Merulinic acid C overcomes gentamicin resistance in Enterococcus faecium.

Enterococci are gram-positive, widespread nosocomial pathogens that in recent years have developed resistance to various commonly employed antibiotics. Since finding new infection-control agents based on secondary metabolites from organisms has proved successful for decades, natural products are potentially useful sources of compounds with activity against enterococci. Herein are reported the results of a natural product library screening based on a whole-cell assay against a gram-positive model organism, which led to the isolation of a series of anacardic acids identified by analysis of their spectroscopic data and by chemical derivatizations. Merulinic acid C was identified as the most active anacardic acid derivative obtained against antibiotic-resistant enterococci. Fluorescence microscopy analyses showed that merulinic acid C targets the bacterial membrane without affecting the peptidoglycan and causes rapid cellular ATP leakage from cells. Merulinic acid C was shown to be synergistic with gentamicin against Enterococcus faecium, indicating that this compound could inspire the development of new antibiotic combinations effective against drug-resistant pathogens.

Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide.

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100­alanyl­hexadecyl­1­amine (BP100-Ala-NH-C16H33) and cyclo(1­4)­d­Cys1, Ile2, Leu3, Cys4-BP100 (Cyclo(1­4)­cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(1­4)­cILC-BP100) was partly α-helical and partly ß-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Violacein Targets the Cytoplasmic Membrane of Bacteria.

Violacein is a tryptophan-derived purple pigment produced by environmental bacteria, which displays multiple biological activities, including strong inhibition of Gram-positive pathogens. Here, we applied a combination of experimental approaches to identify the mechanism by which violacein kills Gram-positive bacteria. Fluorescence microscopy showed that violacein quickly and dramatically permeabilizes B. subtilis and S. aureus cells. Cell permeabilization was accompanied by the appearance of visible discontinuities or rips in the cytoplasmic membrane, but it did not affect the cell wall. Using in vitro experiments, we showed that violacein binds directly to liposomes made with commercial and bacterial phospholipids and perturbs their structure and permeability. Furthermore, molecular dynamics simulations were employed to reveal how violacein inserts itself into lipid bilayers. Thus, our combined results demonstrate that the cytoplasmic membrane is the primary target of violacein in bacteria. The implications of this finding for the development of violacein as a therapeutic agent are discussed.