Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against <i>Burkholderia thailandensis</i>

<div><p><i>Burkholderia thailandensis</i> is a Gram-negative soil bacterium used as a model organism for <i>B</i>. <i>pseudomallei</i>, 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. <i>Burkholderia</i> 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 <i>in vitro</i> against <i>B</i>. <i>thailandensis</i> for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (<i>Naja atra</i>) cathelicidin NA-CATH was significantly antimicrobial against <i>B</i>. <i>thailandensis</i>. 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 <i>B</i>. <i>thailandensis</i>. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of <i>B</i>. <i>thailandensis</i>. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although <i>B</i>. <i>thailandensis</i> 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 <i>B</i>. <i>thailandensis</i>.</p></div

Antimicrobial activity and confidence intervals of the peptide panel.

<p>Scrambled LL-37, ATRA-2, and HNP-2 are not shown as no EC50 could be determined.</p><p>Antimicrobial activity and confidence intervals of the peptide panel.</p

Biofilm activity of cathelicidins against <i>B</i>. <i>thailandensis</i>.

<p>Inhibition of biofilm is demonstrated for the cathelicidins, <b>C.</b> NA-CATH, <b>D.</b> SMAP-29, <b>E.</b> LL-37, and <b>F.</b> D-LL-37, while controls <b>A.</b> the antibiotic ceftazidime, and <b>B.</b> scrambled LL-37 did not show biofilm inhibition. Growth (absorbance at 600 nm) is shown by black bars; growth with no peptide was set to 100%. Biofilm (gray bars) was detected on a polystyrene 96-well plate at 37°C after 48 h of growth in MVBM and detected as absorbance of crystal violet stain (590 nm). Each experiment is representative of 3 individual experiments.</p

Cathelicidin peptide rescues <i>G. mellonella</i> infected with <i>B. anthracis</i>

Cathelicidin peptide rescues <i>G. mellonella</i> infected with <i>B. anthracis</i

Effect of D-enantiomer on antimicrobial activity.

<p><i>B</i>. <i>thailandensis</i> was incubated for 3 h with various peptide concentrations in 10 mM sodium phosphate buffer (pH 7.4); percent (%) survival was calculated as the ratio of CFUs before and after incubation. <b>(A)</b> EC50 for ATRA-1A and for D-ATRA-1A. <b>(B)</b> EC50 for LL-37 and D-LL-37.</p

Biofilm activity of cathelicidins against <i>B</i>. <i>thailandensis</i>.

<p>Inhibition of biofilm is demonstrated for the cathelicidins, <b>C.</b> NA-CATH, <b>D.</b> SMAP-29, <b>E.</b> LL-37, and <b>F.</b> D-LL-37, while controls <b>A.</b> the antibiotic ceftazidime, and <b>B.</b> scrambled LL-37 did not show biofilm inhibition. Growth (absorbance at 600 nm) is shown by black bars; growth with no peptide was set to 100%. Biofilm (gray bars) was detected on a polystyrene 96-well plate at 37°C after 48 h of growth in MVBM and detected as absorbance of crystal violet stain (590 nm). Each experiment is representative of 3 individual experiments.</p

Antimicrobial activity of NA-CATH derivatives against <i>B thailandensis</i>.

<p><i>B</i>. <i>thailandensis</i> was incubated for 3 h with various peptide concentrations in 10 mM sodium phosphate buffer (pH 7.4); percent (%) survival was calculated as the ratio of CFUs before and after incubation. (<b>A</b>) EC50 of ATRA-1 and ATRA-1A. (<b>B</b>)ATRA-2 did not exhibit antimicrobial activity.</p

Antimicrobial activity of a panel of defensins against <i>B</i>. <i>thailandensis</i>.

<p><i>B</i>. <i>thailandensis</i> was incubated for 3 h with various peptide concentrations in 10 mM sodium phosphate buffer (pH 7.4); percent (%) survival was calculated as the ratio of CFUs before and after incubation. EC50 for these peptides could not be calculated because the peptide was ineffective. (<b>A</b>) HNP-1, HNP-2, peptide 4 of hBD3 are depicted. (<b>B</b>) HNP-3 and HNP-4 are shown.</p