Abstract
Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent β-strand of the first “finger” of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50–6.3 μg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.
Highlights
The growing concern regarding increasing microbial antibiotic resistance is occurring worldwide [1,2]
Antimicrobial peptides (AMPs) possess a broad spectrum of antimicrobial activities against Gram-negative and Gram-positive bacteria, viruses, fungi and parasites [7].The main mechanism of action of AMPs against bacteria begins with an electrostatic interaction between cationic portions of antimicrobial peptides and negatively charged structures exposed on the surface of bacterial membranes
The aim of this study is focused on the development and the characterization of novel antimicrobials peptides, designed starting from cardiotoxin 1 (CTX-1) of the Chinese cobra (Naja atra atra), in order to enhance its bactericidal activity and decrease the cytotoxicity showed by the ancestor against eukaryotic cells
Summary
The growing concern regarding increasing microbial antibiotic resistance is occurring worldwide [1,2]. Antibiotic resistance is often associated with marked morbidity and mortality in humans and animals and the number of resistant microorganisms is constantly growing [3,4]. Antimicrobial peptides (AMPs) possess a broad spectrum of antimicrobial activities against Gram-negative and Gram-positive bacteria, viruses, fungi and parasites [7].The main mechanism of action of AMPs against bacteria begins with an electrostatic interaction between cationic portions of antimicrobial peptides and negatively charged structures exposed on the surface of bacterial membranes. In Gram-negative bacteria the mechanism involves anionic phospholipids and LPS-associated phosphate groups exposed on the outer membrane surface. In Gram-positive bacteria, lacking outer membrane or LPS, AMPs are capable to interact with negatively charged teichoic and teichouronic acids of the cell envelope [8,9]
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