The silver cation (Ag+): antistaphylococcal activity, mode of action and resistance studies.

OBJECTIVES: To examine several poorly understood or contentious aspects of the antibacterial activity of silver (Ag(+)), including its cidality, mode of action, the prevalence of resistance amongst clinical staphylococcal isolates and the propensity for Staphylococcus aureus to develop Ag(+) resistance. METHODS: The effects of Ag(+) on the viability, macromolecular synthesis and membrane integrity of S. aureus SH1000 were assessed using established methodology. Silver nitrate MICs were determined for a collection of staphylococcal isolates (n = 1006) collected from hospitals across Europe and Canada between 1997 and 2010. S. aureus biofilms were grown using the Calgary Biofilm Device. To examine the in vitro development of staphylococcal resistance to Ag(+), bacteria were subjected to continuous subculture in the presence of sub-MIC concentrations of Ag(+). RESULTS: Silver was bactericidal against S. aureus in buffered solution, but bacteriostatic in growth medium, and was unable to eradicate staphylococcal biofilms in vitro. Challenge of S. aureus with Ag(+) caused rapid loss of membrane integrity and inhibition of the major macromolecular synthetic pathways. All clinical staphylococcal isolates were susceptible to ≤ 16 mg/L silver nitrate and prolonged exposure (42 days) to Ag(+) in vitro failed to select resistant mutants. CONCLUSIONS: The rapid and extensive loss of membrane integrity observed upon challenge with Ag(+) suggests that the antibacterial activity results directly from damage to the bacterial membrane. The universal susceptibility of staphylococci to Ag(+), and failure to select for resistance to Ag(+), suggest that silver compounds remain a viable option for the prevention and treatment of topical staphylococcal infections.

6-Arylpyrido[2,3-d]pyrimidines as novel ATP-competitive inhibitors of bacterial D-alanine:D-alanine ligase.

BACKGROUND: ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl. METHODOLOGY/PRINCIPAL FINDINGS: Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli. CONCLUSIONS/SIGNIFICANCE: We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem.

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor.

Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the Staphylococcus aureus penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.

Functional and biochemical analysis of the Chlamydia trachomatis ligase MurE.

Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm ligase activity. Recombinant MurE from C. trachomatis (MurE(Ct)) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE(Ct) demonstrated ATP-dependent meso-A(2)pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A(2)pm, D-lysine) were also recognized by MurE(Ct.) However, the activities for these amino acid substrates were weaker than that for meso-A(2)pm. The specificity of MurE(Ct) for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k(cat)/K(m) ratios for UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A(2)pm. However, due to the lack of specificity of MurE(Ct) for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.

The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors.

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening.

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

Discovery of new inhibitors of D-alanine:D-alanine ligase by structure-based virtual screening.

The terminal dipeptide, D-Ala-D-Ala, of the peptidoglycan precursor UDPMurNAc-pentapetide is a crucial building block involved in peptidoglycan cross-linking. It is synthesized in the bacterial cytoplasm by the enzyme d-alanine:d-alanine ligase (Ddl). Structure-based virtual screening of the NCI diversity set of almost 2000 compounds was performed with a DdlB isoform from Escherichia coli using the computational tool AutoDock 4.0. The 130 best-ranked compounds from this screen were tested in an in vitro assay for their inhibition of E. coli DdlB. Three compounds were identified that inhibit the enzyme with K(i) values in micromolar range. Two of these also have promising antibacterial activities against Gram-positive and Gram-negative bacteria.

Synthesis and biological evaluation of N-acylhydrazones as inhibitors of MurC and MurD ligases.

The Mur ligases have an essential role in the intracellular biosynthesis of bacterial peptidoglycan, and they represent attractive targets for the design of novel antibacterials. A series of compounds with an N-acylhydrazone scaffold were synthesized and screened for inhibition of the MurC and MurD enzymes from Escherichia coli. Compounds with micromolar inhibitory activities against both MurC and MurD were identified, and some of them also showed antibacterial activity.

Diazenedicarboxamides as inhibitors of D-alanine-D-alanine ligase (Ddl).

D-Alanine-D-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor D-alanyl-D-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure-activity relationships. Thirteen diazenedicarboxamides were better inhibitors than D-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.

Mechanisms of the post-antibiotic effects induced by rifampicin and gentamicin in Escherichia coli.

OBJECTIVES: The mechanisms by which antibiotics induce a post-antibiotic effect in susceptible bacteria are poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects. METHODS: E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5x MIC for 60 min to induce post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells. The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and leucine into trichloroacetic acid precipitable material. RESULTS: Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect coincided with the recovery of protein synthesis. CONCLUSIONS: These data support the hypothesis that antibiotic molecules retained in the cell mediate the post-antibiotic effect by suppressing the biochemical activity of their molecular targets.

Deletion of the multiple-drug efflux pump AcrAB in Escherichia coli prolongs the postantibiotic effect.

The mechanism of the postantibiotic effect (PAE) was examined in Escherichia coli. Drugs exhibited longer-lasting PAEs in an acrAB mutant, suggesting that intracellular drug concentrations influence the duration of the PAE. With specific assays for tetracycline and erythromycin, a direct link between intracellular persistence of antibiotics and maintenance of the PAE was established.

Assessment of a microplate method for determining the post-antibiotic effect in Staphylococcus aureus and Escherichia coli.

OBJECTIVES: The post-antibiotic effect (PAE) is an important parameter of antibiotic action that is widely used as a predictor of pharmacodynamic activity. Traditionally, PAE has been determined by a labour-intensive method involving determination of viable cell numbers. New methods using spectrophotometric procedures could offer significant advantages for PAE determinations, particularly in terms of speed. A number of such methods have been described in the literature, but extensive comparison with the classical procedure for determining PAEs has not been carried out. We have now compared PAE values obtained using a rapid microplate method with those achieved by the classical viable count procedure. METHODS: We determined PAE values for a variety of antibiotics against Staphylococcus aureus and Escherichia coli following exposure to 5 x MIC drug concentrations for 60 min in Mueller-Hinton Broth (MHB). The duration of the PAE was obtained by following the recovery of bacterial growth in antibiotic-free MHB measured either as colony forming units on Mueller-Hinton agar, or as culture absorbance (600 nm) in a microplate reader. RESULTS: For bacteriolytic agents there was poor correlation between the two methods for both S. aureus (R2=0.096) and E. coli (R2=0.5456). However, when PAEs for bacteriostatic agents and non-lytic bactericidal agents were compared, correlation between the two methods was high for both S. aureus (R2=0.7529) and E. coli (R2=0.7687). CONCLUSIONS: The spectrophotometric microplate method for determining PAEs may be a suitable alternative to the classical method for those antibiotics that do not induce bacterial cell lysis.

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme MurD.

Computer-based molecular design has been used to produce a series of new macrocyclic systems targeted against the bacterial cell wall biosynthetic enzyme MurD. Following their preparation, which involved a novel metathesis-based cyclisation as the key step, these systems were found to show good inhibition when assayed against the MurD enzyme.