关键词: 刚地弓形虫, m⁶A修饰, 转录本, 脑包囊, 纳米孔三代测序

Abstract:

Objective To analyze the effect of Toxoplasma gondii infection on the level of N6-methyladenosine （m⁶A） methylation of brain transcripts in mice. Methods C57BL/6J female mice (n = 20) were randomly divided into TgCtwh6 infection group (n = 7), LHG infection group (n = 7) and control group (n = 3 for the control of TgCtwh6 infection and LHG infection group, respectively). The TgCtwh6 infection group and the LHG infection group were inoculated by gavage with 0.2 ml brain tissue suspension (20 cysts/mouse) from the mice infected with T. gondii Chinese Ⅰ genotype wh6 strain or Chinese Ⅲ genotype LHG strain, respectively. The control group was given with the same amount of normal saline. At 15, 30 and 45 days post-infection, one mouse was randomly selected by drawing lots from each infected group, which was sacrificed under anesthesia to collect brain tissue for microscopic examination and counting of the cyst number in the cerebral cortex, hippocampus and olfactory bulb. At 45 days post-infection, the whole brain tissues of 3 mice in each group were collected, the total RNA was extracted, the genome library was prepared, the transcriptome was sequenced, differential methylation loci (DML) were screened, and differential m⁶A methylation sites and transcripts of mRNA in infected group and control group were recorded. The transcripts with methylation sites were analyzed by gene ontology functional annotation (GO), Kyoto Encyclopedia of Gene and Genome (KEGG), and methylated differential transcripts by gene set enrichment analysis (GSEA). Three m⁶A methylation modification protein genes, methyltransferase like 3 (METTL3), fat mass and obesity-associated protein (FTO) and YTH domain family 3 (YTHDF3), were selected, and their relative transcription levels were detected by real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) using the glyceraldehyde-3-phosphate dehydrogenase gene as an internal reference. Results All the mice in the infection group were infected successfully. On day 45 after infection, the cysts in cerebral cortex, hippocampus and olfactory bulb were (5 676 ± 10), (4 773 ± 9) and (243 ± 10), respectively. A total of 760 650 methylation sites were detected in the infection group and control group. Among them, the methylation levels of four m⁶A motifs, GGACA, GGACC and ACGAT, accounted for 16.8% (127 923/760 650), 4.6% (35 164/760 650), 3.8% (28 983/760 650) and 0.4% (3 122/760 650), respectively. A total of 127 016 transcripts with high methylation sites were detected, including 71 727, 27 754, 24 556 and 2 979 transcripts with GGACA, GGACC, GGACT and ACGAT, respectively. The total number of DML sites for four m⁶A motifs, including ACGAT, GGACA, GGACC and GGACT, in the transcriptome of the infected and control group mice brain tissue was 9 233, including 4 832 high methylation sites and 4 851 low methylation sites. The GO analysis showed that the transcripts where DML was located were significantly enriched in cellular processes of biological processes, cell of cellular components, and binding in molecular functions. The KEGG enrichment analysis of the transcripts where DML was located showed that the intermolecular interaction network was mainly enriched in signaling pathway such as lysosome, spliceosomes, dopamine synapses. The GO enrichment analysis of biological processes showed that in the transcripts where DML was located, some transcripts related to biological processes were mainly enriched in processes such as protein glycosylation, negative regulation of neuronal apoptosis and proteion import into nucleus. The GSEA analysis showed the differential methylation between the control group and the two infection groups was highly enriched in transcriptome subsets related to the negative regulation pathway of fibroblast proliferation and Hippo signaling pathway. Among them, four key transcripts, ENSMUST00000105393, ENSMUST00000006523, ENSMUST00000055261 and ENSMUST00000038658, were screened, which encode costimulatory factor ligand (ICOSL), cysteine-rich intestinal protein 1 (CRIP1), MOB kinase activator 1A201 (MOB1A201) and MOB1A202, respectively. Compared with the control group, the expression of these four genes in TgCtwh6 infection group and LHG infection group were all up-regulated. The results of qRT-PCR showed that the relative expression level of mettl3 mRNA in brain tissue of TgCtwh6 infection group and LHG infection group was 5.47 ± 1.09, 1.63 ± 0.06, respectively, which was significantly different from that of the control group (1.01 ± 0.11) (t = 4.05, 5.03; both P < 0.05). The relative expression level of ythdf3 mRNA in the control group was 3.57 ± 0.08 and 1.80 ± 0.25, respectively, which was significantly different from that in the control group (1.01 ± 0.11) (t = 18.95, 2.85; both P < 0.05). The relative expression level of fto mRNA was 0.41 ± 0.04, 0.60 ± 0.12, respectively, which was significantly different from the control group (1.00 ± 0.06) (t = 7.67, 2.99; both P < 0.05). The qRT-PCR results were consistent with the transcriptional trend obtained by transcriptional sequencing. Conclusion Chronic T. gondii infection may enhance the m⁶A methylation in mouse brain transcripts, leading to changes in the biological processes and signal pathways related to host mental behavioural through modifying and regulating key differential transcripts including icosl, crip1 and mob1a.

Key words: Toxoplasma gondii, m⁶A methylation modification, Transcript, Brain cysts, Nanopore third generation sequencing