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

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

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

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

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

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

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

Chitinases Are Negative Regulators of <i>Francisella novicida</i> Biofilms

<div><p>Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of <i>Francisella tularensis.</i> Chitinases of <i>F. tularensis</i> ssp. <i>novicida</i> (<i>Fn</i>) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that <i>Fn</i> chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the <i>chi</i> mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. <i>Fn</i> biofilm was sensitive to chitinase, proteinase and DNase, suggesting that <i>Fn</i> biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of <i>Fn</i> biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in <i>Francisella</i> species.</p></div

Figure 4

<p>(<b>A</b>) EPS contents of the cells and (<b>B</b>) culture supernatants of the strains. EPS contents were determined by phenol extraction followed by phenol-sulfuric acid method for carbohydrates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093119#s4" target="_blank">Materials and Methods</a>. (<b>C</b>) Lectin binding assay to biofilms. FITC-Con A and FITC-WGA lectins were used for biofilm binding. Lectin binding capacity to biofilms was measured by a fluorescence plate reader and calculated relative fold to WT binding. Fluorescence microscopic images of biofilms of WT, <i>chi</i>A and <i>chi</i>B grown in TC plate are shown in the top panel. Biofilms in the TC plate were shown by CV staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093119#pone.0093119.s001" target="_blank">Fig. S1C</a>). Scale bar, 100 μm.</p