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Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16.

ABSTRACT: Brilacidin (PMX30063) has shown potent bactericidal activity against drug-resistant and -susceptible strains of multiple Gram-negative and Gram-positive pathogens. In this study, we demonstrate that brilacidin causes membrane depolarization in the Gram-positive bacterium Staphylococcus aureus, to an extent comparable to that caused by the lipopeptidic drug daptomycin. Transcriptional profiling of Staphylococcus aureus by deep sequencing shows that the global response to brilacidin treatment is well correlated to those of treatment with daptomycin and the cationic antimicrobial peptide LL37 and mostly indicates abrogation of cell wall and membrane functions. Furthermore, the upregulation of various chaperones and proteases by brilacidin and daptomycin indicates that cytoplasmic protein misfolding stress may be a contributor to the mechanism of action of these drugs. These stress responses were orchestrated mainly by three two-component systems, GraSR, VraSR, and NsaSR, which have been implicated in virulence and drug resistance against other clinically available antibiotics.

SUBMITTER: Mensa B

PROVIDER: S-EPMC4135847 | biostudies-other | 2014 Sep

REPOSITORIES: biostudies-other

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Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16.

Mensa Bruk B Howell Gabriella L GL Scott Richard R DeGrado William F WF

Antimicrobial agents and chemotherapy 20140616 9

Brilacidin (PMX30063) has shown potent bactericidal activity against drug-resistant and -susceptible strains of multiple Gram-negative and Gram-positive pathogens. In this study, we demonstrate that brilacidin causes membrane depolarization in the Gram-positive bacterium Staphylococcus aureus, to an extent comparable to that caused by the lipopeptidic drug daptomycin. Transcriptional profiling of Staphylococcus aureus by deep sequencing shows that the global response to brilacidin treatment is w ...[more]

PMID: 24936592

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Project description:Owing to their potential applicability against multidrug-resistant bacteria, antimicrobial peptides (AMPs) or host defense peptides (HDPs) gain increased attention. Besides diverse immunomodulatory roles, their classical mechanism of action mostly involves membrane disruption of microbes. Notably, their unbalanced overexpression has also been associated with host cell cytotoxicity in various diseases. Relatedly, AMPs can be subject to aggregate formation, either via self-assembly or together with other compounds, which has demonstrated a modulation effect on their biological functions, thus highly relevant both for drug targeting projects and understanding their in vivo actions. However, the molecular aspects of the related assembly formation are not understood. Here, we focused in detail on an experimentally studied AMP-drug system, i.e., CM15-suramin, and performed all-atom and coarse-grain (CG) simulations. Results obtained for all systems were in close line with experimental observations and indicate that the CM15-suramin aggregation is an energetically favorable and dynamic process. In the presence of bilayers, the peptide-drug assembly formation was highly dependent on lipid composition, and peptide aggregates themselves were also capable of binding to the membranes. Interestingly, longer CG simulations with zwitterionic membranes indicated an intermediate state in the presence of both AMP-drug assemblies and monomeric peptides located on the membrane surface. In sharp contrast, larger AMP-drug aggregates could not be detected with a negatively charged membrane, rather the AMPs penetrated its surface in a monomeric form, in line with previous in vitro observations. Considering experimental and theoretical results, it is promoted that in biological systems, cationic AMPs may often form associates with anionic compounds in a reversible manner, resulting in lower bioactivity. This is only mildly affected by zwitterionic membranes; however, membranes with a negative charge strongly alter the energetic preference of AMP assemblies, resulting in the dissolution of the complexes into the membrane. The phenomenon observed here at a molecular level can be followed in several experimental systems studied recently, where peptides interact with food colors, drug molecules, or endogenous compounds, which strongly indicates that reversible associate formation is a general phenomenon for these complexes. These results are hoped to be exploited in novel therapeutic strategies aiming to use peptides as drug targets and control AMP bioactivity by directed assembly formation.

Project description:Daptomycin is an extensively used anti-staphylococcal agent due to the rise in methicillin-resistant Staphylococcus aureus, but the mechanism(s) of resistance is poorly understood. Comparative genome sequencing, transcriptomics, ultrastructure, and cell envelope studies were carried out on two relatively higher level (4 and 8 µg/ml(-1)) laboratory-derived daptomycin-resistant strains (strains CB1541 and CB1540 respectively) compared to their parent strain (CB1118; MW2). Several mutations were found in the strains. Both strains had the same mutations in the two-component system genes walK and agrA. In strain CB1540 mutations were also detected in the ribose phosphate pyrophosphokinase (prs) and polyribonucleotide nucleotidyltransferase genes (pnpA), a hypothetical protein gene, and in an intergenic region. In strain CB1541 there were mutations in clpP, an ATP-dependent protease, and two different hypothetical protein genes. The strain CB1540 transcriptome was characterized by upregulation of cap (capsule) operon genes, genes involved in the accumulation of the compatible solute glycine betaine, ure genes of the urease operon, and mscL encoding a mechanosensitive chanel. Downregulated genes included smpB, femAB and femH involved in the formation of the pentaglycine interpeptide bridge, genes involved in protein synthesis and fermentation, and spa encoding protein A. Genes altered in their expression common to both transcriptomes included some involved in glycine betaine accumulation, mscL, ure genes, femH, spa and smpB. However, the CB1541 transcriptome was further characterized by upregulation of various heat shock chaperone and protease genes, consistent with a mutation in clpP, and lytM and sceD. Both strains showed slow growth, and strongly decreased autolytic activity that appeared to be mainly due to decreased autolysin production. In contrast to previous common findings, we did not find any mutations in phospholipid biosynthesis genes, and it appears there are multiple pathways to and factors in daptomycin resistance.

Dataset Information

Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16.

Publications

Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16.

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