Heterotrophic fermentation of a robust human defensin in Chlamydomonas reinhardtii provides a stable and potent antibacterial.

backgroundThe growing threat of antibiotic resistance underscores the urgent need for novel therapeutic agents. Antimicrobial peptides (AMPs), such as human defensins, are promising candidates due to their broad-spectrum activities, but their widespread application is severely hindered by high production costs and limited stability. Among these, human neutrophil peptide-2 (HNP2), a member of the α-defensin family, is a particularly potent AMP that kills microorganisms by disrupting their membrane integrity. The eukaryotic microalga Chlamydomonas reinhardtii (C. reinhardtii) has emerged as a low-cost and efficient bioreactor for exogenous protein production, yet its potential for expressing human defensins remains largely unexplored. This study aimed to express a tandem trimer of the HNP2 mature peptide (3×HNP2) in C. reinhardtii and to characterize its stability, safety, and antibacterial functions.

resultsIn this study, a gene encoding 3×HNP2 was successfully expressed in the C. reinhardtii strain CC-5325, yielding a fusion protein of approximately 22 kDa. The recombinant protein's expression remained stable for over five months of continuous subculturing. Obtained via fermentation technology and affinity purification, the purified 3×HNP2 demonstrated potent antibacterial activity against the Gram-negative pathogens Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The protein exhibited high thermal stability (up to 90 °C), broad pH tolerance (pH 2-10), and resistance to degradation by proteinase K and papain. Mechanistic investigations using propidium iodide (PI) staining and scanning electron microscopy (SEM) confirmed that 3×HNP2 acts by disrupting bacterial membrane integrity. Furthermore, qRT-PCR analysis revealed that 3×HNP2 significantly downregulated the expression of key virulence-associated genes (lecA, phzA2, csgA, and rpoS). Biosafety assays showed that the peptide had minimal hemolytic activity and low cytotoxicity against mammalian cell lines.

conclusionsThis work establishes a green platform for producing a functional human defensin in C. reinhardtii. It demonstrates that algae-derived 3×HNP2 is a stable, safe, and effective antimicrobial agent, suggesting its potential as a candidate for further development in antimicrobial therapeutics or food safety applications.