Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells’ protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.此前我们已鉴定出溶血素（lysenin）是赤子爱胜蚓（Eisenia fetida）体腔细胞分泌组（secretome）的关键蛋白组分，并揭示其在介导细胞与纳米银周围蛋白冠（protein corona）之间相互作用时的核心调控作用。鉴于蛋白微环境的改变可直接影响蛋白冠的组成，纳米颗粒对细胞蛋白分泌谱的影响程度始终是一个极具研究价值却鲜有关注的科学问题。本研究以低细胞毒性浓度的代表性纳米银材料NM-300K（粒径15 nm）处理赤子爱胜蚓体腔细胞，以2、4、8及24小时的时间梯度探究其转录响应，并重点结合溶血素的表达变化，分析分泌蛋白转录谱的时序变化意义。实验发现，NM-300K可在细胞内及细胞表面积累；溶血素在经历瞬时诱导表达后，随培养时间延长逐渐被抑制，提示存在负反馈循环。该过程或可限制溶血素在蛋白冠中的进一步富集，进而降低溶血素介导的纳米颗粒摄取效率。其余差异表达基因涉及金属应激通路（与硝酸银处理的细胞应激反应一致）及Toll样受体（Toll-like receptor，TLR）信号通路。本研究为蚯蚓体内纳米银的病理生理机制提供了新颖视角，其中保守的模式识别受体TLRs可能在此过程中发挥效应子功能。