toward the discovery of novel drugs to treat these often deadly infections.

The Tie family of endothelial-specific receptor tyrosine kinases is essential for cell proliferation, migration, and survival during angiogenesis. Despite considerable similarity, experiments with Tie1- or Tie2-deficient mice highlight distinct functions for these receptors in vivo. The Tie2 receptor is further unique with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists; angiopoietin-1 and -4 are constitutive agonists. To address the role of Tie1 in angiopoietin-mediated Tie2 signaling and determine the basis for the behavior of the individual angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and -2 localization and association. We provide evidence for Tie1-Tie2 complex formation on the cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by angiopoietin-1 and -2. Based on the available data, we propose a unified model for angiopoietin-induced Tie2 signaling.