两栖动物新型孔道形成毒素类似蛋白ALPs的生物学功能研究

Pore-forming toxins (PFTs) are the most common virulence factors produced by bacteria, especially many pathogenic bacteria. PFTs function to perforate membranes of host cells, predominantly the plasma membrane but also intracellular organelle membranes. Aerolysin is produced by Aeromonas species. The distribution of aerolysin-like proteins (ALPs) was subsequently extended from bacteria to vertebrates, based on the analysis of conserved motifs named aerolysin-like domain. Some creative findings were obtained from our previous study: βγ-CAT，a naturally existing 72 kDa complex of aerolysin-like protein (Bm-ALP1) and trefoil factor（TFF）, was identified from frog Bombina maxima skin secretions. The C-ternal of Bm-ALP1 possess an aerolysin-like domain, so it is an novel ALP from amphibians and this is first report about ALP in the form of complex. In the subsequent functional analysis, we also acquired some fascinating and interesting results, differd with the classical PFTs, βγ-CAT could stimulate innate immune responses and protect the host against pathogen invasion (PNAS. 2014.cover story). Moreover, another aerolysin-like proteins, named Bm-ALP2 and Bm-ALP3, as the homolog of Bm-APL1, were also isolated and purified from Bombina maxima in our previous study. However, the molecular forms and functions of these two proteins were not clear. In our present study, we aimed at: 1) elucidate the molecular forms of Bm-ALP2 and TFF complex, the biological functions, especially the regulation of innate immunity of Bm-ALP2. Moreover, according to study compared with Bm-ALP1, revealed the functional similarity and diversity with Bm-ALP1; 2) study the biological functions of Bm-ALP3, especially the possible regulatory and inhibitory function to Bm-ALP1 or Bm-ALP2 after Bm-ALP3 with Bm-ALP1 or Bm-ALP2 formed complex. This project will provide some scientific evidence and clues for revealing the biological functions and novel innate immune regulatory strategies of novel pore-forming toxin aerolysin-like proteins from vertebrates.

孔道形成毒素是致病菌产生的重要毒力因子, 如产气单胞菌溶素aerolysin。目前发现细菌 aerolysin类似蛋白（ALPs）也广泛表达于脊椎动物中，但其生物学功能未知。我们前期在两栖动物大蹼铃蟾（Bombina maxima）中发现了第一个宿主分泌的ALP（Bm-ALP1）与三叶因子（TFF）复合物βγ-CAT，其具有刺激天然免疫保护宿主抵御微生物感染的重要功能（PNAS，2014，封面故事）；前期也揭示了Bm-ALP1同源蛋白Bm-ALP2和单结构域Bm-ALP3的存在。本项研究旨在，1)阐明Bm-ALP2与TFF形成复合物的分子形式，在激活和调节天然免疫中的功能，并与Bm-ALP1比较研究，揭示它们功能的共性和差异性；2）研究Bm-ALP3与Bm-ALP1或Bm-ALP2结合并抑制和调节后两者的功能。项目为揭示脊椎动物ALP蛋白的生物学功能和天然免疫调节新策略提供线索和科学依据。

Aerolysin孔道形成毒素类似蛋白（ALPs）广泛表达于脊椎动物中，但其生物学功能未知。我们前期在两栖动物大蹼铃蟾（B. maxima）中发现第一个宿主.ALP（BmALP1）与三叶因子（TFF）复合物βγ-CAT，具有刺激天然免疫保护宿主抵御微生物感染的重要功能；前期也揭示了大蹼铃蟾中BmALP1同源蛋白BmALP2和BmALP3的存在，然而其生物学功能未知。因此，本项目要解决的关键科学问题是两栖动物新型孔道形成毒素类似蛋白ALPs（BmALP1， BmALP2和BmALP3）的生物学功能及机制。. 针对以上科学问题，本项目开展以下3个主要研究内容：（1）、BmALP1，BmALP2和BmALP3的分子存在形式鉴定；（2）、BmALP2、BmALP3的生物学功能及与BmALP1功能比较分析；（3）、BmALP3对BmALP1和BmALP2功能的调节抑制作用研究。. 项目按计划执行，进展顺利。分子存在形式鉴定发现BmALP1和BmALP2都是以βγ晶状体蛋白结构域和孔道形成结构域共同组成的融合蛋白形式存在，而BmALP3则是仅由孔道形成结构域组成。生物学功能研究发现BmALP1和TFF复合物βγ-CAT可以内吞入胞并调节内吞溶酶体的pH，从而促进包含胞内菌的内吞囊泡的外排，最终发挥抵御胞内细菌感染的效应（J Infec Dis，2017,215:1753-1763）；进一步的功能研究发现βγ-CAT可促进组织修复（FASEB J， 2019,33:782-795）；βγ-CAT内吞入胞的机制研究发现细胞表面的酸性糖鞘脂可介导其内吞入胞并参与调控了内吞溶酶体的功能（Commun Biol, 2019, 2:59）;新近研究发现BmALP3在氧化条件下可以抑制βγ-CAT的活性进而发挥负调控作用。综上，该项目的开展为揭示脊椎动物来源的 ALP 家族蛋白与 TFF 相互作用， ALP 家族蛋白之间相互作用，生物学功能及调节机制奠定基础。. 目前已发表标注该基金资助SCI期刊论文9篇，发表于Mol Cell Proteomics，J Infect Dis，FASEB J及Commun Biol等专业期刊。该项目的主要成果之一——“云南有毒动物新型肽类毒素发现与功能解析”已获得2018年云南省自然科学二等奖。