Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and Pelodiscus sinensis ovotransferrins.

In light of the increasing threat of bacterial drug resistance to human health on a global scale, research and development of antimicrobial peptides as a novel class of potent antibiotics has gained considerable attention. The present study focuses on the structural evaluation and membrane interaction of two new cationic antimicrobial peptides, cOT2 and sOT2, derived from Siamese crocodile (Crocodylus siamensis) and Chinese softshell turtle (Pelodiscus sinensis) ovotransferrins. Here, cOT1 (+3) and sOT1 (+3) were derived from reptile ovotransferrins by chromatographic purification and characterized by mass spectrometry and N-terminal sequencing analysis. In order to increase the antimicrobial efficacy, two novel peptides, cOT2 (+6) and sOT2 (+5), were designed and synthesized as "naturally-engineered" by primary amino acid sequence extension of cOT1 and sOT1, respectively. These rational designs of modified peptides were assayed in term of antimicrobial activity. These peptides display strong antimicrobial activity against several bacterial strains, e.g. Vibrio cholerae, Bacillus megaterium, and Bacillus pumilus TISTR 905, with MICs of 7-16.1µM. In terms of structural conformation in mimic environments, CD spectroscopic analysis of the secondary peptides structure features revealed fairly the similarity on α-helical content with magainin II. Hence, the modes of actions have been speculated as toroidal and carpet model. Furthermore, the disruption of intact bacterial cells induced by cOT2 and sOT2 was investigated by SEM and AFM. The results provided evidence that cOT2 and sOT2 have the potential to cause different morphological changes of bacterial cells and that these effects can be enhanced by increasing the peptide concentration.

Identification of five reptile egg whites protein using MALDI-TOF mass spectrometry and LC/MS-MS analysis.

Proteomics of egg white proteins of five reptile species, namely Siamese crocodile (Crocodylus siamensis), soft-shelled turtle (Trionyx sinensis taiwanese), red-eared slider turtle (Trachemys scripta elegans), hawksbill turtle (Eretmochelys imbricate) and green turtle (Chelonia mydas) were studied by 2D-PAGE using IPG strip pH 4-7 size 7 cm and IPG strip pH 3-10 size 24 cm. The protein spots in the egg white of the five reptile species were identified by MALDI-TOF mass spectrometry and LC/MS-MS analysis. Sequence comparison with the database revealed that reptile egg white contained at least seven protein groups, such as serpine, transferrin precursor/iron binding protein, lysozyme C, teneurin-2 (fragment), interferon-induced GTP-binding protein Mx, succinate dehydrogenase iron-sulfur subunit and olfactory receptor 46. This report confirms that transferrin precursor/iron binding protein is the major component in reptile egg white. In egg white of Siamese crocodile, twenty isoforms of transferrin precursor were found. Iron binding protein was found in four species of turtle. In egg white of soft-shelled turtle, ten isoforms of lysozyme were found. Apart from well-known reptile egg white constituents, this study identified some reptile egg white proteins, such as the teneurin-2 (fragment), the interferon-induced GTP-binding protein Mx, the olfactory receptor 46 and the succinate dehydrogenase iron-sulfur subunit.