Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis

Abstract

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.

Author Summary

Burkholderia species such as B. pseudomallei, which causes melioidosis, and the model organism B. thailandensis are extremely resistant to antibiotics, including cyclic peptide antibiotics such as polymyxin B. Treatment for Burkholderia infections is impeded by this resistance, and new approaches are needed. We hypothesized that the cathelicidin NA-CATH from the Chinese cobra, Naja atra, and smaller derivative peptides (ATRA peptides) may have antimicrobial activity against Burkholderia. We therefore tested the bactericidal effects of the cathelicidin and its derivative peptides. We also wanted to determine whether the antimicrobial peptides exert anti-biofilm activity, although the role of biofilm as a critical virulence factor of Burkholderia has not yet been established. We found that the peptide ATRA-1A, as well as the stereo-isomer D-ATRA-1A, were able to kill B. thailandensis, and the full-length snake cathelicidin NA-CATH was able to both kill B. thailandensis and inhibit its biofilm formation, unlike the human-alpha defensin peptides HNP-1 and HNP-2, and the small peptide derived from hBD3. These results show that the NA-CATH antimicrobial peptide possess bactericidal and anti-biofilm activity against B. thailandensis, and suggest that these compounds should be tested for their effect against the more virulent strains of Burkholderia.