Inhibitors Of Cell Wall Biosynthesis Research Articles

An attenuated mutant of the Rv1747 ATP-binding cassette transporter ofMycobacterium tuberculosisand a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-relatediniBACoperon

The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence.

Interaction of Streptomyces sp. VITSTK7 compounds with selected antifungal drug target enzymes by in silico molecular docking studies

Antifungal drugs are inhibitors either of fungal cell wall biosynthesis or essential reaction steps of fungal metabolic pathways. In silico studies have proved to be very effective on screening small molecules to be used as drugs and identifying essential reactions and pathways as targets. The aim of the present study was to predict the interactions of compounds present in the ethyl acetate extract of Streptomyces sp. VITSTK7 against selected fungal drug target enzymes. The ethyl acetate extract of the isolate showed significant anti-Aspergillus activity against the selected Aspergillus pathogens. Presence of the three compounds (C22H37NO7, C17H24N4O6 and C24H28N2O5) in the extract was identified by GC-MS spectra and matched with reference compounds available in the MS spectra library, NIST (National Institute for Standards and Technology). These compounds were analysed for the interaction with five selected fungal target proteins 1AFR, 1EA1, 1LKP, 1ZHX and 3PD73i3E. Docking was done using Patch dock beta 1.3 version and analysed by pymol 1.3 version. The tested compounds C22H37NO7, C17H24N4O6 and C24H28N2O5 showed least binding energy of -254.64 kcal/mol, -248.71 kcal/mol and -338.57 kcal/mol respectively with 1ZHX. The result of this study revealed that all the three compounds from the strain had higher interaction with 1ZHX protein than with the other proteins. It shows that this strain could be the promising source for the antifungal drug.

Molecular Mechanisms and Clinical Impact of Acquired and Intrinsic Fosfomycin Resistance.

Bacterial infections caused by antibiotic-resistant isolates have become a major health problem in recent years, since they are very difficult to treat, leading to an increase in morbidity and mortality. Fosfomycin is a broad-spectrum bactericidal antibiotic that inhibits cell wall biosynthesis in both Gram-negative and Gram-positive bacteria. This antibiotic has a unique mechanism of action and inhibits the initial step in peptidoglycan biosynthesis by blocking the enzyme, MurA. Fosfomycin has been used successfully for the treatment of urinary tract infections for a long time, but the increased emergence of antibiotic resistance has made fosfomycin a suitable candidate for the treatment of infections caused by multidrug-resistant pathogens, especially in combination with other therapeutic partners. The acquisition of fosfomycin resistance could threaten the reintroduction of this antibiotic for the treatment of bacterial infection. Here, we analyse the mechanism of action and molecular mechanisms for the development of fosfomycin resistance, including the modification of the antibiotic target, reduced antibiotic uptake and antibiotic inactivation. In addition, we describe the role of each pathway in clinical isolates.

Cloning and analysis of the planosporicin lantibiotic biosynthetic gene cluster of Planomonospora alba.

The increasing prevalence of antibiotic resistance in bacterial pathogens has renewed focus on natural products with antimicrobial properties. Lantibiotics are ribosomally synthesized peptide antibiotics that are posttranslationally modified to introduce (methyl)lanthionine bridges. Actinomycetes are renowned for their ability to produce a large variety of antibiotics, many with clinical applications, but are known to make only a few lantibiotics. One such compound is planosporicin produced by Planomonospora alba, which inhibits cell wall biosynthesis in Gram-positive pathogens. Planosporicin is a type AI lantibiotic structurally similar to those which bind lipid II, the immediate precursor for cell wall biosynthesis. The gene cluster responsible for planosporicin biosynthesis was identified by genome mining and subsequently isolated from a P. alba cosmid library. A minimal cluster of 15 genes sufficient for planosporicin production was defined by heterologous expression in Nonomuraea sp. strain ATCC 39727, while deletion of the gene encoding the precursor peptide from P. alba, which abolished planosporicin production, was also used to confirm the identity of the gene cluster. Deletion of genes encoding likely biosynthetic enzymes identified through bioinformatic analysis revealed that they, too, are essential for planosporicin production in the native host. Reverse transcription-PCR (RT-PCR) analysis indicated that the planosporicin gene cluster is transcribed in three operons. Expression of one of these, pspEF, which encodes an ABC transporter, in Streptomyces coelicolor A3(2) conferred some degree of planosporicin resistance on the heterologous host. The inability to delete these genes from P. alba suggests that they play an essential role in immunity in the natural producer.

Tuning the Moenomycin Pharmacophore To Enable Discovery of Bacterial Cell Wall Synthesis Inhibitors

New antibiotic drugs need to be identified to address rapidly developing resistance of bacterial pathogens to common antibiotics. The natural antibiotic moenomycin A is the prototype for compounds that bind to bacterial peptidoglycan glycosyltransferases (PGTs) and inhibit cell wall biosynthesis, but it cannot be used as a drug. Here we report the chemoenzymatic synthesis of a fluorescently labeled, truncated analogue of moenomycin based on the minimal pharmacophore. This probe, which has optimized enzyme binding properties compared to moenomycin, was designed to identify low-micromolar inhibitors that bind to conserved features in PGT active sites. We demonstrate its use in displacement assays using PGTs from S. aureus, E. faecalis, and E. coli. 110,000 compounds were screened against S. aureus SgtB, and we identified a non-carbohydrate based compound that binds to all PGTs tested. We also show that the compound inhibits in vitro formation of peptidoglycan chains by several different PGTs. Thus, this assay enables the identification of small molecules that target PGT active sites, and may provide lead compounds for development of new antibiotics.

Antimicrobial mechanism of lantibiotics

Lantibiotics are ribosomally synthesized antimicrobial peptides that commonly target the cell wall precursor lipid II during their antimicrobial mechanism and exert their inhibitory activity by (i) inhibition of cell wall biosynthesis, and (ii) stable pore formation in the target membrane. Type-A(I) (i.e. nisin) and two-component (i.e. lacticin 3147) lantibiotics initially interact with lipid II to stabilize the complex, which then proceeds to inhibit cell wall biosynthesis and pore formation. Type-A(II) (i.e. nukacin ISK-1) and type-B (i.e. mersacidin) lantibiotics also use lipid II as a docking molecule, but can only inhibit cell wall biosynthesis without forming pores. In the present paper, we review the antimicrobial mechanism of different types of lantibiotics, their current progress and future prospect.

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized α-helical and β-sheet (CSαβ) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wall biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76μM. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.

The Putative Lactococcal Extracytoplasmic Function Anti-Sigma Factor Llmg2447 Determines Resistance to the Cell Wall-Active Bacteriocin Lcn972

Lactococcin 972 (Lcn972) is a cell wall-active bacteriocin that inhibits cell wall biosynthesis in Lactococcus lactis. In this work, the transcriptomes of the Lcn972-resistant (Lcn(r)) mutant L. lactis D1 and its parent strain were compared to identify factors involved in Lcn972 resistance. Upregulated genes included members of the cell envelope stress (CesSR) regulon, the penicillin-binding protein pbpX gene and gene llmg2447, which may encode a putative extracytoplasmic function (ECF) anti-sigma factor. The gene llmg2447 is located downstream of the nonfunctional ECF gene sigX(pseudo). Nisin-controlled expression of llmg2447 led to high Lcn972 resistance in L. lactis, with no cross-resistance to other cell wall-active antimicrobials. Upregulation of llmg2447 in L. lactis D1 (Lcn(r)) was linked to the integration of insertion element IS981 into the llmg2447 promoter region, replacing the native -35 box and activating the otherwise silent promoter P(2447). This is the first example of an orphan ECF anti-sigma factor involved in bacteriocin resistance. This new role in neutralizing cell wall-active compounds (e.g., Lcn972) could have evolved from a putative primary function of Llmg2447 in sensing cell envelope stress.

Isolation of Lactococcus lactis Mutants Simultaneously Resistant to the Cell Wall-Active Bacteriocin Lcn972, Lysozyme, Nisin, and Bacteriophage c2

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972, Lactococcus lactis D1 and D1-20, with high (>320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover, L. lactis lacking a functional D,D-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein gene pip. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance.

Further insights into the mode of action of the lipoglycopeptide telavancin through global gene expression studies.

Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a decylaminoethyl side chain that is active against Gram-positive bacteria, including Staphylococcus aureus strains resistant to methicillin or vancomycin. A dual mechanism of action has been proposed for telavancin involving inhibition of peptidoglycan biosynthesis and membrane depolarization. Here we report the results of genome-wide transcriptional profiling of the response of S. aureus to telavancin using microarrays. Short (15-min) challenge of S. aureus with telavancin revealed strong expression of the cell wall stress stimulon, a characteristic response to inhibition of cell wall biosynthesis. In the transcriptome obtained after 60-min telavancin challenge, in addition to induction of the cell wall stress stimulon, there was induction of various genes, including lrgA and lrgB, lysine biosynthesis operon (dap) genes, vraD and vraE, and hlgC, that have been reported to be induced by known membrane-depolarizing and active agents, including carbonyl cyanide m-chlorophenylhydrazone, daptomycin, bacitracin, and other antimicrobial peptides These genes were either not induced or only weakly induced by the parent molecule vancomycin. We suggest that expression of these genes is a response of the cell to mitigate and detoxify such molecules and is diagnostic of a membrane-depolarizing or membrane-active molecule. The results indicate that telavancin causes early and significant induction of the cell wall stress stimulon due to strong inhibition of peptidoglycan biosynthesis, with evidence in support of membrane depolarization and membrane activity that is expressed after a longer duration of drug treatment.

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity.

Haloduracin α Binds the Peptidoglycan Precursor Lipid II with 2:1 Stoichiometry

The two-peptide lantibiotic haloduracin is composed of two post-translationally modified polycyclic peptides that synergistically act on Gram-positive bacteria. We show here that Halα inhibits the transglycosylation reaction catalyzed by PBP1b by binding in a 2:1 stoichiometry to its substrate lipid II. Halβ and the mutant Halα-E22Q were not able to inhibit this step in peptidoglycan biosynthesis, but Halα with its leader peptide still attached was a potent inhibitor. Combined with previous findings, the data support a model in which a 1:2:2 lipid II:Halα:Halβ complex inhibits cell wall biosynthesis and mediates pore formation, resulting in loss of membrane potential and potassium efflux.

Interaction of 5-(2, 4-dimethylbenzyl) pyrrolidin-2-one with selected antifungal drug target enzymes by in silico molecular docking studies

Currently the criteria used for selecting optimal new antifungal drug candidates include inhibitors of fungal cell wall biosynthesis, essential reaction and pathways. In silico approach resulting in the identification of essential reactions and pathways spreads across several parts of metabolism. The aim of the present study was to study the interaction of the isolated anti-Aspergillus compound, 5-(2, 4-dimethylbenzyl) pyrrolidin-2-one (DMBPO) from a novel Streptomyces VITSVK5 spp. with 6 selected antifungal drug target enzymes by in silico molecular docking approach. The compound DMBPO showed minimum binding energy (-6.66 kcal/mol) with 14 alpha-sterol demethylase (cyp51), (-5.65 kcal/mol) with Rubythrine, and (-4.43 kcal/mol) with β-1-3 Glucan binding protein. Two enzymes 14 alpha-sterol demethylase (cyp51) and β-1-3 Glucan were reported to be mostly responsible for drug resistance in Aspergillus species. The compound DMBPO interacted with several amino acid residues, of which leucine was found to be common among all the target enzymes for protein and hydrogen bond formation. Our results suggest that DMBPO could target Aspergillus fungal proteins to exhibit anti-Aspergillus activity in drug resistant strains.

Theoretical studies on benzimidazole derivatives as E. coli biotin carboxylase inhibitors

Biotin carboxylase (AccC) protein plays an essential role in cell wall biosynthesis in majority of bacterial genera. Inhibition of cell wall biosynthesis might be an ideal way to control the bacterial multiplication in the host system. AccC is one of the promising targets for the antibacterial drugs production. The benzimidazole derivatives are hopeful biotin carboxylase inhibitors, which sensitizes to the Escherichia coli (E. coli) and many other bacterial species too. In steam of developing better benzimidazole derivatives, we describe a quantitative pharmacophore model of benzimidazole derivatives using Phase module of Schrodinger LLC. This model suggested that the following features are essential for ligand binding, i.e., two aromatic rings, two hydrogen bond donors, one hydrogen bond acceptor, and one hydrophobic group. Further, atom-based 3D-QSAR model was constructed using training set of 37 inhibitors. The constructed QSAR model has cross validated co-efficient value of (Q2) 0.736 and regression co-efficient value of (R2) 0.937. The external validation indicated that our QSAR model possessed high predicted powers with \( r_{o}^{2} \) value of 0.933, \( r_{\text m}^{2} \) value of 0.876. The best active and least active compounds were docked into the active site of receptor using Glide and hotspots of the active site were analyzed. The QSAR elucidated here for benzimidazole derivatives combined with their binding information will provide an opportunity to explore the chemical space to promote the potency of AccC inhibitors.

Echinocandins in Children

Echinocandins in Children

Mechanism of Inhibition of Bacillus anthracis Spore Outgrowth by the Lantibiotic Nisin

The lantibiotic nisin inhibits growth of vegetative Gram-positive bacteria by binding to lipid II, which disrupts cell wall biosynthesis and facilitates pore formation. Nisin also inhibits the outgrowth of bacterial spores, including spores of Bacillus anthracis, whose structural and biochemical properties are fundamentally different from those of vegetative bacteria. The molecular basis of nisin inhibition of spore outgrowth had not been identified, as previous studies suggested that inhibition of spore outgrowth involved either covalent binding to a spore target or loss of membrane integrity; disruption of cell wall biosynthesis via binding to lipid II had not been investigated. To provide insights into the latter possibility, the effects of nisin were compared with those of vancomycin, another lipid II binding antibiotic that inhibits cell wall biosynthesis but does not form pores. Nisin and vancomycin both inhibited the replication of vegetative cells, but only nisin inhibited the transition from a germinated spore to a vegetative cell. Moreover, vancomycin prevented nisin’s activity in competition studies, suggesting that the nisin-lipid II interaction is important for inhibition of spore outgrowth. In experiments with fluorescently labeled nisin, no evidence was found for a covalent mechanism for inhibition of spore outgrowth. Interestingly, mutants in the hinge region (N20P/M21P and M21P/K22P) that still bind lipid II but cannot form pores had potent antimicrobial activity against vegetative B. anthracis cells but did not inhibit spore outgrowth. Therefore, pore formation is essential for the latter activity but not the former. Collectively, these studies suggest that nisin utilizes lipid II as the germinated spore target during outgrowth inhibition and that nisin-mediated membrane disruption is essential to inhibit spore development into vegetative cells.

Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.

ChemInform Abstract: Synthesis of Reactive γ-Lactams Related to Penicillins and Cephalosporins.

γ-Lactams with structures analogous to both penicillin (1) and cephalosporin (2) antibiotics have been synthesised. These compounds show high reactivity towards modest nucleophiles in keeping with their design as potential inhibitors of bacterial cell wall biosynthesis. Reactive γ-lactam analogues 27 and 29 of β-lactamase inhibitors such as 22 have also been prepared. An X-ray crystal structure of the cephalosporin analogue 8 has allowed structural comparisons to be made with the classical β-lactam antibiotic cephaloridine (30).

Human β-Defensin 3 Inhibits Cell Wall Biosynthesis in Staphylococci

Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.

An oldie but a goodie – cell wall biosynthesis as antibiotic target pathway

Bacterial cell wall biosynthesis represents the target pathway for penicillin, the first antibiotic that was clinically applied on a large scale. Penicillin, by means of its beta-lactam ring, inhibits a number of enzymes which participate in inserting monomeric cell wall building blocks into the cell wall polymer and which have been termed penicillin-binding proteins (PBPs). Ever since the introduction of penicillin, hundreds of beta-lactam antibiotics have been developed and details of their molecular activities elaborated. Meanwhile, various additional classes of antibiotics have been described, which inhibit the same pathway, yet use target molecules others than the PBPs. Such classes include the glycopeptide antibiotics, lipopeptide and lipodepsipeptide antibiotics, the lantibiotics and various other natural product antibiotics with comparatively complex structures. They usually target the membrane-bound steps of the biosynthesis pathway and the highly conserved lipid-bound intermediates of the building block such as lipid II, which represents a particular “Achilles’ heel” for antibiotic attack. With in-depth analysis of the activity of more recently identified inhibitors and with the availability of novel techniques for studying prokaryotic cell biology, new insights were obtained into the molecular organisation of the cell wall biosynthesis machinery and its interconnections with other vital cellular processes such as cell division. This, in turn, provides hints for new targets to be exploited and for the development of novel cell wall biosynthesis inhibitors.