Chlamydomonas reinhardtii-derived triple CruFc distorts membrane integrity for inhibiting bacterial growth

Abstract Background Antimicrobial resistance (AMR) has become an escalating global public health crisis. As promising alternatives to conventional antibiotics, antimicrobial peptides (AMPs) have received extensive attention due to their low propensity to trigger bacterial resistance and favorable safety profiles. A crustin-like protein (CruFc), identified from the plasma of Fenneropenaeus chinensis , can effectively inhibit the growth of Gram-positive bacteria but shows limited inhibitory effect on Gram-negative bacteria. Results In this study, we expressed a recombinant polypeptide 3×CruFc-HA-6×His in Chlamydomonas reinhardtii , which consists of three copies of CruFc, a hemagglutinin (HA) tag, and a 6×histidine (6×His) tag. We verified the inhibitory activity of 3×CruFc-HA-6×His against both Gram-negative and Gram-positive bacteria, with its minimum inhibitory concentration (MIC) ranging from 10 to 50 µg/mL. Additionally, 3×CruFc-HA-6×His exhibited stability under different temperature and pH conditions, as well as excellent resistance to digestion by various proteases. When tested on different cell lines, it also showed low cytotoxicity and hemolysis. Furthermore, scanning electron microscopy (SEM) and propidium iodide (PI) staining both confirmed that 3×CruFc-HA-6×His can disrupt bacterial cell membranes, leading to bacterial death. Conclusion In summary, the recombinant trimeric peptide 3×CruFc‑HA‑6×His heterologously expressed in C. reinhardtii displays favorable biosafety and a low risk of triggering bacterial resistance, holding preliminary promise as a candidate antimicrobial agent for further preclinical development.