巨噬细胞是先天性免疫系统的重要组成部分,但无乳链球菌(Streptococcus agalactiae)却能在巨噬细胞内存活并以巨噬细胞为载体入侵中枢神经系统.为解析无乳链球菌在巨噬细胞内的存活机制,将无乳链球菌HN016与RAW264.7共孵育,提取胞内细菌RNA后进行转录组测序(RNA-seq)、Gene Ontology(GO)及Kyoto encyclopedia of genes and genomes(KEGG)富集分析;于体外将HN016与罗非鱼原代巨噬细胞孵育,提取胞内细菌RNA,并利用quantitative real-time PCR(qPCR)验证目标基因(包括:sip、fbsA、cylB、cylD、cylE、neuA、cpsB和cfb)表达情况.结果显示,与无处理组相比,共筛选到1 215个差异表达基因(DEG),其中896个上调基因,319个下调基因.GO富集结果显示,DEG在分子功能、生物学过程和细胞组分3大类中均显著富集.KEGG富集结果显示,显著富集通路主要为ABC转运体、核糖体和群体感应等代谢途径.在DEG中,筛选到无乳链球菌毒力相关基因27个,包括fbsA(+8.65)、sip(+6.28)、cylD(+4.93)和cfb(-4.65)等,并利用qPCR验证RNA-seq结果,二者数据一致.而检测罗非鱼原代巨噬细胞内存活细菌的转录水平发现其目标基因的表达水平与小鼠巨噬细胞的相似.因此推测,无乳链球菌在被巨噬细胞吞入后,巨噬细胞内的有害环境增强了无乳链球菌的信号传导机制,刺激了无乳链球菌的能量运输和对巨噬细胞所产生的次生代谢物代谢的能力,并且提高了毒力相关基因的表达水平.
Transcriptome analysis of Streptococcus agalactiae surviving in mouse macrophages
Macrophage is an essential component of the innate immune system.However,Streptococ-cus agalactiae has the ability to survive within macrophage,allowing it to infiltrate the central nervous sys-tem.To elucidate the mechanisms underlying the survival of S.agalactiae within macrophages,we investi-gated the genome-wide transcriptional changes of S.agalactiae following phagocytosis by RAW264.7 and explored the metabolic pathways and genes associated with the intracellular survival of S.agalactiae.The S.agalactiae strain HN016 was co-incubated with RAW264.7 and the intracellular bacteria were subjected to transcriptome sequencing(RNA-seq),gene ontology(GO),and Kyoto encyclopedia of genes and ge-nomes(KEGG)enrichment analysis.Additionlly,HN016 was incubated with primary tilapia macrophages in vitro and RNA from the intracellular bacteria were extracted.Quantitative real-time PCR(qPCR)was used to verify the expression of the target genes.The results showed that a total of 1 215 differentially ex-pressed genes(DEGs),including 869 up-regulated genes and 319 down-regulated genes,were screened,compared with the untreated group.DEGs were significantly enriched in all three Go terms:molecular func-tion,biological process,and cellular component.KEGG enrichment analysis showed that the main en-riched pathways were ABC transporter,ribosome,quorum sensing,and glycolysis/gluconeogenesis.Among the DEGs,27 virulence-related genes were screened,including fbsA(+8.65),sip(+6.28),cylD(+4.93),and cfb(-4.65).qPCR was used to validate the RNA-seq results,and the agreement be-tween two datasets demonstrated the reliability of the RNA-seq analysis.The transcript levels of surviving bacteria in primary macrophages of tilapia were similar to those in mouse macrophages.Based on these find-ings,it was hypothesized that the noxious environment within macrophage enhanced the signaling mecha-nisms,energy transport capacity,and metabolic capacity of secondary metabolites produced by macro-phages,as well as the expression levels of virulence-related genes of S.agalactiae.
Streptococcus agalactiaemouse leukemia cells of monocyte macrophage RAW264.7in-tracellular survivalsecondary metabolite
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