Bacterial Cell Wall Biosynthesis Research Articles

Studies on macrolides and new antibiotics

After structure elucidation of 16-membered macrolide antibiotic leucomycin, the structures of spiramycin, tylosin, megalomicin, irumamycin and virustomycin were determined by chemical degradation and nuclear magnetic resonance (NMR) spectroscopy. The studies on the chemical modification and structure activity correlation of macrolides led to the elaboration of clinically useful new derivatives.The biosynthetic origin of the carbon skeletone of macrolide aglycone was investigated by means of 13C NMR and the feeding experiments of 13C labeled precursors. We found that an antifungal antibiotic cerulenin is a specific inhibitor of fatty acid and “polyketide” biosyntheses, and applied the antibiotic to the biosynthetic studies of macrolides after formation of the lactone ring and to the production of a new “hybrid” macrolide, chimeramycin. Furthermore, a strain which produces a new “hybrid” antibiotic mederrhodin was breaded from actinorhodin and medermycin producers by gene manipulation.Sixty five or more new antibiotics have been discovered from secondary microbial metabolites using newly devised screening systems. Azureomycins and izupeptins were found as bacterial cell wall biosynthesis inhibitors. In combination with the above screening, several new antibiotics, nanaomycins, frenolicin B, cervinomycins, asukamycin and so on were discovered as antimycoplasmal substances. Diazaquinomycins as antifolate substance, hitachimycin and avermectin as anthelmintics, herbimycin, phosalacine and oxetin as herbicids have been found by the corresponding each screening system. The structure elucidation, biosynthesis and mode of action of these antibiotics have been studied. Among of them, nanaomycin A and avermectin (or ivermectin) have been employed as antifungal and antiparasitic therapeutics, respectively, for veterinary use.

Aztreonam: The first monobactam

A novel screening procedure led to isolation of the structurally unique, bacterially produced, monocyclic beta-lactam antibiotics early in 1979. These naturally occurring “monobactams” were not clinically useful as antibiotics because of their poor antibacterial properties. They were, however, found to interact with certain penicillin-binding proteins of bacteria and thus to interfere with the biosynthesis of bacterial cell walls. The focus of monobactam development then turned toward ihcreasing the binding activity of the beta-lactam ring of the molecule. Aztreonam was the first compound to emerge that fulfilled the objectives of the program. It is relatively inactive against gram-positive and anaerobic bacteria but is extremely effective against aerobic gram-negative bacteria, even in low concentrations. In addition, it is highly resistant to enzymatic hydrolysis by beta-lactamases and demonstrates a high degree of stability against plasmid-mediated gram-negative lactamases. With the chromosomally mediated beta-lactamases, on the other hand, aztreonam can act either as an inhibitor or as a poor substrate. It is unique in that it does not induce production of chromosomally mediated enzymes. Interference with normal gut flora by the use of broad-spectrum antibiotics can result in decreased defense capacity and can lead to intestinal colonization by resistant pathogenic organisms. Therapy directed specifically against the invading pathogen is thus preferred. Such directed therapy is provided by aztreonam. Its narrow spectrum can, if necessary, be broadened by combining it with other antibiotics while continuing to maintain an alternative to the more generalized “shotgun” therapy with its attendant side effects such as disturbances of the natural gut flora, diarrhea, and the emergence of resistant bacteria.

13] Photolabile analogs of the allylic pyrophosphate substrate of prenyltransferases

This chapter designs, prepares and tests photolabile analogs of the allylic pyrophosphate substrate of prenyltransferases as substrates, inhibitors, or covalent modifying agents for the long-chain prenyltransfcrase, C 55 PP synthetase. The synthesis of photolabile analogs of isoprenoid compounds provides a tool for probing the structure of the substrate binding sites of a variety of prenyltransferases. Recently, ones have synthesized two photolabile analogs of the allylic pyrophosphate substrates, geranyl and trans,trans-farnesyl pyrophosphate, which bear the 2-diazo-3-trifluoropropionyloxy (DATFP) moiety. DATFP containing lipids have been used subsequently as effective probes of lipid-lipid and lipid-protein interactions. The availability of photolabile prenyltransferase substrates now offers a number of other opportunities for their application in studying prenyltransferases. The ability to prepare long chain photolabile polyprenyl phosphates such as photoactivatable undecaprenyl- and dolichylmonophosphates would be of significant value in providing a tool to probe enzymes or enzyme complexes, which use the parent prenyl phosphates as glycosyl carriers in bacterial cell wall biosynthesis or mammalian glycoprotein biosynthesis.

The molecular basis for the mode of action of Beta-lactam antibiotics and mechanisms of resistance.

This review on the molecular basis for the mode of action of β-lactam antibiotics and mechanisms of resistance is divided into three main sections. Firstly, a brief introduction to the β-lactam antibiotic family is presented from the standpoint of their natural product origins. The second section is concerned with bacterial cell wall structure and biosynthesis, and the mode of action of β-lactam antibiotics. It includes an attempted rationalization of the multiple enzyme targets of penicillin, the so-called "penicillin binding proteins", into one or two lethal sites of action and the interaction of these enzymes with β-lactams in terms of their analogy to the natural substrate and to the substrate-enzyme transition state. The final part covers the phenomenon of bacterial resistance to β-lactam antibiotic therapy and deals with the two most important manifestations of resistance; permeability and the production of β-lactamases. This latter more crucial factor is then expanded with particular reference to the irreversible inhibition of these enzymes by suicide inactivators; a general theory for irreversible β-lactamase inhibition is discussed and the future prospects within this whole area are briefly overviewed.

Molecular basis for the antimycotic and antibacterial activity of N‐substituted imidazoles and triazoles: The inhibition of isoprenoid biosynthesis

AbstractThe antimycotic N‐substituted imidazoles and triazoles, such as imazalil, ketoconazole and itraconazole, interfere selectively at low concentrations (≥0.01nm) with the 14α‐demethylase system (which is dependent on cytochrome P‐450) of fungal cells, for example, Candida albicans and Penicillium italicum. This results in a decreased availability of ergosterol and the accumulation of 14α‐methyl‐sterols such as lanosterol. Cholesterol synthesis in a subcellular fraction of rat liver, in intact fibroblasts, and in vivo in rat liver, was much less sensitive, for example, to ketoconazole. The imidazole derivatives imazalil, miconazole, ketoconazole and parconazole, and the triazole derivatives propiconazole, terconazole and itraconazole affect the cytochrome P‐450 species of microsomal fractions from Saccharomyces cerevisiae and rat liver. Cytochrome P‐450 of rat‐liver microsomes was much less sensitive to these azole derivatives, in parallel with the lower sensitivity of cholesterol synthesis. Using unilamellar vesicles composed of phosphatidylcholine, phosphatidyl‐ethanolamine and diphosphatidylcholine, multilamellar vesicles of dipalmitoylphos‐phatidylcholine, and intact S. cerevisiae, it was shown that the substitution of ergosterol by lanosterol leads to functional changes in the membranes. It is speculated that the selective interaction of the azole derivatives with the yeast microsomal cytochrome P‐450 leads to the accumulation of 14a‐methyl‐sterols and results in changes in the permeability of the membranes and leakages. The observed inhibition of growth may have its origin in these changes. Miconazole, ketoconazole and deacylated ketoconazole (R‐39519) also affect the growth of Staphylococcus aureus, miconazole being 12.5 and 14 times, respectively, more active than R‐39519 and ketoconazole. The greater antibacterial activity of miconazole coincides with its greater inhibition of the biosynthesis of C‐55 isoprenoid alcohol and vitamin K. The phosphorylated derivative of C‐55 isoprenoid alcohol has functional importance in the biosynthesis of bacterial cell wall and membrane polymers, and the menaquinone vitamin K plays a role in the electron transport of Gram‐positive bacteria. The reduced synthesis of these vital compounds may contribute to the antibacterial activity of miconazole.

Synthesis of analogs of D-Ala-D-Ala as potential inhibitors of bacterial cell wall biosynthesis.

The syntheses of the L,L- and D,D-stereoisomers of N- phenoxyacetyl -X-alanine in which X = Ser, Ala( beta Cl ) or Arg, are described. The antibacterial activity of these peptides and some of their synthetic intermediates has been examined. Four of the intermediates in which X = Ala( beta Cl ) and Arg(NO2), which possess C-terminal benzyl ester groups, were active against viridans streptococci and Streptococcus agalactiae. The D,D-enantiomers were more active than the corresponding L,L-isomers. None of the compounds were active against beta-lactamase producing bacteria or acted as beta-lactamase inhibitors.

Antibacterial activity and mechanism of action of phosphonopeptides based on aminomethylphosphonic acid.

Phosphonopeptides based on aminomethylphosphonic acid as the C-terminal residue linked to L-amino acids possessed antibacterial activity in vitro and in vivo. Analogs in this series were generally less potent than corresponding compounds based on L-1-aminoethylphosphonic acid such as alafosfalin (L-alanyl-L-1-aminoethylphosphonic acid). Significant differences in antibacterial spectra were observed. The mechanism of action involved active transport of the peptide mimetics into the bacterial cells, followed by intracellular release of high concentrations of aminomethylphosphonic acid which inhibited bacterial cell wall biosynthesis. Aminomethylphosphonic acid behaved as a mimetic of both D- and L-alanine and inhibited D-Ala-D-Ala synthetase (EC 6.3.2.4.), alanine racemase (EC 5.1.1.1.), and UDP-N-acetylmuramyl-L-alanine synthetase (EC 6.3.2.8.). The minimal inhibitory concentration of L-norvalyl-aminomethylphosphonic acid was essentially unaffected by the presence of D-alanine, whereas the activity of the corresponding L-norvalyl derivative of L-1-aminoethylphosphonic acid was markedly decreased. Substantial differences in the inhibitory and lytic activity of the L-norvalyl derivatives of aminomethylphosphonic and L-1-aminoethylphosphonic acids were also observed when these agents were combined with other inhibitors of bacterial cell wall biosynthesis.

Structure of the Antibiotic Moenomycin A

The coupling of the (known) structural units, A‐B‐C and D‐E‐F‐G‐H‐I in moenomycin, has been explained by a combination of degradation, and high resolution mass‐ and 1H‐NMR‐spectroscopic studies. Moenomycin is one of the most active inhibitors of bacterial cell wall biosynthesis; now that the structure is known the determination of the important characteristics of its antibiotic activity should follow.

Penicillins and cephalosporins are active site-directed acylating agents: evidence in support of the substrate analogue hypothesis.

Penicillin and related beta-lactam antibiotics are known to exert their bactericidal effects by inhibiting the cross-linking step (transpeptidation) of bacterial cell wall biosynthesis. Evidence is presented in support of the hypothesis that this inhibition results from covalent modification of the active site of sensitive enzymes as a consequence of the structural similarity between penicillin and the acyl-D-alanyl-D-alanine terminus of nascent peptidoglycan strands. Several predictions of this proposal have been verified experimentally. Penicillin-sensitive enzymes are inactivated, with the formation of a covalent, stoichiometric penicilloyl-enzyme complex in vitro. Acylenzyme intermediates have been trapped with several of these enzymes by using cell wall-related substrates. Sequence analysis of the peptides derived from active site-labelled enzymes has established that both penicilloyl and an acyl moiety derived from substrate are covalently bound to the same site, as an ester of serine 36, as predicted by the substrate analogue hypothesis. Sequences near the active site serine are homologous to sequences found in four beta-lactamases, supporting the proposal that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are evolutionarily related. Structural features important for the specific and potent inhibitory properties of beta-lactam antibiotics are discussed in terms of the original substrate analogue hypothesis.

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases.

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.

From penicillin-binding proteins to the lysis and death of bacteria: a 1979 view.

The mechanism by which interference with the biosynthesis of bacterial cell wall causes death and lysis of bacteria appears more complex than originally thought. In an earlier model of the mode of action of beta-lactams, it was assumed that, in the presence of the antibiotics, bacteria synthesize a mechanically weak (poorly cross-linked) cell wall that ruptured under the osmotic-mechanical pressure of the normally growing cytoplasmic mass. However, recent findings suggest a much more complex picture. Lysis and, in at least some bacteria, loss of viability as well, seem to be catalyzed by autolytic enzymes (murein hydrolases), the destructive activity of which is triggered in the beta-lactam-treated bacterium via a poorly understood mechanism. Furthermore, different species of bacteria respond quite differently to treatment with the same beta-lactam: some bacteria are both killed and lysed, others only lose viability, whereas still other species respond mainly by a reversible inhibition of growth (beta-lactam-tolerant bacteria). In addition, structurally different beta-lactams may cause quite different biochemical, morphological, and antibacterial effects, even within the same bacterial species. It is conceivable, therefore, that there is more than one mechanism for loss of viability and/or lysis. Most of the bacteria examined so far contain a number (four to eight) of different penicillin-binding proteins. Genetic and physiological evidence obtained in E. coli indicate that these proteins play essential roles in a variety of physiological functions, such as maintenance of structural integrity, shape, and cell division. Pneumococci with a suppressed autolytic system are resistant to he lytic (and, partially at least, to the bactericidal) effect of beta-lactams. Interference with cell wall synthesis seems to trigger autolysin activity by upsetting the cellular control of autolytic enzyme. It is suggested that the irreversible antimicrobial effect of beta-lactams may have an indirect mechanism in other bacteria as well.

Phosphonopeptides as antibacterial agents: rationale, chemistry, and structure-activity relationships.

Peptide mimetics with C-terminal residues simulating natural amino acids have been designed as inhibitors of bacterial cell wall biosynthesis. The phosphonopeptide series consisting of various l and d residues of natural amino acids combined with 1-aminoalkyl (and aryl-alkyl-) phosphonic acid residues had the most interesting antibacterial properties when the C-terminal residue was l-1-aminoethylphosphonic acid. The in vitro antibacterial activities of representative phosphonodi- to phosphonohexapeptides were investigated. The antibacterial action of the active compounds has been explained in terms of transport into the bacterial cell and intracellular release of the alanine mimetic, which interferes with the biosynthesis of the peptidoglycan of the bacterial cell wall.

Isolation of the L-phase variant from toxigenic Corynebacterium diphtheriae C7(beta).

The L-phase variant was isolated from lysogenic strain C7(beta) of Corynebacterium diphtheriae by the penicillin disk tecnnique on an agar medium containing 3.7% brain heart infusion, 3% NaCl, 1.25% Grycine, 1% Noble agar, and 10% horse serum. The L-phase formed a "fried egg" colony; it was resistant to antibiotics that inhibit biosynthesis of bacterial cell walls but was susceptible to antibiotics that act on the protein-synthesizing system. After several transfers of the L-phase initially isolated, the L-phase of strain C7(beta) became stable, forming a typical L-phase colony on the agar even without penicillin. The L-phase variant retained lysogenicity and toxigenicity. The toxin production by this L-phase variant was, like that by the bacterial phase, inhibited by excess iron.

Gardimycin, a New Antibiotic Inhibiting Peptidoglycan Synthesis

Gardimycin, a new antibiotic, at 100 mug/ml, specifically inhibited cell wall synthesis and induced accumulation of uridine 5'-diphosphate-N-acetylmur-amylpentapeptide in whole cells of Bacillus subtilis. The antibiotic was active in a particulate enzyme preparation from Bacillus stearothermophilus: 60 mug/ml caused 50%, and 200mug/ml caused 100%, inhibition of peptidoglycan synthesis. Suppression of peptidoglycan synthesis was accompanied by parallel accumulation of the lipid intermediate. This mechanism of action is discussed in comparison with those of other antibiotics that are known to inhibit bacterial cell wall biosynthesis.

The significance of D-alanyl-D-alanine termini in the biosynthesis of bacterial cell walls and the action of penicillin, vancomycin and ristocetin

Abstract

Chemical synthesis of pyrophosphodiesters of carbohydrates and isoprenoid alcohols. Lipid intermediates of bacterial cell wall and antigenic polysaccharide biosynthesis.

Chemical synthesis of pyrophosphodiesters of carbohydrates and isoprenoid alcohols. Lipid intermediates of bacterial cell wall and antigenic polysaccharide biosynthesis.

Effect of D-cycloserine on transformation in group H streptococcus, strain Challis.

d-Cycloserine (d-CS), a selective inhibitor of bacterial cell wall biosynthesis, inhibited transformation in group H streptococcus, strain Challis, by preventing the development of the competent state. The incubation of strain Challis cells with d-CS resulted in the production of a substance which inhibited the action of competence factor on these cells. d-CS had an enhancing effect on transformation when deoxyribonucleic acid uptake or phenotypic expression was allowed to occur in its presence.

How penicillin kills bacteria: progress and problems.

Penicillins and cephalosporins are specific inhibitors of the biosynthesis of bacterial cell walls. This discovery was first made in 1957 and was based on two observations. First, penicillins induced the formation of protoplasts or spheroplasts in bacteria (organisms in which the cell wall has been lost or weakened) (Lederberg 1957). Secondly, a uridine nucleotide accumulated in Staphylococcus aureus and other bacteria inhibited by penicillin which had a striking relationship to the composition of the cell wall (Park & Strominger 1957). It was therefore suggested that this nucleotide was an activated precursor of the wall. Over the next decade, a great deal of work was carried out in order to elucidate the structure of the bacterial cell wall and the mechanism of its biosynthesis from the uridine nucleotides and other precursors (reviewed by Strominger 1970; Strominger & Ghuysen 1967; Ghuysen 1968). It was demonstrated that interpeptide cross-links were an important structural feature of the wall. Several kinds of experiments carried out with whole cells indicated that the final step in cell wall synthesis, the crosslinking reaction catalysed by a transpeptidase, was the site of action of penicillin (Wise & Park 1965; Tipper & Strominger 1965 a , b , 1968). Finally, in 1966, the transpeptidase catalysing this cross-linking reaction was obtained in a cell-free system and shown to be a penicillin-sensitive enzyme (Izaki, Matsuhashi & Strominger 1966, 1968). The history of these developments has been reviewed elsewhere (Strominger 1970), and in the present paper, attention will be focused on recent studies of the penicillin-sensitive transpeptidase and other penicillinsensitive activities found in bacterial cell membranes. First, however, it is necessary to describe briefly the structure of the cell wall of bacteria and the nature of the inhibited reactions. The walls of bacteria consist of glycan strands in which two sugars, acetylglucosamine (X) and acetylmuramic acid (Y), strictly alternate (figure 1). Four such glycan strands are represented in figure 1. The acetylmuramic acid residues of the polymer are substituted by a tetrapeptide (represented in the figure by open circles). The peptidoglycan strand (i.e., the glycan substituted by the tetrapeptide) are cross-linked to one another by means of an interpeptide bridge which is to some extent a genus-specific character­istic. In the genus Staphylococcus aureus , the interpeptide bridge is a pentaglycine chain (represented in figure 1 by the closed circles) which extends from the carboxyl group on the terminal D-alanine residue of the tetrapeptide to the ∊-amino group of lysine, the third amino acid in the tetrapeptide chain. The wall of S . aureus is a very tightly knit structure in that virtually every peptide subunit is cross-linked to another subunit by means of this interpeptide bridge. Penicillins and cephalosporins are specific inhibitors of the reaction in which the cross-link is actually formed. This step is the last reaction in wall synthesis.

Compounds formed between nucleotides related to the biosynthesis of bacterial cell wall and vancomycin

Compounds formed between nucleotides related to the biosynthesis of bacterial cell wall and vancomycin

Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions.

In 1929, Fleming discovered penicillins and observed that this group of substances kills gram-positive bacteria more effectively than gram-negative bacteria (thus implying some important physiological difference between the two groups of organisms). I Subsequent knowledge of the bacterial cell wall made it possible to establish both on physiological and chemical grounds that penicillins are specific and highly selective inhibitors of the biosynthesis of cell walls, both in gram-positive and in gram-negative bacteria.2-6 The reason for the relative insensitivity of gramnegative bacteria to most penicillins has remained obscure. More recent knowledge of the structure and biosynthesis of bacterial cell walls has suggested that, in a terminal reaction in cell wall synthesis, linear glycopeptide strands are cross-linked in a transpeptidation, accompanied by release of the terminal D-alanine of the pentapeptide precursor, with formationi of a two- or threedimensional network. Direct chemical analyses of cell walls prepared from cells treated with penicillinll 8 and isotopic studies of wall biosynthesis9' 10 suggested that penicillin was interfering with this hypothetical g]ycopeptide cross-linking reaction. In the presence of penicillin, nascent glycopeptide, an uncross-linked mlonomeric uniit of the wall, accumulated.8 Pulse-labeling experiments have established that this nascent unit is an immediate precursor of the final cross-linked glycopeptide; penicillin completely inhibited its integration into the iietwork.10 Moreover, molecular models of penicillin resembled the acyl-D-alanyl-D-alanine in the ]inear glycopeptide and it was possible to suggest a molecular mlechanism for the transpeptidation and its inhibition by penicillin.8 However, the actual reaction or reactions inhibited had not been demoonstrated. In this paper we wish to report that a cell-free enzymatic system has been obtained from ce]ls of Escherichia coli which catalyzes this cross-linking reactiorn. We call the enzyme which catalyzes this step glycopeptide transpeptidase, since it catalyzes a reaction in which the peptide chains of two linear glycopeptide strands are cross-linked by a transpeptidation, in which the terminal D-alanine residue of one of the strands is elimirnated; this peptide bond synthesis proceeds without any other source of energy and is reversible. The terminal D-alanine residue of the other strand is also removed by hydrolysis catalyzed by a D-alanine carboxypeptidase in the preparation (Fig. 1). Both of these reactions are inhibited by low levels of penici]lin G, other penicillins, and a cephalosporin. Materials and Methods.-Preparation of substrates: UDP-MtirNAc L-ala -D-glu H3-mesoDAP D-ala D-ala and UDP-MurNAc L-ala- D-glu meso-DAP C14-D-ala C14-D-ala were prepared enzymatically by sequential addition of amino acids to the appropriate uridine nucleotides. lOa UJDP-GlcNAc-C14 is the same preparation uised previously. Preparation of enzyme: Two particulate enzymes, obtained from cells of E. coli strain Y-1O or E. coli strain B, were employed. The cells were ground with alumina. The fraction of the dis