MBL regulates phagocytosis and bactericidal activity of macrophages by triggering AKT/NF-κB/Rab5A axis occurred early in vertebrate evolution

Mannose-binding lectin (MBL) is a vital and versatile component of innate immunity, which plays an essential role in host defense. In mammals, MBL performs multiple functions, including pattern recognition, complement activation, and phagocytosis. Previous studies revealed that OnMBL can promote the opsonophagocytosis of macrophages through its interaction with calreticulin (CRT) in a complement activation-independent manner in Nile tilapia (Oreochromis niloticus). However, the oligomer structural characteristics of MBL and the pathways involved in immune defense mechanisms remain poorly understood. In this study, we identified different oligomer forms of OnMBL in tilapia serum, with significant increases in trimer and tetramer levels present following immunization with Streptococcus agalactiae. Further investigation demonstrated that a higher degree of OnMBL oligomerization enhanced its ability to bind and phagocytose bacteria. Notably, OnMBL promoted the formation of phagolysosomes, which are responsible for degrading and eliminating ingested bacteria. Additionally, OnMBL knockdown caused a marked downregulation of the CD91a postinfection. Moreover, it is confirmed that OnMBL interacted with OnCRT and OnCD91a, with OnCD91a being essential for the OnMBL/CRT complex to facilitate phagocytosis and bacterial clearance. Mechanistic studies revealed that OnMBL/CRT complex enhanced phagocytosis through collaboration with OnCD91a, triggering a positive feedback loop mediated by the AKT/NF-κB/Rab5A signaling axis, thereby boosting macrophage activities and antibacterial immune responses. Therefore, this study elucidates the antibacterial response mechanism of oligomer OnMBL and its receptor in early vertebrates. These findings add to the knowledge regarding the regulatory mechanisms of C-type lectins in fish and provide valuable insights into the evolution of innate immunity.