Green algae-derived triple CATH_BRALE multimer protein potently inhibits bacterial growth by permeabilizing the bacterial cell membrane

Salmonoid cathelicidin (termed CATH_BRALE) is a fish-derived antimicrobial peptide (AMP) isolated from Brachymystax lenok. In this study, CATH_BRALE was expressed in Chlamydomonas reinhardtii as a format of three repeats (3xCATH_BRALE) attached with hemagglutinin (HA) and 6xHis at its C-terminus, producing a recombinant peptide with a molecular weight of similar to 18 kDa. The recombinant 3xCATH_BRALE-HA-6xHis remains to be expressed stably in Chlamydomonas cells even after passaging continuously for five months and yields up to 0.21% of the total Chlamydomonas soluble proteins. 3xCATH_BRALE-HA-6xHis showed broad-spectrum antibacterial activity against bacteria, with MIC values ranging from 40 to 50 mu g/ml for Gram-negative bacteria and 30-40 mu g/ml for Gram-positive bacteria. This recombinant peptide had strong thermostability and pH stability, and its antibacterial activity was rarely altered when temperature and pH are changed. It resisted to the digestion of several tested proteases to certain extents. Besides, 3xCATH_BRALE-HA-6xHis is biologically safe as it did not hemolyze rat erythrocytes nor caused cytotoxicity on Vero, BHK21, HEK293, and MDBK cells. Its antibacterial action was achieved by penetrating the cell membrane to disrupt the membrane of the target bacterial cell. In sum, our data showed that C. reinhardtii can be used as a heterologous expression host to produce biologically active CATH_BRALE.

Automated summary

In this study, a fish-derived antimicrobial peptide (AMP), CATH_BRALE, was expressed in Chlamydomonas reinhardtii as a recombinant peptide with stable expression and broad-spectrum antibacterial activity. It showed strong thermostability and pH stability, and did not exhibit toxicity on different cell lines. The results demonstrate the potential of using C. reinhardtii as a host for producing biologically active CATH_BRALE.