Antibacterial properties and mechanisms of anti-lipopolysaccharide factor 1 from Procambarus clarkii

Antimicrobial peptides (AMPs) are tiny effectors in host defense by directly targeting bacteria or by indirectly regulating immune responses. Anti-lipopolysaccharide factors (ALFs) are crucial antimicrobial peptides in crustaceans, exhibiting broad-spectrum antimicrobial activity. However, the sequence characteristics, antimicrobial properties, and antimicrobial mechanisms of some key ALF members in Procambarus clarkii have not yet been well clarified. In the present study, anti-lipopolysaccharide factor 1 from P. clarkii (PcALF1) was firstly identified, then its antibacterial characteristics and potential antibacterial mechanism was investigated. PcALF1 is 1271 bp in full length and encodes 122 amino acids, and contains a conserved lipopolysaccharides binding domain (LBD) consisting of 24 amino acids. Pc‌ALF1 is widely expressed in various tissues of P. clarkii, particularly in immune-related tissues, such as hemocytes and hepatopancreas. Upon bacterial infection, PcALF1 expression significantly increases, with a lower response level to G^- bacteria compared to G⁺ bacteria. Both recombinant PcALF1 (rPcALF1) or synthetic LBD peptides (LBD1) showed broad antimicrobial activity towards two G⁺ bacteria and three G^- bacteria, additionally, their bactericidal effect were more potent against G^- species compared to G⁺ counterparts. PcALF1 knockdown impairs host defense against Aeromonas hydrophila, whereas exogenous supplementation of either rPcALF1 or LBD1 restores immune function. Surprisingly, PcALF1 exerted significant regulatory effects on the intestinal microbiota, leading to a shift in the composition of the diseased intestinal microbiota towards a healthy state. We further investigated the bactericidal mechanisms of rPcALF1 and LBD1 against A. hydrophila. The antibacterial effect against A. hydrophila was characterized by irreversible membrane damage, resulting in cytoplasmic leakage and bacterial deformation, as observed via electron microscopy. Notably, transcriptomic analysis indicated a multi-target action on central metabolism and transport, pointing to a mechanism that may inherently circumvent traditional resistance pathways. In summary, PcALF1 is expected to become an effective antibacterial agent, particularly crucial for treating P. clarkii infected with A. hydrophila.