Molecular and functional characterization of a bactericidal/ permeability-increasing protein 2 (BPI2) from American eel (Anguilla rostrata)

Bactericidal/permeability-increasing protein (BPI) is an antimicrobial protein showing bactericidal, opsonic and anti-inflammatory activities with therapeutic potential. However, the potential role of BPI on immunological properties in teleost is not fully understood. In this study, a BPI2-like gene was successfully cloned from American eel (Anguilla rostrata), which contains an open reading frame (ORF) of 1422 bp encoding 473 amino acids. The ArBPI2 consists of a signal peptide of 19 amino acids, an N-terminus LPS-binding domain, a C-terminal domain, and a basic pI of 9.0, typical attribute of BPI proteins found in mammals. The N-terminal domain of ArBPI2 (27–249 amino acids, ArBPI2-ND) was produced as a recombinant protein (rArBPI2-ND) and its antimicrobial activity was performed. ArBPI2-ND exhibited a strong affinity for LPS, and had a broad binding capacity for both Gram-negative and Gram-positive bacteria. The minimum inhibitory concentration (MIC) assays revealed that rArBPI2-ND exerted stronger antibacterial activity on Gram-negative bacteria than on Gram-positive bacteria. Further study revealed that rArBPI2-ND exerted its bactericidal activity against Aeromonas veronii by inhibiting the formation of bacterial biofilms, increasing the outer membrane permeability, destroying the cell membrane structure and binding to genomic DNA, leading to cytoplasmic leakage and bacterial death. This is the first report on the molecular characteristics, biological functions, and bactericidal mechanism of the N-terminal domain of a BPI protein from A. rostrata.