Inhibition Of Cell Wall Synthesis Research Articles

Epopromycins, Novel Cell Wall Synthesis Inhibitors of Plant Protoplast Produced by Strcptomyces sp. NK04000.

Epopromycins, Novel Cell Wall Synthesis Inhibitors of Plant Protoplast Produced by Strcptomyces sp. NK04000.

A vancomycin-inducible lacZ reporter system in Bacillus subtilis: induction by antibiotics that inhibit cell wall synthesis and by lysozyme.

We have constructed a Bacillus subtilis strain in which expression of a vanH::lacZ gene fusion is regulated by VanR and VanS of Enterococcus faecium. This construct allows a nonpathogenic bacterial strain to be used as a model system for studying regulation of vancomycin resistance. Antibiotics and enzymes that affect cell wall biosynthesis and stability were tested for the ability to induce lacZ expression. As a result, fosfomycin and D-cycloserine were added to the group of peptidoglycan synthesis inhibitors shown to induce expression from the vanH promoter. Induction by cell wall hydrolytic enzymes, as well as by antibiotics whose actions may lead to the accumulation of chemically different peptidoglycan precursors, raises the possibility that models that postulate induction by peptidoglycan [correction of peptidodoglycan] precursors are wrong.

Glycosyltransferases as targets for inhibition of cell wall synthesis in M. tuberculosis and M. avium

A series of threo- and erythro-configured open-chain trihydroxy ketones was synthesized starting from l- and d-ascorbic acid respectively as the starting material, through the use of Grignard reactions for the requisite CC bond formations. The lipophilic ketones were screened against Mycobacteriumtuberculosis for anti-proliferative activity. The lipophilic ketones with tetradecyl alkyl side chains were found to be moderately active against cell proliferation.

Gibberellic acid promotes growth and cell wall synthesis in Avena internodes regardless of the orientation of cell expansion

Segments cut from the next‐to‐last (peduncular‐1) internode of Avena sativa L. cv. Victory (oat) shoots elongate as much as 10‐fold in response to gibberellic acid (GA3). The objective of the present investigation was to differentiate the effects of GA3 on growth from its effects on wall synthesis (measured gravimetrically and through the incorporation of [14C]‐glucose) by using several cell wall synthesis inhibitors with widely varying mechanisms of action. Four compounds, viz. monensin, cycloheximide, lanthanum, and galactose. caused (1) relatively little inhibition of either cell wall synthesis or elongation in segments without GA3, (2) roughly proportionate, dose‐dependent inhibition of elongation and wall synthesis in GA3‐treated segments and (3) generally greater inhibition of GA3‐promoted uptake of radioactivity than of wall incorporation or elongation. Two other compounds, colchicine and 2,6‐dichlorobenzonitrile (DCB). (1) inhibited GA3‐induced elongation considerably more than cell wall synthesis and (2) caused swelling (radial expansion). especially of GA3‐treated segments. DCB‐treated internodal cells apparently compensated for inhibited cellulose synthesis by greater synthesis of matrix polysaccharide (beginning between 3 and 6 h). While normal cellulose synthesis was not required for short‐term (up to 6 h) GA3‐induced elongation or for long‐term hormone‐promoted radial expansion, it was required for sustained GA3‐induced elongation. These results indicate that GA3‐promoted cell wall loosening (manifested as radial expansion) and cell wall synthesis in Avena internodes occur at least partially independently of any hormonal effect on the orientation of microtubules and microfibrils.

Mechanism of action of antifungals and combination therapy

Biochemical studies have identified a number of potential targets for antifungal chemotherapy, including cell wall synthesis, membrane sterol biosynthesis, nucleic acid synthesis, metabolic inhibition and macromolecular biosynthesis. The inhibition of cell wall synthesis would be in theory highly specific since only fungal cells build their cell wall with chitin and glucan. Despite this defined specificity, none of the clinically available drugs act there. Most antifungals interfere with enzymatic steps involved in the complicated synthesis of ergosterol. Allylamines interfere with the squalene epoxidation leading to an intracellular accumulation of squalene. The imidazole and triazole derivatives interact with the cytochrom P-450-dependent C14-demethylase system. C14-methyl-sterols like lanosterol are accumulated within the fungal cell. Under treatment of morpholine derivatives like amorolfine, ignosterol a sterol containing a Δ14 double bond is accumulated. Amorolfine inhibits two steps in the pathway of ergosterol biosynthesis namely the Δ14 reductase and the Δ7,8 isomerase.Amorolfine possesses a broad antimycotic spectrum against fungi pathogenic to plants and humans. The drug not only inhibits the fungal growth, it also shows a strong fungicidal activity which is both concentration- and time-dependent. The fungicidal activity against dermatophytes is in the same order as the one of terbinafine. Amorolfine also shows good fungicidal activity against yeasts, dimorphic and dematiaceous fungi.Amorolfine exerts synergistic activity in combination with several other antifungals such as ketoconazole, terbinafine, itraconazole and griseofulvin. The fungistatic activity of all these antifungals against dermatophytes is slightly increased by addition of amorolfine. The synergistic effect seen in vitro is, however, more pronounced in an animal model. Combination therapy of amorolfine with terbinafine, itraconazole and griseofulvin was studied in a murine trichophytosis model. Murine trichophytosis is self-healing, the course of infection reaches a plateau on day 7 and declines thereafter throughout the second week. Mice infected with trichophyton and treated with various antifungal drugs generally responded to the treatment in a dose-dependent manner, showing a significant reduction in the appearance of mycotic foci in comparison to untreated animals. The combinations of amorolfine with griseofulvin, terbinafine, itraconazole and fluconazole were always more efficient than the individual drug treatment alone, provided that the single therapy is not overwhelmingly efficacious per se. Antagonism was never observed.

Inhibition of cell wall synthesis — is this the mechanism of action of penicillins?

Penicillins have been shown to inhibit bacterial cell wall synthesis, and interact with penicillin binding proteins, leading to bacterial lysis. These two mechanisms, the former more than the latter are believed to be responsible for their therapeutic potential. It has further been demonstrated that only actively multiplying cells are susceptible to bactericidal effects of the antibiotic, which is in accordance with the suggested mechanism of action. Bacterial growth takes place in terms of size and number, both requiring additional cell wall. An increase in bacterial size is due to an icrease in the volume of cytosol and area of the cell wall. Presently there is no proof that the former is the cause of the latter or vice versa. Penicillin by inhibiting cell wall synthesis would inhibit both growth and multiplication. Since the antibiotic is bactericidal to rapidly multiplying cells, its effect on cell wall would interfere with its bactericidal action. As per the present understanding penicillin acts principally by inhibiting cell wall synthesis. There is however a discrepancy between its observed effects and what should logically be expected, which forces us to reexamine the mechanism of action of penicillin. We believe that the present understanding of the action of penicillin is incomplete if not outright faulty. It would be expedient to radically modify the same in view of its implication, for example on drug development.

Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis

The pneumocandins are natural lipopeptide products of the echinocandin class which inhibit the synthesis of 1,3-beta-D-glucan in susceptible fungi. The lack of a corresponding pathway in mammalian hosts makes this mode of action an attractive one for treating systemic infections. Substitution by an aminoethyl ether at the hemiaminal and dehydration and reduction of the glutamine of pneumocandin B0 produced a semisynthetic compound (L-733,560) with intrinsic water solubility, significantly increased potency, and a broader antifungal spectrum. To evaluate the mechanism for the improved antifungal efficacy, we determined that L-733,560 was a more potent inhibitor of glucan synthase activity in vitro, did not affect the other membrane-bound enzymes tested, conferred susceptibility to lysis in the absence of osmotic support, and did not disrupt currents in liposomal bilayers or 86Rb+ fluxes from liposomes. In Aspergillus species L-733,560 also produced the same morphological alterations as pneumocandin B0. A stereoisomer of L-733,560 with poor antifungal activity was a weak inhibitor of glucan synthase. All of these results support the notion that the enhanced antifungal activity of L-733,560 is achieved by superior inhibition of glucan synthesis and not by nonspecific membrane effects or a second mode of action.

Electric fields induce curved growth of Enterobacter cloacae, Escherichia coli, and Bacillus subtilis cells: implications for mechanisms of galvanotropism and bacterial growth

Directional growth in response to electric fields (galvanotropism) is known for eukaryotic cells as diverse as fibroblasts, neurons, algae, and fungal hyphae. The mechanism is not understood, but all proposals invoke actin either directly or indirectly. We applied electric fields to bacteria (which are inherently free of actin) to determine whether actin was essential for galvanotropism. Field-treated (but not control) Enterobacter cloacae and Escherichia coli cells curved rapidly toward the anode. The response was both field strength and pH dependent. The direction of curvature was reversed upon reversal of field polarity. The directional growth was not due to passive bending of the cells or to field-induced gradients of tropic substances in the medium. Field-treated Bacillus subtilis cells also curved, but the threshold was much higher than for E. cloacae or E. coli. Since the curved morphology must reflect spatial differences in the rates of cell wall synthesis and degradation, we looked for regions of active wall growth. Experiments in which the cells were decorated with latex beads revealed that the anode-facing ends of cells grew faster than the cathode-facing ends of the same cells. Inhibitors of cell wall synthesis caused spheroplasts to form on the convex regions of field-treated cells, suggesting that the initial curvature resulted from enhanced growth of cathode-facing regions. Our results indicate that an electric field modulates wall growth spatially and that the mechanism may involve differential stimulation of wall growth in both anode- and cathode-facing regions. Electric fields may therefore serve as valuable tools for studies of bacterial wall growth. Use of specific E. coli mutants may allow dissection of the galvanotropic mechanism at the molecular level.

Therapeutic Effect of Fosfomycin on Experimentally Induced Pseudotuberculosis in Yellowtail.

In vitro antibacterial activity of fosfomycin (FOM), a cell-wall synthesis inhibitor, was investigated against Pasteurella piscicida, the causative agent of pseudotuberculosis in yellowtail Seriola quinqueradiata. Minimum inhibitory concentrations (MIC) of FOM against 68 isolates of P. piscicida, including strains resistant to other antibiotics, ranged from 1.56 to 3.13 μg/ml. Bactericidal activity of FOM was evident at the MIC level.Therapeutic effect of FOM was studied in yellowtail experimentally infected with P. piscicida. The cumulative mortalities among infected fish after oral administration of FOM at doses of 0, 5, 10, 10, 30 and 40 mg/kg/day for 5 consecutive days from 1 h after infection were 60, 45, 45, 15, 5 and 0%, respectively.Based on the results obtained in this study, FOM may be a new therapeutic agent against pseudotuberculosis in yellowtail.

Influence of growth rate and nutrient limitation on monobactam production and peptidoglycan synthesis in Pseudomonas aeruginosa.

The effects of growth rate and nutrient limitation on monobactam production, peptidoglycan content and mean cell length in Pseudomonas aeruginosa was studied using continuous culture techniques. All three parameters increased progressively with growth rate, a greater response being shown under carbon limitation compared to that occurring under nitrogen limiting conditions. Interestingly, monobactum production mirrored peptidoglycan synthesis. In addition, the monobactam exhibited a broad range of antibacterial activity and bound preferentially to PBP 1A in the producing organism. Moreover, addition of the monobactam to a growing culture inhibited cell wall synthesis. These results are discussed in relation to the control and regulation of peptidoglycan synthesis.

A kinetic study on the regeneration of protoplasts in the presence of cell wall synthesis inhibitors

A kinetic study on the regeneration of protoplasts in the presence of cell wall synthesis inhibitors

A kinetic study on the regeneration of Candida albicans protoplasts in the presence of cell wall synthesis inhibitors.

Aculeacin A and papulacandin B block cell wall regeneration in Candida albicans protoplasts at an intermediate step in which the protoplasts have not yet synthesized the rigid structure of the cell wall and are therefore still osmotically sensitive. In the presence of the antibiotics, total synthesis of glucan is not significantly lowered with respect to control cells, although most of it appears either in the culture medium or in the regenerating wall as alkali-soluble glucan. Thus, it is proposed that echinocandins (such as aculeacin A) and papulacandins may not inhibit glucan synthesis per se but instead inhibit its incorporation into the supramolecular organization of the cell wall.

Increased transformation levels in intact cells of Saccharomyces cerevisiae aculeacin A‐resistant mutants

Several Saccharomyces cerevisiae mutants resistant to the cell wall synthesis inhibitor aculeacin A exhibit higher transformation levels than the parental strain. Mutant acr2 has been studied in more detail. It is transformed up to ten-fold more efficiently than the wild-type strain with episomal, centromeric and integrative plasmids, and dimethyl sulfoxide has an additive effect improving transformation efficiency. Transformation with linear DNA molecules is not as much affected in acr2 cells. The observed effects may be caused by the altered plasma membrane composition of the mutants.

Effect of antibiotics on endotoxin release from gram-negative bacteria

Antibiotics may inhibit bacterial growth or may kill bacteria by inhibiting cell wall synthesis or protein synthesis. The amount of endotoxin released during antibiotic action has been found to be clinically important. Nine antibiotics, representing seven classes, were studied for the amounts of endotoxin released during their action on susceptible strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. Staphylococcus aureus, which produces no endotoxin, was used as a control organism. Aztreonam induced the highest release of endotoxin, whereas other antibiotics such as imipenem and the quinolones induced the lowest release of endotoxin. Although the quantities of endotoxin released are not easily explained from the established mechanisms of antibiotic action, our findings may have implications for therapy of the acutely ill, septic patient in whom release of large quantities of endotoxin may be catastrophic.

Patent Evaluation: Novel bacterial cell wall synthesis inhibitors

Patent Evaluation: Novel bacterial cell wall synthesis inhibitors

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.

In vitro Antibacterial Activity of Bicozamycin against Pasteurella piscicida.

In vitro antimicrobial activity of bicozamycin (BCM), a cell wall synthesis inhibitor, was investigated against Pasteurella piscicida. Minimum inhibitory concentrations (MIC) of BCM against 263 clinical isolates of P. piscicida ranged from 1.56 to 6.25μg/ml. High incidence of drug-resistant strains agains other drugs such as β-lactamam antibiotics and pyridonecarboxylic acids were recognized. No cross resistance between BCM and other antibacterial agents were observed. BCM displayed bactericidal activity at its MIC level.P.piscicida strains showed a slowly increasing and stepwise pattern of artificial developmen of resistance against BCM.From the results obtained, it may be suggested that BCM would be applied as a new type of therapeutic agent among the cell wall synthesis inhibitors for the treatment of pseudotuberculosis.

Galactose inhibits auxin-induced growth of Avena coleoptiles by two mechanisms.

Galactose inhibits auxin-induced growth of Avena coleoptiles by at least two mechanisms. First, it inhibits auxin-induced H(+)-excretion needed for the initiation of rapid elongation. Galactose cannot be doing so by directly interfering with the ATPase since fusicoccin-induced H(+)-excretion is not affected. Secondly, galactose inhibits long-term auxin-induced growth, even in an acidic (pH 4.5) solution. This may be due to an inhibition of cell wall synthesis. However, galactose does not reduce the capacity of walls to be loosened by H+, given exogenously or excreted in response to fusicoccin.

Regeneration from Sweetpotato Protoplasts and Assessment of Growth Conditions for Flow-sorting of Fusion Mixtures

Petiole protoplasts of the sweetpotato [Ipomoea batatas (L.) Lam.] cultivars Red Jewel and Georgia Jet formed cell walls within 24 hours and divided in 2 to 3 days. Pretreating enzyme solutions with activated charcoal increased the viability and division frequency of protoplasts. Culture of protoplast-donor plants in a medium containing STS did not affect plant growth, protoplasm yield, or viability, but did increase the division frequency. Culture of protoplasts for 24 hours in a medium containing DB, a cell wall synthesis inhibitor, or staining of protoplasts with FDA did not significantly affect division frequency. The division frequency of protoplasts cultured in liquid medium was significantly higher than that of protoplasts cultured in agarose-solidified medium. Cell cycle analysis of petioles and freshly isolated protoplasts showed that the latter has a significantly higher proportion of nuclei in G1 phase. Protoplasts did not initiate DNA synthesis or mitosis within the first 24 hours of culture. Low-frequency regeneration of shoots from protoplast-derived callus was accomplished on MS medium containing 1.0 mg ldnetin/liter when preceded by MS medium modified to contain only (in mg·liter-1) 800 NH4NO3, 1400 KNO3, 0.5 2,4-D, 0.5 kinetin, and 1.0 ABA. Roots produced from protoplast-derived callus formed adventitious shoots after 4 weeks on MS medium containing 2% sucrose, 0.02 mg kinetin/liter and 0.2% Gelrite. Secondary shoot formation from regenerated roots will be a more effective means of obtaining plants from protoplasts than direct shoot regeneration from callus. Chemical names used: silver thiosulfate (STS): 2.6-dichlorobenzonitrile (DB); fluorescein diacetate (FDA): 2.4-diacetate (FDA); 2.4 dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA).

Reevaluation of the factors involved in the efficacy of new beta-lactams against Enterobacter cloacae

The roles of outer membrane permeability, beta-lactamase stability, and inhibition of penicillin-binding proteins in the activity of new beta-lactams against Enterobacter cloacae were reappraised by using several methodological improvements. Outer membrane permeability in intact cells was determined by using a high-pressure liquid chromatography (HPLC)-based technique that avoided certain possible artifacts of the traditional methods. Vmax values were calculated from the numbers of enzyme molecules produced per cell and from catalytic constant (Kcat) values, which were obtained with purified beta-lactamase. Minimal periplasmic antibiotic concentrations needed to inhibit bacterial cell wall synthesis were estimated from the Zimmermann-Rosselet equation. All the beta-lactams tested formed relatively stable complexes with purified beta-lactamase. The antibiotics that exhibited low affinity for beta-lactamase apparently needed higher periplasmic concentrations to inhibit cell wall synthesis, suggesting a possible correlation between the affinity of beta-lactamase and the affinity of penicillin-binding proteins for the new beta-lactams. By using these estimates of outer membrane permeability, beta-lactamase hydrolysis, and cell wall-inhibiting concentrations, MIC could be theoretically predicted to within 1 dilution for five beta-lactams in three isogenic E. cloacae strains with differences in antibiotic susceptibility due to different porin or beta-lactamase contents.