Extreme antimicrobial peptide and polymyxin B resistance in the genus Burkholderia

The opportunistic bacteria of the Burkholderia cepacia complex (Bcc) are extremely pathogenic to cystic fibrosis (CF) patients, and acquisition of Bcc bacteria is associated with a significant increase in mortality. Treatment of Bcc infections is difficult because the bacteria are multidrug resistant and able to survive in biofilms. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an innate defense protein that is secreted by the upper airways and pharynx. While SPLUNC1 is known to have antimicrobial functions, its effects on Bcc strains are unclear. We therefore tested the hypothesis that SPLUNC1 is able to impair Bcc growth and biofilm formation. We found that SPLUNC1 exerted bacteriostatic effects against several Bcc clinical isolates, including B. cenocepacia strain J2315 (50% inhibitory concentration [IC 50 ] ‫؍‬ 0.28 M), and reduced biofilm formation and attachment (IC 50 ‫؍‬ 0.11 M). We then determined which domains of SPLUNC1 are responsible for its antimicrobial activity. Deletions of SPLUNC1's N terminus and ␣6 helix did not affect its function. However, deletion of the ␣4 helix attenuated antimicrobial activity, while the corresponding ␣4 peptide displayed antimicrobial activity. Chronic neutrophilia is a hallmark of CF lung disease, and neutrophil elastase (NE) cleaves SPLUNC1. However, we found that the ability of SPLUNC1 to disrupt biofilm formation was significantly potentiated by NE pretreatment. While the impact of CF on SPLUNC1-Bcc interactions is not currently known, our data suggest that understanding this interaction may have important implications for CF lung disease.

Section: Resultsmentioning

confidence: 68%

Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex

Ahmad

Tyrrell

Walton

et al. 2016

“…Bcc organisms resist killing in vitro by CGD neutrophils but not normal neutrophils (43). This correlates with the ability of Bcc organisms to resist killing in vitro by high concentrations of cationic peptides and polymyxin B (25).…”

mentioning

confidence: 78%

“…The intrinsic resistance of Bcc species to polycationic antimicrobials is due to features of their LPS and outer membrane structure (23,25,30,33). This study suggests a role for membrane hopanoids in modulating B. multivorans membrane permeability that contributes to high levels of polymyxin B and colistin resistance in an LPS-independent manner.…”

Section: Discussionmentioning

confidence: 99%

Identification of Hopanoid Biosynthesis Genes Involved in Polymyxin Resistance in Burkholderia multivorans

Malott

Steen-Kinnaird

Lee

et al. 2012

A major challenge to clinical therapy of Burkholderia cepacia complex (Bcc) pulmonary infections is their innate resistance to a broad range of antimicrobials, including polycationic agents such as aminoglycosides, polymyxins, and cationic peptides. To identify genetic loci associated with this phenotype, a transposon mutant library was constructed in B. multivorans ATCC 17616 and screened for increased susceptibility to polymyxin B. Compared to the parent strain, mutant 26D7 exhibited 8-and 16-fold increases in susceptibility to polymyxin B and colistin, respectively. Genetic analysis of mutant 26D7 indicated that the transposon inserted into open reading frame (ORF) Bmul_2133, part of a putative hopanoid biosynthesis gene cluster. A strain with a mutation in another ORF in this cluster, Bmul_2134, was constructed and named RMI19. Mutant RMI19 also had increased polymyxin susceptibility. Hopanoids are analogues of eukaryotic sterols involved in membrane stability and barrier function. Strains with mutations in Bmul_2133 and Bmul_2134 showed increased permeability to 1-N-phenylnaphthylamine in the presence of increasing concentrations of polymyxin, suggesting that the putative hopanoid biosynthesis genes are involved in stabilizing outer membrane permeability, contributing to polymyxin resistance. Results from a dansyl-polymyxin binding assay demonstrated that polymyxin B does not bind well to the parent or mutant strains, suggesting that Bmul_2133 and Bmul_2134 contribute to polymyxin B resistance by a mechanism that is independent of lipopolysaccharide (LPS) binding. Through this work, we propose a role for hopanoid biosynthesis as part of the multiple antimicrobial resistance phenotype in Bcc bacteria.

“…Thus, cationic antimicrobial peptides target the bacterial cell with low affinity through several coincident microbicidal mechanisms (4,14). Bacterial biofilms have been found to be particularly resistant to cationic antibiotics, possibly due to the presence, in the biofilm matrix, of negatively charged polymers that bind and deactivate these antibiotics (19,29,32,39). However, recently we made the breakthrough observation that the natural human cathelicidin peptide LL-37 is able to block Pseudomonas aeruginosa biofilm growth and accelerate disintegration of preformed biofilms (41).…”

mentioning

confidence: 99%

Inhibition of Bacterial Biofilm Formation and Swarming Motility by a Small Synthetic Cationic Peptide

Fuente-Núñez

Korolik

Bains

et al. 2012

406

329

dBiofilms cause up to 80% of infections and are difficult to treat due to their substantial multidrug resistance compared to their planktonic counterparts. Based on the observation that human peptide LL-37 is able to block biofilm formation at concentrations below its MIC, we screened for small peptides with antibiofilm activity and identified novel synthetic cationic peptide 1037 of only 9 amino acids in length. Peptide 1037 had very weak antimicrobial activity, but at 1/30th the MIC the peptide was able to effectively prevent biofilm formation (>50% reduction in cell biomass) by the Gram-negative pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia and Gram-positive Listeria monocytogenes. Using a flow cell system and a widefield fluorescence microscope, 1037 was shown to significantly reduce biofilm formation and lead to cell death in biofilms. Microarray and follow-up studies showed that, in P. aeruginosa, 1037 directly inhibited biofilms by reducing swimming and swarming motilities, stimulating twitching motility, and suppressing the expression of a variety of genes involved in biofilm formation (e.g., PA2204). Comparison of microarray data from cells treated with peptides LL-37 and 1037 enabled the identification of 11 common P. aeruginosa genes that have a role in biofilm formation and are proposed to represent functional targets of these peptides. Peptide 1037 shows promise as a potential therapeutic agent against chronic, recurrent biofilm infections caused by a variety of bacteria.