中国Ⅲ型弓形虫潜伏性感染前后宿主脑转录组差异分析

Analysis of host brain transcriptome differences before and after latent infection of Toxoplasma gondii Chinese Ⅲ genotype strain

目的分析中国III型弓形虫虫株感染小鼠脑组织前后转录组表达差异,鉴定与小鼠行为变化相关的差异表达基因(differentially expressed genes,DEGs)。方法取感染和未感染中国III型弓形虫LHG株的小鼠脑组织,镜下观察脑包囊;提取总RNA,构建转录组文库,采用Oxford Nanopore Technologies单分子实时测序平台对感染组和对照组文库进行第三代纳米孔高通量测序,以Fold Change≥1.5且P<0.05作为筛选标准,从测序结果中筛选DEGs并对其进行Gene Ontology(GO)富集分析,Kyoto Encyclopedia of Genes and Genomes(KEGG)富集分析和Protein-Protein interaction network(PPI network)蛋白互作网络分析。结果共检测到722个DEGs,其中708个表达上调,14个表达下调。GO功能注释表明DEGs主要富集在宿主的突触刺激应答和免疫应答过程,同时也与行为调控、成瘾、化学突触传递等生物学过程有关。KEGG富集分析表明,多数DEGs富集在寄生虫和病毒感染相关通路。将基因表达差异幅度大、GO注释和KEGG富集排名均靠前的DEGs取交集,共筛选到10个关键上调基因(Cd74、Ccl5及H2家族的H2-Aa、H2-Ab1、H2-Eb1、H2-D1、H2-Q7、H2-K1、H2-DMb1、H2-Qa基因)和5个关键下调基因(Gng4、Frzb、Hes5、Mfge8、Cartpt基因)。对关键DEGs进行PPI互作分析,H2蛋白家族及CD74的蛋白互作连接度大,节点多。结论MHC类分子可能是弓形虫LHG株感染小鼠过程中宿主抵抗其感染的主要因子,弱毒弓形虫株可能通过干扰宿主的神经元干细胞功能和兴奋性神经递质代谢通路导致宿主出现精神行为异常。

Objective To analyze the transcriptome expression differences in the brain tissues of mice before and after infection with the Chinese strain of Toxoplasma gondii III,and to identify the differentially expressed genes(DEGs)related to the behavioral changes ofmice.Methods The brain tissues of mice infected and uninfected with the LHG strain of T.gondii were collected,and the brain cysts were observed under a microscope.TotalRNA was extracted to construct a transcriptome library.The third-generation Nanopore high-throughput sequencing was performed on the libraries of the infected and control groups using the Oxford Nanopore Technologiessingle-molecule real-time sequencing platform.Fold Change≥1.5 and P<0.05 were used as screening criteria.DEGs were screened from the sequencing results andGene Ontology(GO)enrichment analysis was performed.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis and Protein-Protein interaction network(PPI network)Protein interaction networkanalysis.Results A total of 722 DEGs were detected,of which 708 were up-regulated and 14 were down-regulated.GO functional annotation showed that DEGs were mainly enriched in the host synaptic stimulus response and immune response,and were also related to biological processes such as behavioral regulation,addiction,and chemical synapti transmission.KEGG enrichment analysis showed that most DEGs were enriched in parasite and viral infection-related pathways.The intersection of DEGs with large differences in gene expression andtop GO annotation and KEGG enrichment ranking was taken.A total of 10 key up-regulated genes(Cd74,Ccl5 and H2-Aa,H2-Ab1,H2-Eb1,H2-D1,H2-Q7,H2-K1,H2-DMb1,H2-Qa genes)and 5 key down-regulated genes(Gng4,Frzb,Hes5,Mfge8,Cartpt)were screenedGenes.PPI interaction analysis of key DEGs showed that H2 protein family and CD74 protein interaction was highly connected and had many nodes.Conclusion MHC molecules may be the main factor ofhost resistance to T.gondii infection in mice during the infection with the LHG strain of T.gondii.The attenuated T.gondii strain may cause mental and behavioral abnormalities in the host by interfering with the neuronal stem cell function and excitatory neurotransmitter metabolism pathway.