The Structure And Function Of Bombina Maxima Aerolysin-like Protein Bm-ALP3

Pore-forming protein toxins exist widely in the nature, mainly come from bacterial proteins which can form channel in the cell surface. The pore-forming toxin family is the most widely distributed toxins and maximum toxins in bacterial toxins. They are usually important virulence factors. Aerolysins are important virulence factors belonging to the bacterial β-pore forming toxin superfamily produced by Aeromonas species. Aerolysin-like proteins are the proteins shared similar domains with aerolysins. Aerolysin-like proteins distributed widely from bacteria to vertebrates. Bacteria produce PFTs either to kill other bacteria, or in the case of pathogenic bacteria, to affect their hosts to promote colonization and spreading.In previous studies, we found the first aerolysin-like protein, Bm-ALP1, identified from Bombina maxima skin secretions. Bm-ALP1 and TFF form a complex, which named Py-CAT. The study found that Py-CAT could activate the NLRP3 inflammasome, cause IL-1β that rely on Caspase 1 secretion, then protect the host against microbial infection. But the effect of Py-CAT must be tightly regulated in the host, implying the necessary existence of the inhibitory factors of βγ-CAT pathway in the Bombina maxima. Upon infection, the down-regulation of these unknown specific inhibitory factors may facilitate Py-CAT to exert its functions.Recently, we also identified a homologous protein of Bm-ALP1 from Bombina maxima skin secretions:Bm-ALP3 (18 kD, only contains C-domain), for Bm-ALP3, what functions does it possess in Bombina maxima.To answer this question, we used two approaches:(1) the function research of Bm-ALP3. We mainly express Bm-ALP3 protein in vitro (2) using prokaryotic expression system to preparing natural Bm-ALP3 antibody. We found that, compared with Bm-ALP1, the expression level of Bm-ALP3 is different on immune-related tissues.We used two different challenging methods, we cultivated the frogs in the environment with bacteria, then used quantitative PCR analysis, and showed that the expression level of Bm-ALP3 was down-regulated on skin and blood. A peritonitis model was also used to test the expression level of Bm-ALP3, which revealed that after challenging, the expression level was down-regulated in vivo. These results were also confirmed in subsequent study. In vitro, we expressed Bm-ALP3 and we found Bm-ALP3 could inhibit the activity of Bm-ALPl. Taken together, these findings suggest the existence of a hitherto unknown regulatory pathway in the host innate immunity.