Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program

Background

Large amounts of microarray expression data have been generated for the Apicomplexan parasite Toxoplasma gondii in an effort to identify genes critical for virulence or developmental transitions. However, researchers’ ability to analyze this data is limited by the large number of unannotated genes, including many that appear to be conserved hypothetical proteins restricted to Apicomplexa. Further, differential expression of individual genes is not always informative and often relies on investigators to draw big-picture inferences without the benefit of context. We hypothesized that customization of gene set enrichment analysis (GSEA) to T. gondii would enable us to rigorously test whether groups of genes serving a common biological function are co-regulated during the developmental transition to the latent bradyzoite form.

Results

Using publicly available T. gondii expression microarray data, we created Toxoplasma gene sets related to bradyzoite differentiation, oocyst sporulation, and the cell cycle. We supplemented these with lists of genes derived from community annotation efforts that identified contents of the parasite-specific organelles, rhoptries, micronemes, dense granules, and the apicoplast. Finally, we created gene sets based on metabolic pathways annotated in the KEGG database and Gene Ontology terms associated with gene annotations available at http://www.toxodb.org. These gene sets were used to perform GSEA analysis using two sets of published T. gondii expression data that characterized T. gondii stress response and differentiation to the latent bradyzoite form.

Conclusions

GSEA provides evidence that cell cycle regulation and bradyzoite differentiation are coupled. Δgcn5A mutants unable to induce bradyzoite-associated genes in response to alkaline stress have different patterns of cell cycle and bradyzoite gene expression from stressed wild-type parasites. Extracellular tachyzoites resemble a transitional state that differs in gene expression from both replicating intracellular tachyzoites and in vitro bradyzoites by expressing genes that are enriched in bradyzoites as well as genes that are associated with the G1 phase of the cell cycle. The gene sets we have created are readily modified to reflect ongoing research and will aid researchers’ ability to use a knowledge-based approach to data analysis facilitating the development of new insights into the intricate biology of Toxoplasma gondii.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-515) contains supplementary material, which is available to authorized users.     

Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein

The bradyzoite stage of Toxoplasma gondii is a key step in the parasite life cycle. For a better understanding of this stage, a sensitive system to detect the tissue cysts would be required. In this study, we generated the T. gondii cyst-forming strain PLK expressing green fluorescent protein (GFP) under control of the dense granule protein 1 promoter, which works at both the tachyzoite and the bradyzoite stages. The bradyzoites with GFP fluorescence within both small and large cysts were detectable in the brain of mice infected with the recombinant PLK. Indeed, the bradyzoites expressing GFP had infectivity to mice. This study shows that transfection of the cyst-forming strain with GFP gene under control of the GRA1 promoter could be a useful approach for the study of the bradyzoite stage of T. gondii.     

Primary culture of skeletal muscle cells as a model for studies of Toxoplasma gondii cystogenesis

Toxoplasma gondii is a protozoan pathogen of birds and mammals, including humans. The infective stage, the bradyzoite, lives within cysts, which occur predominantly in cells of the central nervous system and skeletal and cardiac muscles, characterizing the chronic phase of toxoplasmosis. In the present study, we employed for the first time primary mouse culture of skeletal muscle cells (SkMC) infected with bradyzoites, as a cellular model for cystogenesis. The interconversion of bradyzoite and tachyzoite was analyzed by immunofluorescence using 2 stage-specific antibodies, i.e., anti-bradyzoite (anti-BAG1) and anti-tachyzoite (anti-SAG1). After 24 hr of interaction only bradyzoites were multiplying, as revealed by anti-BAG1 incubation; interconversion to tachyzoites was not observed. After 48 hr of infection, 2 types of vacuoles were seen, i.e., BAG1+ and SAG1+, indicating the presence of bradyzoites as well as their interconversion to tachyzoites. After 96 hr of infection, BAG1+ vacuoles presented a higher number of parasites when compared to 48 hr, indicating multiplication of bradyzoites without interconversion. Using ultrastructural analysis, bradyzoites were found to adhere to the cell membranes via both the apical and posterior regions or were associated with SkMC membrane expansions. During bradyzoite invasion of SkMC, migration of the rough endoplasmic reticulum (RER) profiles to the parasite invasion site was observed. Later, RER profiles were localized between the mitochondria and parasitophorous vacuole membrane (PVM) that contained the parasite. After 31 days of parasite-host cell infection, RER profiles and mitochondria were not observed in association with the cyst wall. Alterations of the PVM, including increased thickness and electrondensity gain on its inner membrane face, were observed 48 hr after infection. Cystogenesis was complete 96 hr after infection, resulting in the formation of the cyst wall, which displayed numerous membrane invaginations. In addition, an electron-dense granular region enriched with vesicles and tubules was present, as well as numerous intracystic bradyzoites. These results show that the in vitro T. gondii model and SkMC are potential tools for both the study of cystogenesis using molecular approaches and the drug screening action on tissue cysts and bradyzoites.     

Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant

Growth rate is a major pathogenesis factor in the parasite Toxoplasma gondii; however, how cell division is controlled in this protozoan is poorly understood. Herein, we show that centrosomal duplication is an indicator of S phase entry while centrosome migration marks mitotic entry. Using the pattern of centrosomal replication, we confirmed that mutant ts11C9 undergoes a bimodal cell cycle arrest that is characterized by two subpopulations containing either single or duplicated centrosomes which correlate with the bipartite genome distribution observed at the non-permissive temperature. Genetic rescue of ts11C9 was performed using a parental RH strain cDNA library, and the cDNA responsible for conferring temperature resistance (growth at 40 degrees C) was recovered by recombination cloning. A single T. gondii gene encoding the protein homologue of XPMC2 was responsible for genetic rescue of the temperature-sensitive defect in ts11C9 parasites. This protein is a known suppressor of mitotic defects, and in tachyzoites, TgXPMC2-YFP localized to the parasite nucleus and nucleolus which is consistent with the expected subcellular localization of critical mitotic factors. Altogether, these results demonstrate that ts11C9 is a conditional mitotic mutant containing a single defect which influences two distinct control points in the T. gondii tachyzoite cell cycle.     

Toxoplasma gondii: characterization of temperature-sensitive tachyzoite cell cycle mutants

We mutagenized RH delta hxgprt strain tachyzoites of Toxoplasma gondii using N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutants) that were unable to grow in cell culture at 40 degrees C. These isolates grew normally at 34 degrees C, but showed variable growth at temperatures between 34 and 39 degrees C. The inability to grow at 40 degrees C was also correlated with a loss of virulence in mice for those mutants examined. We further characterized the temperature-sensitive (ts) isolates using flow cytometry and propidium iodide staining and identified three types of cell cycle-related mutations. Regardless of temperature, in the isolates ts1C12, ts7B4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (congruent with 85%) than that observed for RH delta hxgprt (congruent with 60%). Four other isolates, ts4F6, ts6C11, ts8G10, and ts11F5, contained G1-related mutations, and in each case, the DNA distribution among parasites at the permissive temperature was similar to that of the parental strain, but at 40 degrees C only a single population containing a 1N nuclear DNA complement was evident. Furthermore, there was no evidence of nuclear division or cytokinesis at 40 degrees C, and these parasites demonstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populations with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 parasites contained a single nucleus, and a major subfraction of the 2N population contained abnormal and incompletely formed daughters-indicating that the initiation of daughter formation can occur in the absence of nuclear division.     

Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii

Apicomplexa are obligate intracellular pathogens that have fine-tuned their proliferative strategies to match a large variety of host cells. A critical aspect of this adaptation is a flexible cell cycle that remains poorly understood at the mechanistic level. Here we describe a forward genetic dissection of the apicomplexan cell cycle using the Toxoplasma model. By high-throughput screening, we have isolated 165 temperature sensitive parasite growth mutants. Phenotypic analysis of these mutants suggests regulated progression through the parasite cell cycle with defined phases and checkpoints. These analyses also highlight the critical importance of the peculiar intranuclear spindle as the physical hub of cell cycle regulation. To link these phenotypes to parasite genes, we have developed a robust complementation system based on a genomic cosmid library. Using this approach, we have so far complemented 22 temperature sensitive mutants and identified 18 candidate loci, eight of which were independently confirmed using a set of sequenced and arrayed cosmids. For three of these loci we have identified the mutant allele. The genes identified include regulators of spindle formation, nuclear trafficking, and protein degradation. The genetic approach described here should be widely applicable to numerous essential aspects of parasite biology.     

The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii

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 in Toxoplasma that, in turn, causes an increase in cytoplasmic [Ca(2+)] in the parasite. The latter appears to be necessary and sufficient for inducing egress, as buffering of cytoplasmic Ca(2+) blocks egress and calcium ionophores circumvent the need for a reduction of host cell [K(+)] and parasite phospholipase C activation. The increase in [Ca(2+)](C) brings about egress by the activation of at least two signaling pathways: the protein kinase TgCDPK1 and the calmodulin-dependent protein phosphatase calcineurin.     

Redescription of Hammondia hammondi and its differentiation from Toxoplasma gondii

Hammondia hammondi is a protozoan parasite that, until 1975, was misidentified as Toxoplasma gondii. Recently, the validity of H. hammondi has been questioned. In this article, the authors redescribe the parasite and its life cycle, provide accession numbers to its specimens deposited in a museum, and distinguish it structurally and biologically from T. gondii. Hammondia hammondi was found to be structurally, biologically, and molecularly different from T. gondii.     

The expression of lactate dehydrogenase is important for the cell cycle of Toxoplasma gondii

In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (10(4)), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection.     

Toxoplasma gondii triggers secretion of interleukin-12 but low level of interleukin-10 from the THP-1 human monocytic cell line

Previous studies using both in vitro and in vivo mouse models have demonstrated that a subtle balance between pro- and anti-inflammatory cytokines, among which interleukin-12 (IL-12) and interleukin-10 (IL-10), respectively is crucial to control Toxoplasma infection. However, the few studies performed with human cell lines highlighted important host-related differences in the immune response to Toxoplasma gondii. The goal of our work was thus to study the production of both IL-12 and IL-10 by the THP-1 human monocytic cell line in response to Toxoplasma. We demonstrated that infection by live parasites (RH strain) triggers secretion of IL-12, but low level of IL-10. IL-12 secretion appeared within 8 h, up to 48 h. We also showed that infection by live parasites is not mandatory since heat-killed parasites, crude tachyzoite lysate as well as excreted/secreted antigens induced significant, yet reduced production of IL-12.     

DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression

Budding yeast cells suffering a single unrepaired DNA double-strand break (DSB) trigger the ATR (Mec1)-dependent DNA damage checkpoint and arrest prior to anaphase for 12–15 h, following which they adapt and resume cell division. When the DNA lesion can be repaired, the checkpoint is extinguished and cells “recover” and resume mitosis. In this autophagic punctum, we report that hyperactivation of autophagy—specifically via the cytoplasm-to-vacuole targeting (Cvt) pathway—prevents both adaptation to, and recovery from, DNA damage, resulting in the permanent arrest of cells in G₂/M. We show that Saccharomyces cerevisiae deleted for genes encoding the Golgi-associated retrograde protein transport (GARP) complex are both adaptation- and recovery-defective. GARP mutants such as vps51Δ exhibit mislocalization of the key mitotic regulator, securin (Pds1), and its degradation by the vacuolar protease Prb1. In addition, separase (Esp1), is excluded from the nucleus, accounting for pre-anaphase arrest. Pds1 is degraded via the Cvt pathway. Many of the same defects seen by deleting GARP genes can be mimicked by hyperactivation of the Cvt pathway by overexpressing an unphosphorylatable form of ATG13 or by adding the TORC1 inhibitor rapamycin. These results suggest that nuclear events such as DNA damage can have profound effects on cytoplasmic processes and further expand the burgeoning connections between DNA damage and autophagy.     

Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.     

Activation of the P2X7 receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages

Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which is widespread throughout the world. After active penetration, the parasite is enclosed within a parasitophorous vacuole and survives in the host cell by avoiding, among other mechanisms, lysosomal degradation. A large number of studies have demonstrated the importance of ATP signalling via the P2X₇ receptor, as a component of the inflammatory response against intracellular pathogens. Here we evaluate the effects of extracellular ATP on T. gondii infection of macrophages. ATP treatment inhibits the parasite load and the appearance of large vacuoles in the cytoplasm of intracellular parasites. ROS and NO assays showed that only ROS production is involved with the ATP effects. Immunofluorescence showed colocalization of Lamp1 and SAG1 only after ATP treatment, suggesting the formation of phagolysosomes. The involvement of P2X₇ receptors in T. gondii clearance was confirmed by the use of P2X₇ agonists and antagonists, and by infecting macrophages from P2X₇ receptor-deficient mice. We conclude that parasite elimination might occur following P2X₇ signalling and that novel therapies against intracellular pathogens could take advantage of activation of purinergic signalling.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.