Inhibitors Of Cell Wall Biosynthesis Research Articles

The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling

Cell integrity in Saccharomyces cerevisiae is ensured by a rigid cell wall whose synthesis is controlled by a highly conserved MAP kinase signal transduction cascade. Stress at the cell surface is detected by a set of sensors and ultimately transmitted through this cascade to the transcription factor Rlm1, which governs expression of many genes encoding enzymes of cell wall biosynthesis. We here report on a number of versatile reporter constructs which link activation of a hybrid, Rlm1-lexA, by the MAP kinase Mpk1/Slt2 to the expression of the bacterial lacZ gene. This system was adapted to automated microwell screening and shown to be activated by a number of compounds inhibiting cell wall biosynthesis or interfering with plasma membrane function. In addition, we tested tea tree oil and two of its purified constituents (alpha-terpineol, terpinen-4-ol) for their effects on growth and on cell integrity signalling using such reporter strains. Tea tree oil was found to inhibit growth of wild-type and slg1/wsc1 mutant cells at a threshold of approximately 0.1% v/v, with the purified compounds acting already at half these concentrations. A mid2 deletion displayed hyper-resistance. Tea tree oil also induces the signalling pathway in a dose-dependent manner.

Sesquiterpene farnesol inhibits recycling of the C55 lipid carrier of the murein monomer precursor contributing to increased susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus

The sesquiterpene farnesol, a natural plant metabolite, is known to intensify the effect of antimicrobial agents. However, the mode of action of its antimicrobial synergism has remained poorly understood. In this study, we investigated farnesol's synergistic effects on commonly used antimicrobials, beta-lactams in particular, to explore its potential inhibitory effect on cell wall synthesis. We investigated farnesol's effects on: (i) antimicrobial susceptibilities of methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) to ampicillin, oxacillin, cefoxitin, bacitracin, teicoplanin, amikacin, ciprofloxacin and clarithromycin by MIC determination using the Etest; (ii) penicillin-binding protein PBP2' (2a) expression by western-blot analysis; (iii) beta-lactamase secretion and activity by in vivo and in vitro farnesol inhibition assays; (iv) staphyloxanthin production by thin-layer chromatography (TLC); and (v) cell wall synthesis by [14C]GlcNAc (where GlcNAc stands for N-acetylglucosamine) and [14C]mevalonate incorporation assays, and TLC-based lipid extract profile analysis. Farnesol induced variable degrees of increased susceptibility to all antimicrobials except clarithromycin in both MSSA and MRSA. A remarkable increase in susceptibilities to ampicillin, oxacillin and cefoxitin was observed in both MRSA strains, N315 and COL, whereas a moderate increase in susceptibility to bacitracin was observed in all the strains. Although no apparent suppression of PBP2' expression was observed, beta-lactamase secretion and beta-lactamase activity were significantly reduced by farnesol. In addition, farnesol completely suppressed staphyloxanthin production. Farnesol reduced the incorporation of GlcNAc, but significantly increased that of mevalonate. Farnesol induced accumulation of C55-PP, lipid I and lipid II. Farnesol increased beta-lactam susceptibility of MRSA by inhibition of cell wall biosynthesis through reduction of free C55 lipid carrier with subsequent retardation of murein monomer precursor transport across the cell membrane.

The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques

Nisin, a peptide antibiotic, efficiently kills bacteria through a unique mechanism which includes inhibition of cell wall biosynthesis and pore formation in cytoplasmic membranes. Both mechanisms are based on interaction with the cell wall precursor lipid II which is simultaneously used as target and pore constituent. We combined two biosensor techniques to investigate the nisin activity with respect to membrane binding and pore formation in real time. Quartz crystal microbalance (QCM) allows the detection of nisin binding kinetics. The presence of 0.1 mol% lipid II strongly increased nisin binding affinity to DOPC ( k D 2.68 × 10 − 7 M vs. 1.03 × 10 − 6 M) by a higher association rate. Differences were less pronounced while using negatively charged DOPG membranes. However, lipid II does not influence the absolute amount of bound nisin. Cyclic voltammetry (CV) data confirmed that in presence of 0.1 mol% lipid II, nanomolar nisin concentrations were sufficient to form pores, while micromolar concentrations were necessary in absence of lipid II. Both techniques suggested unspecific destruction of pure DOPG membranes by micromolar nisin concentrations which were prevented by lipid II. This model membrane stabilization by lipid II was confirmed by atomic force microscopy. Combined CV and QCM are valuable to interpret the role of lipid II in nisin activity.

The mode of action of the lantibiotic lacticin 3147 – a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.

Brucella abortus Rough Mutants Induce Macrophage Oncosis That Requires Bacterial Protein Synthesis and Direct Interaction with the Macrophage

Previous studies suggest that smooth Brucella organisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucella organisms in vitro has been reported, which may in part explain the failure of some rough organisms to thrive. To characterize these potential macrophage killing mechanisms, J774.A1 murine macrophages were infected with Brucella abortus S2308-derived rough mutant CA180. Electron microscopic analysis and polyethylene glycol protection assays revealed that the cells were killed as a result of necrosis and oncosis. This killing was shown to be unaffected by treatment with carbenicillin, an inhibitor of bacterial cell wall biosynthesis and, indirectly, replication. In contrast, chloramphenicol treatment of macrophages infected at multiplicities of infection exceeding 10,000 prevented cell death, despite internalization of large numbers of bacteria. Similarly, heat-killed and gentamicin-killed CA180 did not induce cytopathic effects in the macrophage. These results suggested that killing of infected host cells requires active bacterial protein synthesis. Cytochalasin D treatment revealed that internalization of the bacteria was necessary to initiate killing. Transwell experiments demonstrated that cell death is not mediated by a diffusible product, including tumor necrosis factor alpha and nitric oxide, but does require direct contact between host and pathogen. Furthermore, macrophages preinfected with B. abortus S2308 or pretreated with B. abortus O polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucella rough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.

Pulvinones as Bacterial Cell Wall Biosynthesis Inhibitors.

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Control of the C. albicans Cell Wall Damage Response by Transcriptional Regulator Cas5

The fungal cell wall is vital for growth, development, and interaction of cells with their environment. The response to cell wall damage is well understood from studies in the budding yeast Saccharomyces cerevisiae, where numerous cell wall integrity (CWI) genes are activated by transcription factor ScRlm1. Prior evidence suggests the hypothesis that both response and regulation may be conserved in the major fungal pathogen Candida albicans. We have tested this hypothesis by using a new C. albicans genetic resource: we have screened mutants defective in putative transcription factor genes for sensitivity to the cell wall biosynthesis inhibitor caspofungin. We find that the zinc finger protein CaCas5, which lacks a unique ortholog in S. cerevisiae, governs expression of many CWI genes. CaRlm1 has a modest role in this response. The transcriptional coactivator CaAda2 is also required for expression of many CaCas5-dependent genes, as expected if CaCas5 recruits CaAda2 to activate target gene transcription. Many caspofungin-induced C. albicans genes specify endoplasmic reticulum and secretion functions. Such genes are not induced in S. cerevisiae, but promote its growth in caspofungin. We have used a new resource to identify a key C. albicans transcriptional regulator of CWI genes and antifungal sensitivity. Our gene expression findings indicate that both divergent and conserved response genes may have significant functional roles. Our strategy may be broadly useful for identification of pathogen-specific regulatory pathways and critical response genes.

Pulvinones as bacterial cell wall biosynthesis inhibitors

Pulvinones were synthesized (>180) in arrays and evaluated as inhibitors of early stage cell wall biosynthesis enzymes MurA–MurD. Several pulvinones inhibited Mur enzymes with IC 50’s in the 1–10 μg/mL range and demonstrated antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphyloccus aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

4‐Alkyl and 4,4′‐Dialkyl 1,2‐Bis(4‐chlorophenyl)pyrazolidine‐3,5‐dione Derivatives as New Inhibitors of Bacterial Cell Wall Biosynthesis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases

Protein N-glycosylation in eukaryotes and peptidoglycan biosynthesis in bacteria are both initiated by the transfer of a D-N-acetylhexosamine 1-phosphate to a membrane-bound polyprenol phosphate. These reactions are catalyzed by a family of transmembrane proteins known as the UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-phosphate transferases. The sole eukaryotic member of this family, the d-N-acetylglucosamine 1-phosphate transferase (GPT), is specific for UDP-GlcNAc as the donor substrate and uses dolichol phosphate as the membrane-bound acceptor. The bacterial translocases, MraY, WecA, and WbpL, utilize undecaprenol phosphate as the acceptor substrate, but differ in their specificity for the UDP-sugar donor substrate. The structural basis of this sugar nucleotide specificity is uncertain. However, potential carbohydrate recognition (CR) domains have been identified within the C-terminal cytoplasmic loops of MraY, WecA, and WbpL that are highly conserved in family members with the same UDP-N-acetylhexosamine specificity. This review focuses on the catalytic mechanism and substrate specificity of these bacterial UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-P transferases and may provide insights for the development of selective inhibitors of cell wall biosynthesis.

4-Alkyl and 4,4′-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives as new inhibitors of bacterial cell wall biosynthesis

Over 195 4-alkyl and 4,4-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives were synthesized, utilizing microwave accelerated synthesis, for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good activity against MurB in vitro and low MIC values against Gram-positive bacteria, particularly penicillin-resistant Streptococcus pneumoniae (PRSP). Derivative 7l demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria. Derivatives 7f and 10a also demonstrated potent nanomolar Kd values in their binding to MurB.

Mammalian defensins: structures and mechanism of antibiotic activity

Antibiotic peptides are important effector molecules in host-parasite interactions throughout the living world. In vertebrates, they function in first-line host defense by antagonizing a wide range of microbes including bacteria, fungi, and enveloped viruses. The antibiotic activity is thought to be based on their cationic, amphipathic nature, which enables the peptides to impair vital membrane functions. Molecular details for such activities have been elaborated with model membranes; however, there is increasing evidence that these models may not reflect the complex processes involved in the killing of microbes. For example, the overall killing activity of the bacterial peptide antibiotic nisin is composed of independent activities such as the formation of target-mediated pores, inhibition of cell-wall biosynthesis, formation of nontargeted pores, and induction of autolysis. We studied the molecular modes of action of human defense peptides and tried to determine whether they impair membrane functions primarily and whether additional antibiotic activities may be found. We compared killing kinetics, solute efflux kinetics, membrane-depolarization assays, and macromolecular biosynthesis assays and used several strains of Gram-positive cocci as test strains. We found that membrane depolarization contributes to rapid killing of a significant fraction of target cells within a bacterial culture. However, substantial subpopulations appear to survive the primary effects on the membrane. Depending on individual strains and species and peptide concentrations, such subpopulations may resume growth or be killed through additional activities of the peptides. Such activities can include the activation of cell-wall lytic enzymes, which appears of particular importance for killing of staphylococcal strains.

Mechanism of action of the mannopeptimycins, a novel class of glycopeptide antibiotics active against vancomycin-resistant gram-positive bacteria.

The naturally occurring mannopeptimycins (formerly AC98-1 through AC98-5) are a novel class of glycopeptide antibiotics that are active against a wide variety of gram-positive bacteria. The structures of the mannopeptimycins suggested that they might act by targeting cell wall biosynthesis, similar to other known glycopeptide antibiotics; but the fact that the mannopeptimycins retain activity against vancomycin-resistant organisms suggested that they might have a unique mode of action. By using a radioactive mannopeptimycin derivative bearing a photoactivation ligand, it was shown that mannopeptimycins interact with the membrane-bound cell wall precursor lipid II [C(55)-MurNAc-(peptide)-GlcNAc] and that this interaction is different from the binding of other lipid II-binding antibiotics such as vancomycin and mersacidin. The antimicrobial activities of several mannopeptimycin derivatives correlated with their affinities toward lipid II, suggesting that the inhibition of cell wall biosynthesis was primarily through lipid II binding. In addition, it was shown that mannopeptimycins bind to lipoteichoic acid in a rather nonspecific interaction, which might facilitate the accumulation of antibiotic on the bacterial cell surface.

Cell wall analysis

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)β-glucan and (1,3)α-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.

Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon.

In response to sublethal concentrations of antibiotics, bacteria often induce an adaptive response that can contribute to antibiotic resistance. We report the response of Bacillus subtilis to bacitracin, an inhibitor of cell wall biosynthesis found in its natural environment. Analysis of the global transcriptional profile of bacitracin-treated cells reveals a response orchestrated by two alternative sigma factors (sigmaB and sigmaM) and three two-component systems (YvqEC, YvcPQ and BceRS). All three two-component systems are located next to target genes that are strongly induced by bacitracin, and the corresponding histidine kinases share an unusual topology: they lack about 100 amino acids in their extracellular sensing domain, which is almost entirely buried in the cytoplasmic membrane. Sequence analysis indicates that this novel N-terminal sensing domain is a characteristic feature of a subfamily of histidine kinases, found almost entirely in Gram-positive bacteria and frequently linked to ABC transporters. A systematic mutational analysis of bacitracin-induced genes led to the identification of a new bacitracin-resistance determinant, bceAB, encoding a putative ABC transporter. The bcrC bacitracin resistance gene, which is under the dual control of sigmaX and sigmaM, was also induced by bacitracin. By comparing the bacitracin and the vancomycin stimulons, we can differentiate between loci induced specifically by bacitracin and those that are induced by multiple cell wall-active antibiotics.

2‐Phenyl‐5,6‐dihydro‐2H‐thieno[3,2‐c]pyrazol‐3‐ol Derivatives as New Inhibitors of Bacterial Cell Wall Biosynthesis.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Analogues of the mycobacterial arabinogalactan linkage disaccharide as cell wall biosynthesis inhibitors

The mycobacterial arabinogalactan linkage disaccharide [α- l-Rha-(1→3)-α- d-GlcNAc] provides a basis for the design of new antitubercular drugs, since it supports a key skeletal structure in the bacterial cell wall. A series of analogues of the linker was synthesized by coupling appropriate thiorhamnosyl donors modified at their 4-positions, with an N-acetyl glucosamine acceptor. In a cell-free enzyme inhibition assay, three analogues inhibited the activity of the galactosyltransferase that adds a Gal f residue to the linkage disaccharide. Although the compounds were modest inhibitors, these data confirm the viability of this approach to anti-mycobacterial agents. It is especially significant that the three effective compounds are modified at the site of the acceptor atom in the natural substrate.

SigM, an extracytoplasmic function sigma factor of Bacillus subtilis, is activated in response to cell wall antibiotics, ethanol, heat, acid, and superoxide stress.

The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (P(A)) is sigma A dependent, and the other (P(M)) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the P(M) fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 micro g per ml), and by heat shock of 50 degrees C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 micro M paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H(2)O(2), the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 micro M monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 micro g of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival-a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.

2-Phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives as new inhibitors of bacterial cell wall biosynthesis.

Twenty-five 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives were synthesized for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good inhibitory activity against Staphylococcus aureus MurB, MurC and MurD enzymes in vitro and antimicrobial activity against gram-positive bacteria including MRSA, VRE and PRSP. However, when they were tested in the presence of 4% bovine serum albumin, the MIC values increased to greater than 128 microg/mL against PRSP. None of the compounds demonstrated activity against gram-negative bacteria at MIC <32 microg/mL.

Structure-based design approaches to cell wall biosynthesis inhibitors.

This review summarizes some of the published attempts to incorporate protein and NMR structures in the design of new antibiotics that specifically target Cell Wall biosynthesis. Most of the steps involved in peptidglycan synthesis have been investigated as potential strategies against cell wall inhibition. Structural information has been most useful in the design of molecules in the Mur enzyme pathway, penicillin binding proteins and lactamases, as well as proteins that are part of the final steps of transglycosylation - in particular, d-Ala-d-Ala ligase. Several unique issues exist in the design of effective antibacterials, such as the significant differences in protein structure between organisms, such as the case of MurB in which a large amino acid loop that occupies the active site of the E. Coli is gone in the Staph aureus enzyme. Additionally, bacterial resistance is an important issue, and in some cases, structural information can be used to understand the source of this resistance. For example, mutations within the d-Ala-d-Ala ligases lead to the inability of Vancomycin antibiotics to bind.