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

Abstract

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 (3×CATH_BRALE) attached with hemagglutinin (HA) and 6×His at its C-terminus, producing a recombinant peptide with a molecular weight of ∼18 kDa. The recombinant 3×CATH_BRALE-HA-6×His 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. 3×CATH_BRALE-HA-6×His showed broad-spectrum antibacterial activity against bacteria, with MIC values ranging from 40 to 50 µg/ml for Gram-negative bacteria and 30–40 µg/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, 3×CATH_BRALE-HA-6×His 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.