Identification of the first Crocodylus siamensis cathelicidin gene and RN15 peptide derived from cathelin domain exhibiting antibacterial activity

Abstract

Cathelicidins are effector molecules of vertebrate immunity that play vital roles against microbial invasion. They are widely identified in mammals, but few have been reported in Crocodilians, which are considered to be species with a powerful immune system. In the present study, we identified and characterized a novel cathelicidin from the blood of the Siamese crocodile, Crocodylus siamensis. A cDNA sequence (501 base pair) encoded a predicted 166-residue prepropeptide of C. siamensis cathelicidin (Cs-CATH), which comprised a 21-residue signal peptide, a 109-residue cathelin domain, and a 36-residue mature cathelicidin peptide. Multiple sequence alignment and phylogenetic analysis demonstrated that Cs-CATH shared a high degree of similarity with other crocodilian cathelicidins. Joint consideration of elastase cleavage site, physicochemical properties, and predicted secondary structure demonstrated that RN15 peptide is a candidate antimicrobial peptide derived from Cs-CATH. The synthetic RN15 peptide demonstrates antimicrobial activity against Gram-positive and Gram-negative bacteria. Scanning electron microscopy illustrated RN15-peptide-induced bacteria cells exhibited morphological change. Besides, RN15 peptide demonstrates low hemolytic activity against human erythrocytes and low cytotoxic activity against normal human dermal fibroblasts. This is the first cathelicidin identified from C. siamensis, and it is highlighted that its derived peptide from cathelin domain promises potent novel peptide antibiotics templates.

Potential of crocodile blood as a medication and dietary supplement: A systemic review

“…The physical barriers, such as the skin and gut mucosa, prevent the entry of pathogens, whereas chemokines refer to a category of small proteins involved in cell signalling. The documented cytokines in crocodilians include lysozymes, phospholipase A2 (PLA2), antimicrobial peptides (AMPs) (defensins and cathelicidin), complement proteins, and dipeptidyl peptidase IV (DPP4) 20,29,31,57,59‐61 …”

“…Defensins and cathelicidin are two documented AMPs present in crocodilians 60,61 . The positive charge and secondary structure of AMPs allow for preferential electrostatic interactions with the bacterial membrane, which has a stronger negative charge than the somatic cell membrane due to the high density of lipopolysaccharides (LPS) 62 .…”

“…As mentioned in “The Immunity of Crocodiles”, conserved proteins from animals present in plasma or secreted by leukocytes, including lysozyme, 20 hepcidin, 34 phospholipase A2, 29 cathelicidin, 61 and complement proteins, 31 have a wide spectrum of innate immunity against bacteria. Lysozyme was able to inhibit the growth of Psuedomonas aeruginosa and Staph.…”

“…In addition, crocodile complement proteins were also shown to enhance the phagocytic activities of macrophage (RAW 264.7 cells) on bacteria 31 . However, there is a lack of direct evidence to prove the antimicrobial effects for phospholipase A2 29 and cathelicidin 61 isolated from crocodile blood, although they are widely accepted as antimicrobial peptides in other species 79,80 …”

Physicochemical-guided design of cathelicidin-derived peptides generates membrane active variants with therapeutic potential

“…The propensity to form structures in amphipathic α-helices in membrane-mimicking membrane environments has been demonstrated in several studies as an essential factor for the disruptive activity of AMPs 48 . More recently, a smaller derivative of cathelicidin KP36, designated AMP (RN15), also demonstrated promising activity, mostly against Bacillus bacteria, and low hemolytic and cytotoxicity against dermal human dermal fibroblasts 49 . However, for Gram-negative strains, a slight increase of BotrAMP14 over the parental Btn [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] was observed.…”