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Computational prediction of microRNAs from Toxoplasma gondii potentially regulating the hosts' gene expression.

ABSTRACT: MicroRNAs (miRNAs) were discovered two decades ago, yet there is still a great need for further studies elucidating their genesis and targeting in different phyla. Since experimental discovery and validation of miRNAs is difficult, computational predictions are indispensable and today most computational approaches employ machine learning. Toxoplasma gondii, a parasite residing within the cells of its hosts like human, uses miRNAs for its post-transcriptional gene regulation. It may also regulate its hosts' gene expression, which has been shown in brain cancer. Since previous studies have shown that overexpressed miRNAs within the host are causal for disease onset, we hypothesized that T. gondii could export miRNAs into its host cell. We computationally predicted all hairpins from the genome of T. gondii and used mouse and human models to filter possible candidates. These were then further compared to known miRNAs in human and rodents and their expression was examined for T. gondii grown in mouse and human hosts, respectively. We found that among the millions of potential hairpins in T. gondii, only a few thousand pass filtering using a human or mouse model and that even fewer of those are expressed. Since they are expressed and differentially expressed in rodents and human, we suggest that there is a chance that T. gondii may export miRNAs into its hosts for direct regulation.

SUBMITTER: Sacar MD

PROVIDER: S-EPMC4411416 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Computational prediction of microRNAs from Toxoplasma gondii potentially regulating the hosts' gene expression.

Saçar Müşerref Duygu MD Bağcı Caner C Allmer Jens J

Genomics, proteomics & bioinformatics 20141028 5

MicroRNAs (miRNAs) were discovered two decades ago, yet there is still a great need for further studies elucidating their genesis and targeting in different phyla. Since experimental discovery and validation of miRNAs is difficult, computational predictions are indispensable and today most computational approaches employ machine learning. Toxoplasma gondii, a parasite residing within the cells of its hosts like human, uses miRNAs for its post-transcriptional gene regulation. It may also regulate ...[more]

PMID: 25462155

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Project description:BackgroundToxoplasma gondii is an apicomplexan protozoan parasite that can cause serious clinical illnesses in both humans and animals. microRNAs (miRNAs) are non-protein-coding RNAs that can regulate the expression of target genes. A previous study found that many miRNAs were differentially expressed after T. gondii infection and exert significant effects and revealed that both host survival and the virulence of different strains can be regulated by different miRNAs. Macrophages play an important role in T. gondii infection, but few studies have investigated the relationship between miRNAs and porcine alveolar macrophages infected with T. gondii.MethodsPorcine alveolar macrophages (3D4-21) were infected with the RH (Type I) and Me49 (Type II) strains of T. gondii for 12 h and 24 h and then harvested. miRNA libraries were generated using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA), and the miRNA expression levels were estimated based on transcripts per million reads (TPM).ResultsOur study generated six miRNA expression profiles from macrophages infected with RH and Me49 compared with the control groups. The comparison of the T. gondii-infected and uninfected samples identified 81 differentially expressed miRNAs, including 36 novel miRNAs and 45 mature miRNAs. The target genes of these differentially expressed miRNAs were predicted using miRanda software, and ssc-miR-127 and ssc-miR-143-3p were predicted to regulate nitric oxide synthase 1 (NOS1) and nitric oxide synthase 3 (NOS3), respectively, which play essential roles in synthesizing nitric oxide (NO) by oxidizing L-arginine. These genes were differentially expressed in both the RH- and Me49-infected groups. A KEGG enrichment analysis indicated that the predicted target genes were involved in multiple signaling pathways, including FcγR-mediated phagocytosis, the AMPK signaling pathway, the mTOR signaling pathway, and the FcγRI signaling pathway, all of which are indispensable for the normal functioning of porcine alveolar macrophages.ConclusionsOur results provide data on the miRNA profile of porcine alveolar macrophages infected with T. gondii. To our knowledge, this study provides the first demonstration of the relationship between miRNA and macrophages of swine origin. Understanding the functions of these regulated miRNAs will aid the investigation of T. gondii infectious diseases, and the differentially expressed miRNAs might be candidate drug targets for T. gondii infection in pigs.

Project description:Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Project description:Background: Considerable work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, of Toxoplasma gondii in large part due to the accessible culturing methods for these stages. However, culturing methods for stages beyond the bradyzoite, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. We begin to unravel the molecular aspects of the merozoite stage focusing on gene expression. Results: To initiate this, we harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by combining it with a previously published study that generated array data for the oocyst, tachyzoite, and bradyzoite stages (Fritz HM et al. PLoS One, 2012). Principal component analysis highlights the unique profile of the merozoite samples, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen p30 (SAG1) and many dense granule proteins do not label merozoites, and our microarray data confirms that these genes are not expressed at this stage. Also, the expression for many rhoptry and microneme proteins is drastically reduced while the expression for many surface antigens is increased at the merozoite stage. Gene Ontology and KEGG analysis reveals that genes involved in transcription/translation and many metabolic pathways are upregulated at the merozoite stage, highlighting unique growth requirements of this stage. We also show that an upstream promoter region of a merozoite specific gene is sufficient to control stage specific expression at the merozoite stage. Conclusion: The merozoite represents the first developmental stage within the gut of the definitive host. Determining the correct conditions that coax the parasite into the merozoite stage in vitro may allow the parasite to complete sexual development. The data presented here describe the global gene expression profile of merozoite stage and the creation of transgenic parasite strains that will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate coccidian development.

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Computational prediction of microRNAs from Toxoplasma gondii potentially regulating the hosts' gene expression.

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Computational prediction of microRNAs from Toxoplasma gondii potentially regulating the hosts' gene expression.

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