Antisense peptide nucleic acid­peptide conjugates for functional analyses of genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common and clinically important pathogens because of its resistance to a wide variety of antibiotics. A number of treatments of P. aeruginosa have been developed, but there is still no definitive one. Antisense drugs have a great potential to treat multidrug-resistant P. aeruginosa because this technology, in principle, can inhibit the expression of any essential genes. Nucleic Acid Ther.2012, 22, 323 reported that peptide nucleic acid (PNA) antisenses conjugated to the carrier peptide (RXR)4 and targeted to ftsZ and acpP (essential genes) had antibacterial activity in P. aeruginosa. However, growth inhibition was also found with peptide-PNA antisense conjugates of mismatched sequences (negative controls), and hence there remains a possibility for considerable enhancement of basal level activity due to the general toxicity. To assess the true potential of peptide-PNA conjugates, we measured sequence-dependent knockdown of the (RXR)4-PNA conjugates by using a scrambled sequence as a negative control. In addition, we evaluated (RXR)4-PNA antisenses against three other essential genes (lepB, lptD and mraY) and a non-essential gene (PA1303), and confirmed that multiple sequences targeting only the essential genes showed antimicrobial activity in P. aeruginosa PAO1 cells. We also conducted a rescue experiment and confirmed that the antimicrobial activity of anti-mraY antisenses was an on-target effect, not due to general toxicity. These findings indicate that the (RXR)4­PNA antisense should be a useful tool for target validation of a specific gene and could be a therapeutic platform capable of targeting a variety of genes in P. aeruginosa.

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance.

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (D-alanyl-D-alanine) and its analogous dipeptides (L-alanyl-L-alanine and D-alanyl-D-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the D-alanyl-D-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.

Binding properties of antimicrobial agents to lipid membranes using surface plasmon resonance.

In the present study, we examined the interaction of antimicrobial agents with four model lipid membranes that mimicked mammalian cell membranes and Gram-positive and -negative bacterial membranes and analyzed the binding kinetics using our surface plasmon resonance (SPR) technique. The selective and specific binding characteristics of antimicrobial agents to the lipid membranes were estimated, and the kinetic parameters were analyzed by application of a two-state reaction model. Reproducible analysis of binding kinetics was observed. Vancomyicn, teicoplanin, erythromycin, and linezolid showed little interaction with the four lipid membranes in the SPR system. On the other hand, vancomycin analogues showed interaction with the model lipid membranes in the SPR system. The selective and specific binding characteristics of vancomycin analogues to the lipid membranes are discussed based on data for in vitro antibacterial activities and our data on the binding affinity of the D-alanyl-D-alanine terminus of a pentapeptide cell wall obtained by SPR. The mechanism of antibacterial activity against Staphylococcus aureus and vancomycin-resistant enterococci could be evaluated using the binding affinity obtained with our SPR techniques. The results indicate that the SPR method could be widely applied to predict binding characteristics, such as selectivity and specificity, of many antimicrobial agents to lipid membranes.

Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis.

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

High-throughput screening of inhibitors targeting Agr/Fsr quorum sensing in Staphylococcus aureus and Enterococcus faecalis.

Staphylococcus aureus and Enterococcus faecalis employ cyclic peptide-mediated quorum sensing (QS) systems, termed agr and fsr respectively, to regulate the expression of a series of virulence genes. To identify quorum sensing inhibitors (QSIs) that target agr/fsr systems, an efficient screening system was established. In addition to the gelatinase-induction assay to examine E. faecalis fsr QS, the use of an S. aureus agr reporter strain that carries luciferase and green fluorescence protein genes under the agr P3 promoter facilitated the development of a high-throughput screen (HTS) for QSIs. As a result of screening of 906 actinomycetes culture extracts, four showed QSI activity against the agr and fsr systems without growth inhibitory activity. The extracts were purified on a small scale, and three HPLC peaks were obtained with obvious QSI activity. In sum, the established HTS system is a promising strategy for the discovery of anti-pathogenic agents targeting cyclic peptide-mediated QS in Gram-positive pathogens.

Structure-activity relationship of gelatinase biosynthesis-activating pheromone of Enterococcus faecalis.

The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays in which fluorescence-labeled peptides bound onto the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. Alanine-scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity, while side chains of the tail region were not strictly recognized. The alanine substitution of Phe(7) or Trp(10) almost abolished their receptor-binding abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp(10) substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an pi-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side chain of Trp(10) was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe(7) was extended from the core structure into the solvent and was probably directly involved in receptor binding.

Ambuic acid inhibits the biosynthesis of cyclic peptide quormones in gram-positive bacteria.

Quorum sensing is a cell-density-dependent regulatory system in gram-positive bacteria and is often regulated by cyclic peptides called "quormones," which function as extracellular communication signals. With an aim to discover an antipathogenic agent targeting quorum sensing in gram-positive bacteria, we screened 153 samples of fungal butanol extracts with the guidance of the inhibition of quorum-sensing-mediated gelatinase production in Enterococcus faecalis. Following the screenings, we found that ambuic acid, a known secondary fungal metabolite, inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of E. faecalis. We further demonstrated that ambuic acid targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of antipathogenic drugs that target the quorum-sensing-mediated virulence expression of gram-positive bacteria.

Expression, purification and activities of the entire family of intact membrane sensor kinases from Enterococcus faecalis.

Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.

Development of a peptide antagonist against fsr quorum sensing of Enterococcus faecalis.

Enterococcus faecalis fsr quorum sensing (QS) involves an 11-residue cyclic peptide named gelatinase biosynthesis-activating pheromone (GBAP) that autoinduces two pathogenicity-related extracellular proteases in a cell density-dependent fashion. To identify anti-pathogenic agents that target fsr QS signaling, peptide antagonists of GBAP were created by our unique drug design approach based on reverse alanine scanning. First of all, a receptor-binding scaffold (RBS), [Ala(4,5,6,8,9,11)]Z-GBAP, was created, in which all amino acids within the ring region of GBAP, except for two essential aromatic residues, were substituted to alanine. Next, the substituted alanine residues were changed back to the original amino acid one by one, permitting selection of those peptide combinations exhibiting increased antagonist activity. After three cycles of this reverse alanine scan, [Ala(5,9,11)]Z-GBAP was obtained as a maximally reverted peptide (MRP) holding the strongest antagonist activity. Then, the fifth residue in MRP, which is one of the critical residues to determine agonist/antagonist activity, was further modified by substituting with different types of amino acids including unnatural amino acids. As a result, [Tyr(Bzl)(5), Ala(9,11)]Z-GBAP, named ZBzl-YAA5911, showed the strongest antagonist activity [IC(50) = 26.2 nM and Kd against GBAP receptor (FsrC) = 39.4 nM]. In vivo efficacy of this peptide was assessed with an aphakic rabbit endophthalmitis model. ZBzl-YAA5911 suppressed the translocation of E. faecalis from the aqueous humor into the vitreous cavity by more than 1 order of magnitude and significantly reduced retinal damage. We propose that ZBzl-YAA5911 or its derivatives would be useful as anti-infective agents to attenuate virulence expression in this opportunistic pathogen.

Anti-HIV siamycin I directly inhibits autophosphorylation activity of the bacterial FsrC quorum sensor and other ATP-dependent enzyme activities.

Siamycin I disrupts growth and quorum sensing in Enterococcus faecalis. Using purified intact protein, we demonstrate here that quorum membrane sensor kinase FsrC is a direct target of siamycin I, reducing pheromone-stimulated autophosphorylation activity by up to 91%. Inhibition was non-competitive with ATP as substrate. Other ATP-binding enzymes were also inhibited, including nine other membrane sensor kinases of E. faecalis, Rhodobacter sphaeroides PrrB, porcine Na(+)-dependent ATPase and the catalytic subunit of bovine protein kinase A, but not bacterial ß-galactosidase, confirming targeted inhibition of a wide range of ATP dependent reactions, and elucidating a likely mechanism underlying the lethality of the inhibitor.

Siamycin attenuates fsr quorum sensing mediated by a gelatinase biosynthesis-activating pheromone in Enterococcus faecalis.

The expression of two Enterococcus faecalis virulence-related proteases, gelatinase (GelE) and serine protease (SprE), is positively regulated by a quorum-sensing system encoded by the fsr gene cluster. In this system, E. faecalis secretes an autoinducing peptide, gelatinase biosynthesis-activating pheromone (GBAP), which triggers the FsrC-FsrA two-component regulatory system controlling the expression of two transcripts, fsrBDC and gelE-sprE. In the present study, we screened actinomycete metabolites for inhibitors of fsr quorum sensing. E. faecalis was cultured with each actinomycete culture supernatant tested, and the production of gelatinase and the production of GBAP were examined using the first screening and the second screening, respectively. Culture supernatant of Streptomyces sp. strain Y33-1 had the most potent inhibitory effect on both gelatinase production and GBAP production without inhibiting E. faecalis cell growth. The inhibitor in the culture supernatant was identified as a known peptide antibiotic, siamycin I. Siamycin I inhibited both gelatinase production and GBAP production at submicromolar concentrations, and it inhibited E. faecalis cell growth at concentrations above micromolar concentrations. Quantitative analysis of fsrBDC and gelE-sprE transcripts revealed that siamycin I suppressed the expression of both transcripts at a sublethal concentration. Siamycin I attenuated gelatinase production even when an overdose of GBAP was exogenously added to the culture. These results suggested that siamycin I inhibited the GBAP signaling via the FsrC-FsrA two-component regulatory system in a noncompetitive manner. The sublethal concentrations of siamycin I also attenuated biofilm formation. Treatment with siamycin could be a novel means of treating enterococcal infections.

Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd.

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.