Dataset Information

Transcriptome Profiling of Toxoplasma gondii-Infected Human Cerebromicrovascular Endothelial Cell Response to Treatment with Monensin.

ABSTRACT: 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.

SUBMITTER: Harun MSR

PROVIDER: S-EPMC7356316 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Transcriptome Profiling of Toxoplasma gondii-Infected Human Cerebromicrovascular Endothelial Cell Response to Treatment with Monensin.

Harun Mohammad S R MSR Taylor Mica M Zhu Xing-Quan XQ Elsheikha Hany M HM

Microorganisms 20200604 6

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. ...[more]

PMID: 32512820

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Project description:BACKGROUND:Infection with the apicomplexan protozoan parasite T. gondii can cause severe and potentially fatal cerebral and ocular disease, especially in immunocompromised individuals. The anticoccidial ionophore drug monensin has been shown to have anti-Toxoplasma gondii properties. However, the comprehensive molecular mechanisms that underlie the effect of monensin on T. gondii are still largely unknown. We hypothesized that analysis of T. gondii transcriptional changes induced by monensin treatment can reveal new aspects of the mechanism of action of monensin against T. gondii. METHODS:Porcine kidney (PK)-15 cells were infected with tachyzoites of T. gondii RH strain. Three hours post-infection, PK-15 cells were treated with 0.1 ?M monensin, while control cells were treated with medium only. PK-15 cells containing intracellular tachyzoites were harvested at 6 and 24 h post-treatment, and the transcriptomic profiles of T. gondii-infected PK-15 cells were examined using high-throughput RNA sequencing (RNA-seq). Quantitative real-time PCR was used to verify the expression of 15 differentially expressed genes (DEGs) identified by RNA-seq analysis. RESULTS:A total of 4868 downregulated genes and three upregulated genes were identified in monensin-treated T. gondii, indicating that most of T. gondii genes were suppressed by monensin. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of T. gondii DEGs showed that T. gondii metabolic and cellular pathways were significantly downregulated. Spliceosome, ribosome, and protein processing in endoplasmic reticulum were the top three most significantly enriched pathways out of the 30 highly enriched pathways detected in T. gondii. This result suggests that monensin, via down-regulation of protein biosynthesis in T. gondii, can limit the parasite growth and proliferation. CONCLUSIONS:Our findings provide a comprehensive insight into T. gondii genes and pathways with altered expression following monensin treatment. These data can be further explored to achieve better understanding of the specific mechanism of action of monensin against T. gondii.

Project description:Toxoplasma gondii is a ubiquitous obligate intracellular parasite that infects the nucleated cells of warm-blooded animals. From within the parasitophorous vacuole in which they reside, Toxoplasma tachyzoites secrete an arsenal of effector proteins that can reprogram host gene expression to facilitate parasite survival and replication. Gaining a better understanding of how host gene expression is altered upon infection is central for understanding parasite strategies for host invasion and for developing new parasite therapies. Here, we applied ribosome profiling coupled with mRNA measurements to concurrently study gene expression in the parasite and in host human foreskin fibroblasts. By examining the parasite transcriptome and translatome, we identified potential upstream open reading frames that may permit the stress-induced preferential translation of parasite mRNAs. We also determined that tachyzoites reduce host death-associated pathways and increase survival, proliferation, and motility in both quiescent and proliferative host cell models of infection. Additionally, proliferative cells alter their gene expression in ways that are consistent with massive transcriptional rewiring, while quiescent cells were best characterized by reentry into the cell cycle. We also identified a translational control regimen consistent with mechanistic target of rapamycin (mTOR) activation in quiescent cells and, to a lesser degree, in proliferative cells. This study illustrates the utility of the method for dissection of gene expression programs simultaneously in the parasite and host.IMPORTANCEToxoplasma gondii is a single-celled parasite that has infected up to one-third of the world's population. Significant overhauls in gene expression in both the parasite and the host cell accompany parasite invasion, and a better understanding of these changes may lead to the development of new therapeutic agents. In this study, we employed ribosome profiling to determine the changes that occur at the levels of transcription and translation in both the parasite and the infected host cell at the same time. We discovered features of Toxoplasma mRNAs that suggest a means for controlling parasite gene expression under stressful conditions. We also show that differences in host gene expression occur depending on whether they are confluent or not. Our findings demonstrate the feasibility of using ribosomal profiling to interrogate the host-parasite dynamic under a variety of conditions.

Dataset Information

Transcriptome Profiling of Toxoplasma gondii-Infected Human Cerebromicrovascular Endothelial Cell Response to Treatment with Monensin.

Publications

Transcriptome Profiling of <i>Toxoplasma gondii</i>-Infected Human Cerebromicrovascular Endothelial Cell Response to Treatment with Monensin.

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