Transcriptome analysis of mice brain chronically infected with Toxoplasma gondii and validation of the kynurenine pathway associated with depression

doi:10.12140/j.issn.1000-7423.2023.03.002

Abstract

Abstract:

Objective To screen the differentially expressed genes (DEGs) by comparing the transcriptome of the brain tissues between the mice chronically infected with Toxoplasma gondii and normal mice, to analyze the relative transcription level of DEGs in the depression-related kynurenine (KYN) pathway and to provide a theoretical basis for exploring the mechanism of depression-like symptoms caused by Toxoplasma gondii chronic infecttion in mice. Methods SV129 male mice (n = 18) were randomly and equally divided into the infection group and the control group. Mice in the infection group were intraperitoneally injected with 120 tachyzoites of T. gondii ME49 strain (200 μl), and mice in the control group were injected with the same volume of PBS. After 3 months post-infection, mice brain tissues of the two groups were collected for extraction of total RNA to undertake transcriptome sequencing for screening DEG. With the DEGs obtained, cluster analysis, gene ontology (GO) functional annotation analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analysis were performed. Eight DEGs [interferon-γ (IFN-γ), indoleamine 2,3-dioxygenase 1 (IDO1), IDO2, kynurenine-3-monooxygenase (KYNU), kynurenine-3-monooxygenase (KMO), 3-hydroxyanthranilate 3,4-dioxygenase (3-HAO), vimentin (Vim) and brain-derived neurotrophic factor (BDNF)] related to KYN pathway associated with depression were selected to examine each gene’s relative transcription level by quantitative real-time PCR (qRT-PCR), using glyceraldehyde-3-phosphate dehydrogenase gene as an internal reference. Results Transcriptome sequencing found 2 295 DEGs in the brain of the mice from the infection and control groups, of which 2 016 were up-regulated and 279 were down-regulated. GO analysis showed that localisation was the most significantly enriched biological process, with a total of 257 DEGs. The most significantly enriched in cellular components was the protein-containing complex, with a total of 425 DEGs. The most significantly enriched molecular function was molecular transducer activity, with 177 DEGs. The largest number of DEGs enriched in biological process, cell component and molecular function were cell process, cell part and binding, with 1 039, 1 240 and 1 088 DEGs, respectively. KEGG analysis showed that the top three up-regulated metabolic pathways were the immune system, signaling transduction, and viral infectious disease, and the top three down-regulated pathways were signal transduction, signaling molecules and interaction and immune system. Functional enrichment analysis showed that 77 pathways were significantly enriched. The signaling pathways related to depression include tumor necrosis factor signaling pathway, neuroactive ligand-receptor interaction, NF-kappa B signaling pathway, JAK-STAT signaling pathway, necroptosis, apoptosis, chemokine signaling pathway, KYN pathway. The qRT-PCR results showed that the relative transcription levels of IFN-γ, IDO1, IDO2, KYNU, KMO, 3-HAO and Vim genes in the infection group were 3 023.08%, 355.52%, 190.17%, 496.55%, 339.92%, 212.74% and 507.34%, if the relative transcript level of control mice was taken as 100%. Compared with the control group, the transcription was significantly up-regulated (t = 3.782, 3.749, 3.226, 2.908, 2.533, 5.656, 2.948; all P < 0.05 or 0.01). The relative transcription level of BDNF was 63.32%, which was significantly down-regulated (t = 2.398, P < 0.05). The fold change of IFN-γ, IDO1, IDO2, KYNU, KMO, 3-HAO, BDNF, Vim obtained by qRT-PCR was 4.96, 1.74, 0.89, 2.10, 1.60, 1.06, -0.94, 2.18, respectively. The fold change obtained by transcriptome sequencing was 7.30, 0.55, 0.80, 3.83, 2.75, 3.53, -0.86 and 1.93, respectively. The transcriptional trend obtained by qRT-PCR was consistent with that obtained by transcriptome sequencing. Conclusion DEGs from brain tissues of mice chronically infected with T. gondii were screened. Transcriptome analysis revealed that the immune response of central nervous system of the mice with chronic infection of T. gondii was continuously activated. Seven DEGs in KYN pathway related to depression showed up-regulated transcription level.

Key words: Toxoplasma gondii, Mice brain tissue, Transcriptome, Depression, Kynurenine pathway

CLC Number:

R382.5

ZHANG Chi, CHEN Jiating, XIN Zixuan, YANG Lili, YANG Zihan, PENG Hongjuan. Transcriptome analysis of mice brain chronically infected with Toxoplasma gondii and validation of the kynurenine pathway associated with depression[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(3): 270-278.

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基因名称 Gene name	GenBank登录号 GenBank accession No.	引物序列（5'→3'） Primer sequence (5'→3')
甘油醛-3-磷酸脱氢酶 Glyceraldehyde-3-phosphate dehydrogenase	NM_008084.4	F: AGGTCGGTGTGAACGGATTTG R: TGTAGACCATGTAGTTGAGGTCA
γ-干扰素 Interferon-gamma	NM_008337.4	F: GCCACGGCACAGTCATTGA R: TGCTGATGGCCTGATTGTCTT
吲哚胺2,3-双加氧酶1 Indoleamine 2,3-dioxygenase 1	NM_008324.3	F: GCCTCCTATTCTGTCTTATGCAG R: ATACAGTGGGGATTGCTTTGATT
吲哚胺2,3-双加氧酶2 Indoleamine 2,3-dioxygenase 2	NM_145949.2	F: TCAAAGTCAGAGCATGACGCT R: GGCGGTTCTCGATTAAGTGAG
犬尿氨酸酶 Kynureninase	NM_027552.3	F: GTCAAGCCTGCGTTAGTGG R: GGAGGGTTTGAAATTCGGAATCC
犬尿氨酸-3-单加氧酶 Kynurenine 3-monooxygenase	NM_133809.1	F: ATGGCATCGTCTGATACTCAGG R: AGCTTCGTACACATCAACTTGAA
3-羟邻氨苯甲酸加双氧酶 3-hydroxyanthranilate 3,4-dioxygenase	NM_025325.2	F: GAACGCCGTGTGAGAGTGAA R: CCAACGAACATGATTTTGAGCTG
脑源性神经营养因子 Brain derived neurotrophic factor	NM_001048141.1	F: TTACCTGGATGCCGCAAACAT R: TGACCCACTCGCTAATACTGTC
波形蛋白 Vimentin	NM_011701.4	F: TCCACACGCACCTACAGTCT R: CCGAGGACCGGGTCACATA

样品 Sample	过滤后序列 Clean reads	有效碱基数 Clean bases	质量值≥ 30的碱基占比/% Q30/%	GC含量/% GC content/%	比对序列总数（总比对率/%） Total mapped reads（Total mapped rate/%）	唯一比对序列数（唯一比对率/%） Uniquely mapped reads（Uniquely mapped rate/%）
C1	60 348 640	8 633 462 674	95.43	51.57	57 807 295（95.79）	54 855 233（90.90）
C2	61 001 728	8 826 547 002	95.68	51.58	58 482 793（95.87）	55 704 439（91.32）
C3	47 872 426	7 054 290 403	93.07	51.34	45 777 160（95.62）	43 662 798（91.21）
I1	57 527 866	8 375 627 414	95.56	52.33	54 932 387（95.49）	52 527 951（91.31）
I2	65 226 576	9 504 352 717	95.59	51.74	62 245 261（95.43）	59 337 049（90.97）
I3	59 326 802	8 627 270 056	95.68	51.94	5 6301 394（94.90）	53 835 983（90.74）