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。

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.