Global Transcriptome Profiling of Multiple Porcine Organs Reveals Toxoplasma gondii-Induced Transcriptional Landscapes.
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ABSTRACT:
SUBMITTER: He JJ
PROVIDER: S-EPMC6618905 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
Publications
Global Transcriptome Profiling of Multiple Porcine Organs Reveals <i>Toxoplasma gondii</i>-Induced Transcriptional Landscapes.
He Jun-Jun JJ Ma Jun J Wang Jin-Lei JL Zhang Fu-Kai FK Li Jie-Xi JX Zhai Bin-Tao BT Wang Ze-Xiang ZX Elsheikha Hany M HM Zhu Xing-Quan XQ
Frontiers in immunology 20190703
We characterized the porcine tissue transcriptional landscapes that follow <i>Toxoplasma gondii</i> infection. RNAs were isolated from liver, spleen, cerebral cortex, lung, and mesenteric lymph nodes (MLNs) of <i>T. gondii</i>-infected and uninfected (control) pigs at days 6 and 18 postinfection, and were analyzed using next-generation sequencing (RNA-seq). <i>T. gondii</i> altered the expression of 178, 476, 199, 201, and 362 transcripts at 6 dpi and 217, 223, 347, 119, and 161 at 18 dpi in the ...[more]
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Project description:BackgroundToxoplasma gondii is a protozoan parasite which can infect almost all warm-blooded animals and humans. Understanding the differential expression of proteins and transcripts associated with T. gondii infection in its definitive host (cat) may improve our knowledge of how the parasite manipulates the molecular microenvironment of its definitive host. The aim of this study was to explore the global proteomic alterations in the major organs of cats during acute T. gondii infection.MethodsiTRAQ-based quantitative proteomic profiling was performed on six organs (brain, liver, lung, spleen, heart and small intestine) of cats on day 7 post-infection by cysts of T. gondii PRU strain (Genotype II). Mascot software was used to conduct the student's t-test. Proteins with P values < 0.05 and fold change > 1.2 or < 0.83 were considered as differentially expressed proteins (DEPs).ResultsA total of 32,657 proteins were identified in the six organs, including 2556 DEPs; of which 1325 were up-regulated and 1231 were down-regulated. The brain, liver, lung, spleen, heart and small intestine exhibited 125 DEPs, 463 DEPs, 255 DEPs, 283 DEPs, 855 DEPs and 575 DEPs, respectively. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of all proteins and DEPs in all organs showed that many proteins were enriched in binding, cell part, cell growth and death, signal transduction, translation, sorting and degradation, extracellular matrix remodeling, tryptophan catabolism, and immune system. Correlations between differentially expressed proteins and transcripts were detected in the liver (n = 19), small intestine (n = 17), heart (n = 9), lung (n = 9) and spleen (n = 3).ConclusionsThe present study identified 2556 DEPs in six cat tissues on day 7 after infection by T. gondii PRU strain, and functional enrichment analyses showed that these DEPs were associated with various cellular and metabolic processes. These findings provide a solid base for further in-depth investigation of the complex proteotranscriptomic reprogramming that mediates the dynamic interplays between T. gondii and the different feline tissues.
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Project description:Understanding altered expression of proteins and transcripts associated with Toxoplasma gondii infection in cats may improve our understanding of how this parasite manipulates the molecular microenvironment of the definitive host. We performed proteomics analysis of six organs (brain, heart, spleen, liver, lung and small intestine) in cats acutely infected with T. gondii. A total of 32,657 proteins were identified among the six examined organs, including 2,556 differentially expressed proteins (DEPs), of which 1,325 DEPs were up-regulated and 1,231 DEPs were down-regulated. The brain, liver, lung, spleen, heart and small intestine exhibited 125 DEPs, 463 DEPs, 255 DEPs, 283 DEPs, 855 DEPs and 675 DEPs, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed on all proteins and DEPs in all organs, showed that many proteins were enriched in binding, cell part, cell growth and death, signal transduction, translation, sorting and degradation and immune system. Correlation between proteins and transcripts with differential expression patterns were detected in the heart (n = 9), liver (n = 19), lung (n = 9), small intestine (n = 17), and spleen (n = 3). These DEPs were mainly involved in immune response, tryptophan catabolism, and extracellular matrix remodeling. Future investigations are needed to identify the pathophysiological mechanisms underlying the reported associations between the identified proteins and transcripts and T. gondii infection.
2022-10-15 | PXD033778 | Pride
Project description:Toxoplasma gondii's tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo This predilection for neurons suggests that T. gondii's persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells.IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii's persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.
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Project description:BackgroundThe obligate intracellular parasite Toxoplasma gondii establishes a life-long chronic infection within any warm-blooded host. After ingestion of an encysted parasite, T. gondii disseminates throughout the body as a rapidly replicating form during acute infection. Over time and after stimulation of the host immune response, T. gondii differentiates into a slow growing, cyst form that is the hallmark of chronic infection. Global transcriptome analysis of both host and parasite during the establishment of chronic T. gondii infection has not yet been performed. Here, we conducted a dual RNA-seq analysis of T. gondii and its rodent host to better understand host and parasite responses during acute and chronic infection.ResultsWe obtained nearly one billion paired-end RNA sequences from the forebrains of uninfected, acutely and chronically infected mice, then aligned them to the genomic reference files of both T. gondii and Mus musculus. Gene ontology (GO) analysis of the 100 most highly expressed T. gondii genes showed less than half were shared between acute and chronic infection. The majority of the highly expressed genes common in both acute and chronic infection were involved in transcription and translation, underscoring that parasites in both stages are actively synthesizing proteins. Similarly, most of the T. gondii genes highly expressed during chronic infection were involved in metabolic processes, again highlighting the activity of the cyst stage at 28 days post-infection. Comparative analyses of host genes using uninfected forebrain revealed over twice as many immune regulatory genes were more abundant during chronic infection compared to acute. This demonstrates the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription.ConclusionsRNA-seq is a valuable tool to simultaneously analyze host and microbe transcriptomes. Our data shows that T. gondii is metabolically active and synthesizing proteins at 28 days post-infection and that a distinct subset of host genes associated with the immune response are more abundant specifically during chronic infection. These data suggest host and pathogen interplay is still present during chronic infection and provides novel T. gondii targets for future drug and vaccine development.
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Project description:Although microRNAs (miRNAs) play an important role in liver homeostasis, the extent to which they can be altered by Toxoplasma gondii infection is unknown. Here, we utilized small RNA sequencing and bioinformatic analyses to characterize miRNA expression profiles in the liver of domestic cats at 7 days after oral infection with T. gondii (Type II) strain. A total of 384 miRNAs were identified and 82 were differentially expressed, of which 33 were up-regulated and 49 down-regulated. Also, 5690 predicted host gene targets for the differentially expressed miRNAs were identified using the bioinformatic algorithm miRanda. Gene ontology analysis revealed that the predicted gene targets of the dysregulated miRNAs were significantly enriched in apoptosis. Kyoto Encyclopedia of Genes and Genomes analysis showed that the predicted gene targets were involved in several pathways, including acute myeloid leukemia, central carbon metabolism in cancer, choline metabolism in cancer, estrogen signaling pathway, fatty acid degradation, lysosome, nucleotide excision repair, progesterone-mediated oocyte maturation, and VEGF signaling pathway. The expression level of 6 upregulated miRNAs (mmu-miR-21a-5p, mmu-miR-20a-5p, mmu-miR-17-5p, mmu-miR-30e-3p, mmu-miR-142a-3p, and mmu-miR-106b-3p) was confirmed by stem-loop quantitative reverse transcription PCR, which yielded results consistent with the sequencing data. These findings expand our understanding of the regulatory mechanisms of miRNAs underlying T. gondii pathogenesis and contribute new database information on cat miRNAs, opening a new perspective on the prevention and treatment of T. gondii infection.
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Project description:Central to the progression of cerebral toxoplasmosis is the interaction of Toxoplasma gondii with the blood-brain barrier (BBB) endothelial cells. In the present work, we tested the hypothesis that inhibition of Wnt pathway signalling by the monovalent ionophore monensin reduces the growth of T. gondii infecting human brain microvascular endothelial cells (hBMECs) or microglial cells. The anti-parasitic effect of monensin (a Wnt signalling inhibitor) on the in vitro growth of T. gondii tachyzoites was investigated using two methods (Sulforhodamine B staining and microscopic parasite counting). The monensin inhibited T. gondii growth (50% inhibitory concentration [IC50] = 0.61 ?M) with a selective index = 8.48 when tested against hBMECs (50% cytotoxic concentration [CC50] = 5.17 ?M). However, IC50 of monensin was 4.13 ?M with a SI = 13.82 when tested against microglia cells (CC50 = 57.08 ?M), suggesting less sensitivity of microglia cells to monensin treatment. The effect of T. gondii on the integrity of the BBB was assessed by the transendothelial electrical resistance (TEER) assay using an in vitro human BBB model. The results showed that T. gondii infection significantly decreased hBMECs' TEER resistance, which was rescued when cells were treated with 0.1 µM monensin, probably due to the anti-parasitic activity of monensin. We also investigated the host-targeted effects of 0.1 µM monensin on global gene expression in hBMECs with or without T. gondii infection. Treatment of hBMECs with monensin did not significantly influence the expression of genes involved in the Wnt signalling pathway, suggesting that although inhibition of the Wnt signalling pathway did not play a significant role in T. gondii infection of hBMECs, monensin was still effective in limiting the growth of T. gondii. On the contrary, monensin treatment downregulated pathways related to steroids, cholesterol and protein biosynthesis and their transport between endoplasmic reticulum and Golgi apparatus, and deregulated pathways related to cell cycle and DNA synthesis and repair mechanisms. These results provide new insight into the host-modulatory effect of monensin during T. gondii infection, which merits further investigation.
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Project description:Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.
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Project description:Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.
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