Polymyxin B-inspired non-hemolytic tyrocidine A analogues with significantly enhanced activity against gram-negative bacteria: How cationicity impacts cell specificity and antibacterial mechanism.

Naturally occurring cyclic antimicrobial peptides (AMPs) such as tyrocidine A (Tyrc A) and gramicidin S (GS) are appealing targets for the development of novel antibiotics. However, their therapeutic potentials are limited by undesired hemolytic activity and relatively poor activity against Gram-negative bacteria. Inspired by polycationic lipopeptide polymyxin B (PMB), the so called 'last-resort' antibiotic for the treatment of infections caused by multidrug-resistant Gram-negative bacteria, we synthesized and biologically evaluated a series of polycationic analogues derived from Tyrc A. We were able to obtain peptide 8 that possesses 5 positive charges exhibiting potent activities against both Gram-negative and Gram-positive bacteria along with totally diminished hemolytic activity. Intriguingly, antibacterial mechanism studies revealed that, rather than the 'pore forming' model that possessed by Tyrc A, peptide 8 likely diffuses membrane in a 'detergent-like' manner. Furthermore, when treating mice with peritonitis-sepsis, peptide 8 showed excellent antibacterial and anti-inflammatory activities in vivo.

Mimicry of a Non-ribosomally Produced Antimicrobial, Brevicidine, by Ribosomal Synthesis and Post-translational Modification.

The group of bacterial non-ribosomally produced peptides (NRPs) forms a rich source of antibiotics, such as daptomycin, vancomycin, and teixobactin. The difficulty of functionally expressing and engineering the corresponding large biosynthetic complexes is a bottleneck in developing variants of such peptides. Here, we apply a strategy to synthesize mimics of the recently discovered antimicrobial NRP brevicidine. We mimicked the molecular structure of brevicidine by ribosomally synthesized, post-translationally modified peptide (RiPP) synthesis, introducing several relevant modifications, such as dehydration and thioether ring formation. Following this strategy, in two rounds peptides were engineered in vivo, which showed antibacterial activity against Gram-negative pathogenic bacteria susceptible to wild-type brevicidine. This study demonstrates the feasibility of a strategy to structurally and functionally mimic NRPs by employing the synthesis and post-translational modifications typical for RiPPs. This enables the future generation of large genetically encoded peptide libraries of NRP-mimicking structures to screen for antimicrobial activity against relevant pathogens.

The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S.

Cyclic ß-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent.IMPORTANCE Cyclic ß-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of ß-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.

Tyrocidine A Analogues Bearing the Planar d-Phe-2-Abz Turn Motif: How Conformation Impacts Bioactivity.

The d-Phe-Pro ß-turn of the cyclic ß-hairpin antimicrobial decapeptide tyrocidine A, (Tyrc A) was substituted with the d-Phe-2-aminobenzoic acid (2-Abz) motif in a synthetic analogue (1). The NMR structure of 1 demonstrated that compound 1 retained the ß-hairpin structure of Tyrc A with additional planarity, resulting in approximately 30-fold reduced hemolysis than Tyrc A. Although antibacterial activity was partially compromised, a single Gln to Lys substitution (2) restored activity equivalent to Tyrc A against S. aureus, enhanced activity against two Gram negative strains and maintained the reduced hemeloysis of 1. Analysis by transmission electron microscopy (TEM) suggested a membrane lytic mechanism of action for these peptides. Compound 2 also exhibits nanomolar antifungal activity in synergy with amphotericin B. The d-Phe-2-Abz turn may serve as a tool for the synthesis of structurally predictable ß-hairpin libraries. Unlike traditional ß-turn motifs such as d-Pro-Gly, both the 2-Abz and d-Phe rings may be further functionalized.

Stabilizing the monomeric amyloid-ß peptide by tyrocidine A prevents and reverses amyloidogenesis without the accumulation of oligomers.

Amyloid-ß (Aß) oligomers are causative agents triggering AD pathogenesis, but their elimination remains challenging. We herein reported a natural cyclopeptide tyrocidine A prevents and reverses amyloidogenesis without Aß oligomer accumulation by stabilizing the monomeric, but not the oligomeric state of Aß peptides.

Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi.

The tyrocidines and analogues are cyclic decapeptides produced by Brevibacillus parabrevis with a conserved sequence of cyclo(D-Phe1-Pro2-X3-x4-Asn5-Gln6-X7-Val8-X9-Leu10) with Trp3,4/Phe3,4 in the aromatic dipeptide unit, Lys9/Orn9 as their cationic residue and Tyr (tyrocidines), Trp (tryptocidines) or Phe (phenicidines) in position 7. Previous studies indicated they have a broad antifungal spectrum with the peptides containing a Tyr residue in position 7 being more active than those with a Phe or Trp residue in this position. Detailed analysis of antifungal inhibition parameters revealed that Phe3-D-Phe4 in the aromatic dipeptide unit lead to more consistent activity against the three filamentous fungi in this study. These peptides exhibited high membrane activity and fast leakage kinetics against model membranes emulating fungal membranes, with selectivity towards ergosterol containing membranes. More fluid membranes and doping of liposomes with the sphingolipid, glucosylceramide, led to a decreased permeabilising activity. Peptide-induced uptake of membrane impermeable dyes was observed in hyphae of both Fusarium solani and Botrytis cinerea, with uptake more pronounced at the hyphal growth tips that are known to contain ergosterol-sphigolipid rich lipid rafts. Tyrocidine interaction with these rafts may lead to the previously observed fungal hyperbranching. However, the leakage of model membranes and Bot. cinerea did not correlate directly with the antifungal inhibition parameters, indicating another target or mode of action. Proteinase K treatment of target fungi had a minimal influence or even improved the tyrocidine activity, ruling out a mannoprotein target in the fungal cell wall. ß-glucanase treatment of Bot. cinerea did not significantly affect the tyrocidine activity, but there was a significant loss in activity towards the ß-glucanase treated F. solani. This study showed the tyrocidine antifungal membrane activity is selective towards ergosterol and possibly lipid rafts, but also point to additional targets such as the cell wall ß-glucans that could modulate their activity.

Synthesis and antibacterial activities of novel tyrocidine A glycosylated derivatives towards multidrug-resistant pathogens.

Glycosylation can have a multifaceted impact on the properties and functions of peptides and plays a critical role in interacting with or binding to the target molecules. Herein, based on the previously reported method for macrocyclic glycopeptide synthesis, two series of tyrocidine A glycosylated derivatives (1a-f and 2a-f) were synthesized and evaluated for their antibacterial activities to further study the structure and activity relationships (SAR). Biological studies showed that the synthetic glycosylated derivatives had good antibacterial activities towards methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. SAR studies based on various glycans and linkages were used to enhance the biochemical profile, resulting in the identification of several potent antibiotics, such as 1f, with a great improved therapeutic index than tyrocidine A.

Inhibition of agronomically relevant fungal phytopathogens by tyrocidines, cyclic antimicrobial peptides isolated from Bacillus aneurinolyticus.

The tyrocidines, a complex of analogous cyclic decapeptides produced by Bacillus aneurinolyticus, exhibited noteworthy activity against a range of phytopathogenic fungi, including Fusarium verticillioides, Fusarium solani and Botrytis cinerea. The activity of the tyrocidine peptide complex (Trc mixture) and purified tyrocidines exhibited minimum inhibition concentrations below 13 µg ml(-1) (~10 µM) and was significantly more potent than that of the commercial imidazole fungicide, bifonazole. Although the tyrocidines' activity was negatively influenced by the presence of Ca(2+), it remained unaffected by the presence of Mg(2+), Na(+) and K(+). Microscopic analysis revealed significant impact on the morphology of F. solani and Bot. cinerea including retarded germination and hyperbranching of hyphae. Studies with membrane-impermeable dyes, SYTOX green and propidium iodide suggested that the main mode of action of tyrocidines involves the disruption of fungal membrane integrity. Because of the tyrocidines' broad spectrum and potent antifungal activity, possible multiple targets reducing the risk of overt resistance and general salt tolerance, they are promising candidates that warrant further investigation as bio-fungicides.

Synergistic activity of the tyrocidines, antimicrobial cyclodecapeptides from Bacillus aneurinolyticus, with amphotericin B and caspofungin against Candida albicans biofilms.

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.

The high resolution structure of tyrocidine A reveals an amphipathic dimer.

Tyrocidine A, one of the first antibiotics ever to be discovered, is a cyclic decapeptide that binds to membranes of target bacteria, disrupting their integrity. It is active against a broad spectrum of Gram-positive organisms, and has recently engendered interest as a potential scaffold for the development of new drugs to combat antibiotic-resistant pathogens. We present here the X-ray crystal structure of tyrocidine A at a resolution of 0.95Å. The structure reveals that tyrocidine forms an intimate and highly amphipathic homodimer made up of four beta strands that associate into a single, highly curved antiparallel beta sheet. We used surface plasmon resonance and potassium efflux assays to demonstrate that tyrocidine binds tightly to mimetics of bacterial membranes with an apparent dissociation constant (K(D)) of 10 µM, and efficiently permeabilizes bacterial cells at concentrations equal to and below the K(D). Using variant forms of tyrocidine in which the fluorescent probe p-cyano-phenylalanine had been inserted on either the polar or apolar face of the molecule, we performed fluorescence quenching experiments, using both water-soluble and membrane-embedded quenchers. The quenching results, together with the structure, strongly support a membrane association model in which the convex, apolar face of tyrocidine's beta sheet is oriented toward the membrane interior, while the concave, polar face is presented to the aqueous phase.

In situ potentiometric method to evaluate bacterial outer membrane-permeabilizing ability of drugs: example using antiprotozoal diamidines.

We introduced a new assay system, combining tyrocidine A and a K(+)-selective electrode, to evaluate the bacterial outer membrane-permeabilizing ability of drugs. Tyrocidine A, in the presence of an outer membrane permeabilizer, increased the permeability to K(+) of the cytoplasmic membrane of Escherichia coli, because this antibiotic could markedly increase the permeability of phospholipid layers constituting the cytoplasmic membrane, while it acted weakly on the outer membrane. Hence, the novel function of agents increasing the permeability of the outer membrane could be examined directly by monitoring the tyrocidine A-induced leakage of K(+) from the bacterial cytoplasm using a K(+)-selective electrode. We found that antiprotozoal diamidines, such as diminazene, pentamidine, and 4',6-diamidino-2-phenylindole (DAPI), can increase the permeability of the bacterial outer membrane and appropriate lipophilicity is important for diamidines to permeabilize the outer membrane.

Anti-listerial activity and structure-activity relationships of the six major tyrocidines, cyclic decapeptides from Bacillus aneurinolyticus.

Six major tyrocidines, purified from the antibiotic tyrothricin complex produced by Bacillus aneurinolyticus, showed significant lytic and growth inhibitory activity towards the gram+ bacteria, Micrococcus luteus and Listeria monocytogenes, but not against the gram- bacterium, Escherichia coli. The isolated natural tyrocidines were in particular more active against the leucocin A (antimicrobial peptide) resistant strain, L. monocytogenes B73-MR1, than the sensitive L. monocytogenes B73 strain. Remarkably similar structure-activity trends toward the three gram+ bacteria were found between growth inhibition and different physicochemical parameters (solution amphipathicity, theoretical lipophilicity, side-chain surface area and mass-over-charge ratio).

Synthesis and antibacterial activities of N-glycosylated derivatives of tyrocidine A, a macrocyclic peptide antibiotic.

An efficient and practical method for macrocyclic glycopeptide synthesis was developed and utilized to synthesize tyrocidine A and its glycosylated derivatives. The method is based on solid-phase peptide synthesis using 2-chlorotrityl resin as the solid-phase support and glycosyl amino acids as building blocks. After glycopeptides with fully protected glycans and side chains were released from the acid-labile resin, their C- and N-termini were intramolecularly coupled in solution to afford cyclic glycopeptides in quantitative yields. This synthetic method should be generally applicable to various macrocyclic glycopeptides. Biological studies of the synthetic tyrocidine A derivatives showed that linking glycans directly to the Asn residue of tyrocidine A diminished its antibacterial activity, but linking glycans to Asn via a simple spacer did not. These results revealed the important impact of glycans on the activities, and probably the structures, of glycopeptide antibiotics.

Inhibition of malaria parasite blood stages by tyrocidines, membrane-active cyclic peptide antibiotics from Bacillus brevis.

Tyrothricin, a complex mixture of antibiotic peptides from Bacillus brevis, was reported in 1944 to have antimalarial activity rivalling that of quinine in chickens infected with Plasmodium gallinaceum. We have isolated the major components of tyrothricin, cyclic decapeptides collectively known as the tyrocidines, and tested them against the human malaria parasite Plasmodium falciparum using standard in vitro assays. Although the tyrocidines differ from each other by conservative amino acid substitutions in only three positions, their observed 50% parasite inhibitory concentrations (IC(50)) spanned three orders of magnitude (0.58 to 360 nM). Activity correlated strictly with increased apparent hydrophobicity and reduced total side-chain surface area and the presence of ornithine and phenylalanine in key positions. In contrast, mammalian cell toxicity and haemolytic activities of the respective peptides were considerably less variable (2.6 to 28 microM). Gramicidin S, a structurally analogous antimicrobial peptide, was less active (IC(50)=1.3 microM) and selective than the tyrocidines. It exerted its parasite inhibition by rapid and selective lysis of infected erythrocytes as judged by fluorescence and light microscopy. The tyrocidines, however, did not cause an overt lysis of infected erythrocytes, but an inhibition of parasite development and life-cycle progression.

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry.

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.

Chimeric streptogramin-tyrocidine antibiotics that overcome streptogramin resistance.

Streptogramin antibiotics are comprised of two distinct chemical components: the type A polyketides and the type B cyclic depsipeptides. Clinical resistance to the type B streptogramins can occur via enzymatic degradation catalyzed by the lyase Vgb or by target modification through the action of Erm ribosomal RNA methyltransferases. We have prepared through chemical and chemo-enzymatic approaches a series of chimeric antibiotics composed of elements of type B streptogramins and the membrane-active antibiotic tyrocidine that evade these resistance mechanisms. These new compounds show broad antibiotic activity against gram-positive bacteria including a number of important pathogens, and chimeras appear to function by a mechanism that is distinct from their parent antibiotics. These results allow for the development of a brand new class of antibiotics with the ability to evade type B streptogramin-resistance mechanisms.

Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase.

The 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(beta-D-Gal) and Tyr(beta-D-Glc) as well as with Ser-containing beta-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization.

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water.

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50-90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.

Dissociation of antibacterial and hemolytic activities of an amphipathic peptide antibiotic.

Using an alanine-scanning method, we have found that the antibacterial and hemolytic activities of the amphipathic cyclic decapeptide antibiotic tyrocidine A depend on different structural components. Single substitution of glutamine-6 of the natural product with a cationic amino acid results in a therapeutic index enhancement of up to 140-fold. Successful dissociation of the two intimately associated properties should enable discovery of novel analogues with both high bacterial selectivity and antibacterial potency to counter microbial resistance.