How Epigenomics Contributes to the Understanding of Gene Regulation in Toxoplasma gondii1

ABSTRACT. How apicomplexan parasites regulate their gene expression is poorly understood. The complex life cycle of these parasites implies tight control of gene expression to orchestrate the appropriate expression pattern at the right moment. Recently, several studies have demonstrated the role of epigenetic mechanisms for control of coordinated expression of genes. In this review, we discuss the contribution of epigenomics to the understanding of gene regulation in Toxoplasma gondii. Studying the distribution of modified histones on the genome links chromatin modifications to gene expression or gene repression. In particular, coincident trimethylated lysine 4 on histone H3 (H3K4me3), acetylated lysine 9 on histone H3 (H3K9ac), and acetylated histone H4 (H4ac) mark promoters of actively transcribed genes. However, the presence of these modified histones at some non‐expressed genes and other histone modifications at only a subset of active promoters implies the presence of other layers of regulation of chromatin structure in T. gondii. Epigenomics analysis provides a powerful tool to characterize the activation state of genomic loci of T. gondii and possibly of other Apicomplexa including Plasmodium or Cryptosporidium. Further, integration of epigenetic data with expression data and other genome‐wide datasets facilitates refinement of genome annotation based upon experimental data.     

Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers

The apicomplexan parasite Toxoplasma gondii invades tissues and traverses non‐permissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immune‐privileged sites such as the brain parenchyma, after crossing the blood–brain barrier. In the present study, we have established a protocol for high‐purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance. In contrast, challenge with parasite lysate or LPS increased barrier permeability by destabilising intercellular tight junctions (TJs) and accentuated transmigration of T. gondii. Conversely, reduced phosphorylation of the TJ‐regulator focal adhesion kinase (FAK) was observed dose‐dependently upon challenge of monolayers with live T. gondii but not with parasite lysate or LPS. Pharmacological inhibition of FAK phosphorylation reversibly altered barrier integrity and facilitated T. gondii translocation. Finally, gene silencing of FAK by shRNA facilitated transmigration of T. gondii across epithelial and endothelial monolayers. Jointly, the data demonstrate that T. gondii infection transiently alters the TJ stability through FAK dysregulation to facilitate transmigration. This work identifies the implication of the TJ regulator FAK in the transmigration of T. gondii across polarised cellular monolayers and provides novel insights in how microbes overcome the restrictiveness of biological barriers.     

A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages

The apicomplexan parasite Toxoplasma gondii is able to suppress nitric oxide production in activated macrophages. A screen of over 6000 T. gondii insertional mutants identified two clones, which were consistently unable to suppress nitric oxide production from activated macrophages. One strain, called 89B7, grew at the same rate as wild‐type parasites in naïve macrophages, but unlike wild type, the mutant was degraded in activated macrophages. This degradation was marked by a reduction in the number of parasites within vacuoles over time, the loss of GRA4 and SAG1 protein staining by immunofluorescence assay, and the vesiculation and breakdown of the internal parasite ultrastructure by electron microscopy. The mutagenesis plasmid in the 89B7 clone disrupts the promoter of a 3.4 kb mRNA that encodes a predicted 68 kDa protein with a cleavable signal peptide and a patatin‐like phospholipase domain. Genetic complementation with the genomic locus of this patatin‐like protein restores the parasites ability to suppress nitric oxide and replicate in activated macrophages. A haemagglutinin‐tagged version of this patatin‐like protein shows punctate localization into atypical T. gondii structures within the parasite. This is the first study that defines a specific gene product that is needed for parasite survival in activated but not naïve macrophages.     

Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage

Toxoplasma gondii is a widespread intracellular parasite of mammals and birds and an important opportunistic pathogen of humans. Following primary infection, fast‐replicating tachyzoites disseminate within the host and either are subsequently eliminated by the immune system or transform to latent bradyzoites which preferentially persist in brain and muscle tissues. The factors which determine the parasites' tissue distribution during chronic toxoplasmosis are unknown. Here we show that mouse skeletal muscle cells (SkMCs) after differentiation to mature, myosin heavy chain‐positive, polynucleated myotubes, significantly restrict tachyzoite replication and facilitate expression of bradyzoite‐specific antigens and tissue cyst formation. In contrast, proliferating mononuclear myoblasts and control fibroblasts enable vigorous T. gondii replication but do not sustain bradyzoite or tissue cyst formation. Bradyzoite formation correlates with upregulation of testis‐specific Y‐encoded‐like protein‐2 gene expression (Tspyl2) and p21^Waf1/Cip1 as well as downregulation of cyclin B1 and absence of DNA synthesis, i.e. a cell cycle arrest of syncytial myotubes. Following infection with T. gondii, myotubes but not myoblasts or fibroblasts further upregulate the negative cell cycle regulator Tspyl2. Importantly, RNA interference‐mediated knock‐down of Tspyl2 abrogates differentiation of SkMCs to myotubes and enables T. gondii to replicate vigorously but abolishes bradyzoite‐specific gene expression and tissue cyst formation. Together, these data indicate that Tspyl2‐mediated host cell cycle withdrawal is a physiological trigger of Toxoplasma stage conversion in mature SkMCs. This finding might explain the preferred distribution of T. gondii tissue cysts in vivo.     

A comparative study on the heparin‐binding proteomes of Toxoplasma gondii and Plasmodium falciparum

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin‐binding proteome of T. gondii. The parasite‐derived components were affinity‐purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin‐binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion‐related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry‐derived proteins were prominently identified. The profiling of the heparin‐binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin‐mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis.     

表观遗传对炎症的调控机制及其在奶牛乳房炎抗病育种中的应用前景

炎症受遗传和非遗传因素(环境或表观遗传)的共同影响, 其中表观遗传(Epigenetic)在炎症的发生发展过程中发挥重要调控作用。表观遗传修饰是指DNA序列没有改变, 而基因表达却发生了可遗传的变化, 主要包括DNA甲基化和组蛋白修饰等。表观遗传为病原微生物与炎症反应间关系的研究架起了重要桥梁。炎症反应中T辅助细胞的分化, 细胞因子、趋化因子等基因的表达都受到表观遗传的调控。文章主要综述了DNA甲基化、组蛋白修饰等对炎症尤其是乳房炎的调控机制, 并就表观遗传调控在奶牛乳房炎治疗及抗病育种中的应用前景进行了展望。     

Simulated microgravity‐induced epigenetic changes in human lymphocytes

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity‐induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T‐lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity‐induced DNA methylation changes were detected by methylation sensitive‐random amplified polymorphic DNA (MS‐RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real‐time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS‐RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time‐dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity‐induced gene expression changes and associated adverse health effects. J. Cell. Biochem. 111: 123–129, 2010. © 2010 Wiley‐Liss, Inc.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.