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. 2016 May 13;7:721.
doi: 10.3389/fmicb.2016.00721. eCollection 2016.

Dual Identification and Analysis of Differentially Expressed Transcripts of Porcine PK-15 Cells and Toxoplasma Gondii During in Vitro Infection

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Free PMC article

Dual Identification and Analysis of Differentially Expressed Transcripts of Porcine PK-15 Cells and Toxoplasma Gondii During in Vitro Infection

Chun-Xue Zhou et al. Front Microbiol. .
Free PMC article

Abstract

Toxoplasma gondii is responsible for causing toxoplasmosis, one of the most prevalent zoonotic parasitoses worldwide. The mechanisms that mediate T. gondii infection of pigs (the most common source of human infection) and renal tissues are still unknown. To identify the critical alterations that take place in the transcriptome of both porcine kidney (PK-15) cells and T. gondii following infection, infected cell samples were collected at 1, 3, 6, 9, 12, 18, and 24 h post infection and RNA-Seq data were acquired using Illumina Deep Sequencing. Differential Expression of Genes (DEGs) analysis was performed to study the concomitant gene-specific temporal patterns of induction of mRNA expression of PK-15 cells and T. gondii. High sequence coverage enabled us to thoroughly characterize T. gondii transcriptome and identify the activated molecular pathways in host cells. More than 6G clean bases/sample, including >40 million clean reads were obtained. These were aligned to the reference genome of T. gondii and wild boar (Sus scrofa). DEGs involved in metabolic activities of T. gondii showed time-dependent down-regulation. However, DEGs involved in immune or disease related pathways of PK-15 cells peaked at 6 h PI, and were highly enriched as evidenced by KEGG analysis. Protein-protein interaction analysis revealed that TGME49_120110 (PCNA), TGME49_049180 (DHFR-TS), TGME49_055320, and TGME49_002300 (ITPase) are the four hub genes with most interactions with T. gondii at the onset of infection. These results reveal altered profiles of gene expressed by PK-15 cells and T. gondii during infection and provide the groundwork for future virulence studies to uncover the mechanisms of T. gondii interaction with porcine renal tissue by functional analysis of these DEGs.

Keywords: KEGG; PK-15 cells; Toxoplasma gondii; host-pathogen interaction; protein-protein interaction; transcriptome.

Figures

Figure 1
Figure 1
Statistics of single nucleotide polymorphisms (SNPs) and alternative splicing (AS) events identified from the RNA-Seq data. (A) Classification of putative SNPs and INDELs identified from the transcriptomes of T. gondii. The x-axis represents types of SNP events. The y-axis represents number of SNP events. (B) Classification of putative SNPs and INDELs identified from the transcriptomes of PK-15 cells. The x-axis represents types of SNP events. The y-axis represents number of SNP events. (C) Numbers of the AS events in T. gondii. The x-axis represents types of AS events. The y-axis represents number of AS events. (D) Numbers of AS events in PK-15 cells. The x-axis represents types of AS events. The y-axis represents number of AS events. The AS events include transcription start site (TSS), transcription terminal site (TTS), skipped exon (SKIP_ON,SKIP_OFF pair) (SKIP), approximate SKIP (XSKIP_ON,XSKIP_OFF pair) (XSKIP), multi-exon SKIP (MSKIP_ON,MSKIP_OFF pair) (MSKIP), approximate MSKIP (XMSKIP_ON,XMSKIP_OFF pair) (XMSKIP), intron retention (IR_ON, IR_OFF pair) (IR), approximate IR (XIR_ON, XIR_OFF pair) (XIR), multi-IR (MIR_ON, MIR_OFF pair) (MIR), approximate MIR (XMIR_ON, XMIR_OFF pair) (XMIR), alternative exon ends (5′, 3′, or both) (AE), and approximate AE(XAE).
Figure 2
Figure 2
Gene expression profiles during T. gondii infection. (A) Hierarchical clustering of differentially expressed genes of T. gondii during infection. Expression values (FPKM) were log2-transformed and subsequently median-centered by gene. Rows were hierarchically clustered based on average linkage using Pearson correlation coefficients as the distance measure. The expression levels are visualized using gradient color scheme, the scale from least abundant to highest range is from -2.0 to 2.0. Green color indicates low expression, and red color indicates high expression of the detected genes. The left vertical axis represents sample ID. The horizontal axis shows clusters of samples and the above vertical axis shows clusters of DEGs. (B) Hierarchical clustering of differentially expressed genes of T. gondii-infected PK-15 cells. (C) Numbers of DEGs in the parasites during infection process. (D) Numbers of DEGs in the host PK-15 cells during infection process. Up, up-regulated DEGs; Down, down-regulated DEGs; Total, total DEGs.
Figure 3
Figure 3
Venn diagram showing the overlap of DEGs clustered into four comparison groups (T1, T3, T6, and T9) represented by four ellipses. Numbers outside the ecllipses correspond to groups unique to a given infection stage. Numbers in the overlapping parts of different ellipses correspond to the number of DEGs in common from those comparison groups. (A) Veen diagram of identified DEGs in T. gondii. (B) Veen diagram of identified DEGs in PK-15 cells. Numbers in parentheses indicate the total number of DEGs in each data set.
Figure 4
Figure 4
DEGs GO cluster distribution. DEGs were classified into three main categories: biological process, cellular component and molecular function. Shown are the identified functions and the corresponding numbers of DEGs for each GO category. (A) T1/T0-T DEGs GO cluster distribution. (B) T6/T0-T DEGs GO cluster distribution. (C) T1/T0-C DEGs GO cluster distribution. (D) T6/T0-C DEGs GO cluster distribution. The y-axis indicates the number of genes in a specific category. X-axis indicates different GO terms. Top 40 enriched GO terms are listed.
Figure 5
Figure 5
Statistics of KEGG pathway enrichment. The x-axis shows the enrichment factor. The y-axis corresponds to KEGG Pathway. The color of the dot represents q value and size of the dot represents the number of DEGs mapped to the reference pathways. (A) Top 20 enriched pathways in T. gondii at T1. (B) Top 20 enriched pathways in PK-15 cell at T6.
Figure 6
Figure 6
Protein-protein interaction (PPI) networks of the DEGs identified in the comparison between T1 and T0-T. The PPI network with a high combined score >0.9 was prepared using the STRING 10 database program. DEGs are represented as round nodes. The red node indicates upregulated or green node indicates downregulated EDGs. The node size indicates high interaction degree (large) or low degree (small). Proteins that are associated to each other are linked by an edge. The color of the edge indicates the combined interaction score.
Figure 7
Figure 7
Heat maps of the fold changes of immune (A) or disease (B) related genes with significantly changing expression levels across the entire infection course. DEGs have been divided into three parts: the left part indicates genes that are upregulated at most infection times; the middle part indicates genes that are upregulated or downregulated at all infection times; the right part indicates genes that are downregulated at most infection times. The range of expression is represented by a color grade ranging from low (blue) to high (yellow). The x-axis indicates DEGs and y-axis means different samples. (C) Relative abundance of SLC2A2 as determined by RT-PCR and HiSeq analysis. X-axis indicates different samples. Y-axis indicates log2 fold change values. (D) Relative abundance of SERPINB2 as determined by RT-PCR and HiSeq analysis. X-axis indicates different samples. Y-axis indicates log2 fold change values.

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