基于转录组与代谢组联合分析马尔尼菲篮状菌毒力蛋白Mp1p对巨噬细胞的影响

Combined analysis of transcriptome and metabolome on the effect of virulence protein Mp1p from Talaromyces marneffei on macrophages

目的通过转录组学联合代谢组学探索Mp1p对宿主巨噬细胞的影响。方法使用慢病毒构建稳定表达Mp1p的THP-1巨噬细胞株(THP-1-Mp1p^(+))。采用RNA高通量测序(RNA sequencing,RNA-Seq)技术,在转录组学分析中,检测细胞内mRNA的表达量,确定差异表达基因;在代谢组学分析中,通过数据库比对进行代谢物鉴定,对差异代谢物进行通路分析,揭示潜在作用机制。将代谢组学和转录组学结果联合分析,将差异代谢物和差异基因进行联合分析,进一步阐述Mp1p对巨噬细胞的作用机制。结果转录组分析表明,THP-1-Mp1p^(+)组相较于阴性对照组,共有1180个差异基因,上调基因345个、下调基因835个。基因本体论(gene ontology,GO)富集分析显示,差异表达基因有135个,其中BP 105个、CC 28个、MF 2个。京都基因和基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)富集分析结果显示,Mp1p对THP-1巨噬细胞的作用与肿瘤坏死因子信号通路高度相关。代谢组分析发现,正、负离子模式下空白对照组和THP-1-Mp1p^(+)巨噬细胞组在质量控制样本之间都能达到很好的分离。显著差异的阈值为:VIP≥1且T-test P<0.05,筛选出差异代谢物488个,其中正离子模式230个、负离子258个,将鉴定出的代谢产物进行通路富集分析,显著富集到了代谢途径上。联合分析结果显示,发现肿瘤坏死因子信号通路、白介素-17信号通路、NFkappaB信号通路是其中重要的代谢途径。结论毒力因子Mp1p可能通过调节肿瘤坏死因子信号通路、白介素-17信号通路、NF-kappaB信号通路影响宿主巨噬细胞。研究结果有助于进一步理解Mp1p的作用机制,并为未来筛选相关的诊断和治疗靶点提供了可能的方向。

Objective To explore the effect of Mp1p on host macrophages through transcriptomics combined with metabolomics.Methods Firstly,a THP-1 macrophage strain(THP-1-Mp1p^(+))stably expressing Mp1p was constructed using lentivirus.Secondly,using high-throughput RNA sequencing(RNA Seq)technology,the expression level of intracellular mRNA was detected in transcriptomics analysis to determine differentially expressed genes;In metabolomics analysis,metabolite identification was performed through database comparison,and pathway analysis was performed on differential metabolites to reveal potential mechanisms of action.Finally,the results of metabolomics and transcriptomics were combined for analysis,and differential metabolites and genes were analyzed to further elucidate the mechanism of action of Mp1p on macrophages.Results Transcriptome analysis showed that,compared with the negative control group,the THP-1-Mp1p^(+)group had a total of 1180 differentially expressed genes(DEGs),with 345 upregulated genes and 835 downregulated genes.GO enrichment analysis of DEGs showed that there were 135 differentially expressed genes,including 105 in biological processes(BP),28 in cellular components(CC),and 2 in molecular functions(MF).The KEGG analysis results showed that the effect of Mp1p on THP-1 macrophages was highly correlated with the TNF pathway.The metabolomic analysis found that both the blank control group and the THP-1-Mp1p^(+)macrophage group achieved good separation between QC samples in both positive and negative ion modes.The threshold for significant differential metabolites was set at:VIP≥1 and T-test P<0.05,resulting in the identification of 488 differential metabolites,with 230 in the positive ion mode and 258 in the negative ion mode.Pathway enrichment analysis of the identified metabolites pointed to significant enrichment in metabolic pathways.The combined analysis confirmed that the tumor necrosis factor signaling pathway,interleukin-17 signaling pathway,and NFkappaB signaling pathway were important metabolic pathways involved.Conclusions The virulence factor Mp1p may affect host macrophages by modulating the tumor necrosis factor signaling pathway,interleukin-17 signaling pathway,and NFkappaB signaling pathway.The findings contribute to a better understanding of the mechanisms of action of Mp1p and may offer potential directions for the selection of relevant diagnostic and therapeutic targets in the future.