NA-CATH

Naja atra cathelicidin (NA-CATH) is a 34-amino acid highly cationic peptide identified in Chinese cobras to possess potent toxicity against gram-negative and gram-positive bacteria and low toxicity against host cells. Here, we report the NMR solution structure of the full-length NA-CATH peptide and its interaction with liposomes. The structure shows a well-defined α-helix between residues Phe3 to Lys23, on which one surface is lined by the side-chains of one arginine and 11 lysine residues, while the other side is populated by hydrophobic residues. The last eleven amino acids, which are predominately aromatic and hydrophobic in nature, have no defined structure. NMR data reveal that these residues do not interact with the hydrophobic residues of the helix, indicating that the C-terminal residues have random conformations. Fluorescence requenching experiments, in which liposomes serve as a mimic of the bacterial membranes, result in fluorophore leakage that is consistent with a membrane thinning or transient pore formation mechanism. NMR titration studies of the peptide-liposome interaction reveal that the peptide is in fast exchange with the liposome, consistent with the fluorescent studies. These data indicate that full length NA-CATH possesses a helical segment and unstructured C-terminal tail that disrupts the bilayer to induce leakage and lysing.

Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly "highly resistant" to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis.

Our studies presented in this report focus on the behavior of NA-CATH, an α-helical cathelicidin antimicrobial peptide, originally discovered in the Naja atra snake. It has demonstrated high potency against gram-positive and gram-negative bacteria with minimal hemolysis. Here we examine the kinetics, behaviors and potential mechanisms of the peptide in the presence of membrane liposome, modeling Escherichia coli, whose membrane exhibits distinct lipid phases. To understand NA-CATH interactions, the role of lipid phases is critical. We test three different lipid compositions to detangle the effect of phase on NA-CATH's activity using a series of leakage experiments. From these studies, we observe that NA-CATH changes from membrane disruption to pore-based lysing, depending on phases and lipid composition. This behavior also plays a major role in its kinetics.