Advanced Medicine East Inc., 8 Clarke Drive, Cranbury, NJ 08512, USA1
Author for correspondence: Eugene R. Baizman. Tel: +1 609 655 6925. Fax: +1 609 655 6930. e-mail: ebaizman{at}advmedicine.com
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ABSTRACT |
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Keywords: moenomycin, peptidoglycan synthesis, transglycosylation
a Present address: PanTherix Ltd, Unit 6.10, Kelvin Campus, Maryhill Rd, Glasgow G20 0SP, UK.
b Present address: Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5100, Wallingford, CT 06392-7660, USA.
c Present address: National Institutes of Health, NIAID, DEA, Bethesda, MD 20892, USA.
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INTRODUCTION |
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Moenomycin is a natural product inhibitor of bacterial peptidoglycan synthesis, acting at the stage of transglycosylation. Structurally it consists of a pentasaccharide chain linked to a C25 hydrophobic tail, moenocinol, via a phosphoric acid diester and a glycerol acid unit (Fig. 1). Degradation studies reveal that some of the structural elements present in the parent molecule can be removed with retention of varying degrees of biological activity (for review see El-Abadla et al., 1999
). Although trisaccharide analogues of moenomycin are potent inhibitors of the in vitro transglycosylation reaction in Escherichia coli, they are 50-fold less active as antibacterial agents against Staphylococcus aureus (El-Abadla et al., 1999
). In contrast, specific disaccharide analogues maintain the ability to inhibit Esc. coli transglycosylase activity in vitro, while losing almost all (>500-fold against Sta. aureus, and at least 700-fold against Streptococcus pyogenes) antibacterial activity (El-Abadla et al., 1999
).
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Much less is known about the effects of moenomycin on Gram-positive organisms. Given the poor activity of moenomycin on Esc. coli compared to Gram-positive bacteria, it seems likely that moenomycin evolved as an inhibitor of the much more sensitive Gram-positive organisms. Moenomycin does induce the vancomycin-resistance pathway in Enterococcus faecalis (Mani et al., 1998 ) and Enterococcus faecium (Lai & Kirsch, 1996
), consistent with inhibition of cell-wall synthesis; however, there are few additional data on the biochemical and physiological consequence of exposure of Gram-positive organisms to moenomycin.
There is a growing need for new antibacterial agents that are active on drug-resistant Gram-positive pathogens. Our effort to discover novel analogues of moenomycin prepared by combinatorial synthesis (Kakarla et al., 1999 ; Sofia et al., 1999
) led to identification of several related disaccharide analogues with unique antibacterial properties. Synthetic disaccharide analogues were identified that: (1) were bactericidal for Gram-positive organisms; (2) were active against antibiotic-resistant bacteria (including vancomycin-resistant enterococci); (3) killed growing, but not stationary-phase cells; and (4) inhibited peptidoglycan synthesis in intact Ent. faecalis cells.
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METHODS |
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Esc. coli OV58 (pTA9) overexpressing MurG (Ikeda et al., 1992 ) was grown to an OD600 1·0 at 37 °C in a 10 l batch fermenter in BHI/CAA using 1 l min-1 aeration with air and 150 r.p.m. agitation. The pH was not controlled. The cells were chilled on ice and harvested by centrifugation at 6000 g for 20 min. Cells were washed once with 5 mM Tris/HCl, pH 8·0 [50 ml (g wet pellet wt)-1], then resuspended in the same buffer to a concentration of 0·06 g wet wt ml-1. Membranes were prepared following lysis of cells at 20000 p.s.i. (138 MPa) with a French press. The lysate was centrifuged at 6000 g for 10 min, and the resulting supernatant was ultracentrifuged at 200000 g for 1 h. The pellet was resuspended in assay buffer and used as a source of enzymes for evaluation of lipid II formation (see below).
Determination of MIC values.
The MIC of test compounds was determined in 96-well microtitre plates using twofold dilutions in BHI/CAA medium. Exponentially growing cells were diluted to approximately 5x105 c.f.u. ml-1 and subjected to test compounds solubilized and serially diluted in DMSO. At a final concentration of 5% (v/v), DMSO had no effect on cell growth or viability. Following an 18 h incubation at 37 °C, the OD600 values were read on a microplate reader (Dynatec, model MR5000) immediately after plate mixing. The MIC was determined using the following criteria: (OD600 untreated cells OD600 test concentration)/(OD600 untreated cells OD600 media blanks)x100 90%.
Determination of bactericidal activity.
Cells were grown in BHI/CAA at 37 °C and test compounds were added to exponentially growing cells (OD600 0·080·1). Samples were incubated with shaking at 37 °C for 4 h, and viable cells determined over time by plating a dilution series in triplicate onto BHI/CAA agar plates that were incubated at 37 °C for 1836 h prior to counting. Bactericidal activity was also determined using stationary cells prepared by overnight growth in BHI/CAA. Cell growth was shut off with either chloramphenicol (50 µg ml-1), tetracycline (20 µg ml-1) or moenomycin (10 µg ml-1) added to the culture. Viable counts were determined at the time of growth shut-off, then challenge compounds were added. Viable bacteria remaining after a 4 h incubation at 37 °C with shaking were determined by plating for c.f.u.
Preparation of ether permeabilized Esc. coli cells.
Esc. coli ATCC 23226 cells were exposed to diethyl ether as described (Mirelman et al., 1976 ; Vosberg & Hoffmann-Berling, 1971
) with minor modifications. Frozen bacterial cell pellets (grown and harvested as described above) were thawed on ice, washed twice by centrifugation (8000 g for 10 min) in Basic Medium (Vosberg & Hoffmann-Berling, 1971
), then permeabilized by a 1 min ether treatment. Cell suspensions were stored at -80 °C in the presence of DMSO (1%, v/v) in 1 ml aliquots of 510 mg protein ml-1. A new aliquot was thawed immediately prior to use for each assay, washed with and resuspended in peptidoglycan polymerization assay buffer (see below).
Purification of UDP-MurNAc-pentapeptide from bacteria.
Ent. faecium strain MT10 Rev (Billot-Klein et al., 1997 ), or Bacillus cereus ATCC 11778 was grown aerobically in a 10 l fermenter in BHI/CAA medium. The substrate UDP-MurNAc-pentapeptide, containing either L-lysine or meso-diaminopimelic acid, was isolated from these cells as described (Kohlrausch & Holtje, 1991b
), with minor modifications. The concentration of purified nucleotide sugars was determined using the molar extinction coefficient of uridine,
M,262=1x104. The UDP-MurNAc-pentapeptides were authenticated by mass spectrometry, and lyophilized for storage at -80 °C.
Biotin labelling of UDP-MurNAc-pentapeptide.
UDP-MurNAc-pentapeptide (containing L-lysine) was labelled (Men et al., 1998 ) using solid sulfo-NHS-LC Biotin (Pierce). The UDP-MurNAc-pentapeptide-biotin conjugate was purified by HPLC using a Supelcosil C-18 (4·6 mmx25 cm) column eluted at 1·0 ml min-1 with an elution profile of potassium phosphate (10 min), water (5 min), followed by a 30 min linear gradient to 50% methanol/water (v/v) (Branstrom et al., 2000b
).
Peptidoglycan polymerization assay.
Peptidoglycan synthesis was determined in ether-treated bacteria (ETB) as described (Allen et al., 1992 , 1996
), with modifications for adaptation to automated liquid handing equipment. GFC filter-bottom 96-well microplates (Millipore) were used throughout. Assay buffer was prepared fresh daily from 10x stock solutions, and contained: 50 mM Tris/HCl (pH 8·3); 50 mM NH4Cl; 20 mM MgSO4; 0·15 mM D-aspartic acid, 100 µg tetracycline ml-1 and 0·5 mM ß-mercaptoethanol (reagents from Sigma Chemical). Reaction mixtures (100 µl) also contained: 5 mM ATP (Tris salt), 1·0 µM UDP-N-acetyl-[14C]D-glucosamine [UDP-GlcNAc, DuPont/NEN or Amersham; 200300 mCi (7·411·1 GBq) mmol-1], and 1525 µM UDP-MurNAc-pentapeptide (containing meso-diaminopimelic acid). Assay buffer (10 µl), ATP (20 µl), UDP-MurNAc-pentapeptide (10 µl) and UDP-[14C]GlcNAc (20 µl) were mixed and added as a single 60 µl aliquot to all wells with a Tecan Genesis 150 liquid handler, followed by either 20 µl test compound, reference standard or buffer/vehicle. Reactions were initiated by adding 20 µl ETB suspension (2530 µg bacterial protein well-1). Plates were covered, mixed and incubated for 120 min at 37 °C. A 100 µl aliquot of ice-cold 20% (w/v) TCA was added to terminate the reaction, and plates were held at 4 °C for 30 min to ensure complete precipitation of peptidoglycan. Each plate was then filtered under vacuum and washed rapidly 56 times with 200 µl 10% (w/v) ice-cold TCA. A 30 µl aliquot of Optiphase Supermix (EG & G Wallac) was added to each well for overnight equilibration and plates were counted in a Microbeta Trilux LSC (model 1450; EG & G Wallac). Reaction blanks were defined as corrected c.p.m. retained on the filter either in the absence of UDP-MurNAc-pentapeptide or in the presence of a concentration of reference compound (moenomycin) completely suppressing incorporation of radiolabel. Concentration-response curves were analysed by non-linear regression using a four-parameter logistic model fitted and plotted with GraphPad Prism (v. 2.01, GraphPad Software). IC50 values given in the text represent means from 210 separate experiments, each curve using at least 57 concentrations of test compound in duplicate wells.
Effect of drugs on peptidoglycan degradation.
The stability of peptidoglycan made during various time periods of the reaction using ether permeabilized Esc. coli cells was examined in the presence of the disaccharide analogues. Reactions were set as given above, and initiated by addition of warmed ether permeabilized cells (20 µl, 30 µg protein per well) to all reaction wells. Vehicle or test compound (20 µl) was then added sequentially to appropriate wells at t0 (120 min incubation with drug), t30 min (90 min incubation with drug), t60 min (60 min incubation with drug), t90 min (30 min incubation with drug) and t120 min (0 min incubation with drug). At t120 min, reactions were terminated by addition of 100 µl 20% (w/v) TCA to all wells, followed by shaking for 30 s on an orbital plate shaker. Samples were then processed (see above) and radioactivity incorporated into peptidoglycan determined as described above.
Peptidoglycan synthesis in intact cells.
Ent. faecalis ATCC 29212 was grown in BHI/CAA medium at 37 °C and used to measure incorporation of radiolabelled lysine into peptidoglycan as described by Allen et al. (1996) with minor modifications for adaptation to microtitre filtration. Briefly, this involved treatment of cells with 50 µg tetracycline ml-1 plus 100 µg chloramphenicol ml-1 for 30 min to inhibit protein synthesis. Varying concentrations of drugs were added to cells, and reactions were initiated by addition of [14C]L-lysine [0·25 µCi (9·25 kBq) per reaction, 329 mCi (12·2 GBq) mmol-1; Amersham]. Reactions were stopped, samples processed and data analysed as described above for the peptidoglycan polymerization assay. Incorporation was linear for 60 min, and 1500020000 d.p.m. were incorporated into control samples.
Inhibition of transglycosylation and accumulation of lipid intermediates.
Inhibition of mature (cross-linked) and immature (nascent) peptidoglycan, and accumulation of lipid intermediates was determined as described (Ge et al., 1999 ). Briefly, this method uses 1·0 mg penicillin G ml-1 to inhibit cross-linking of peptidoglycan into mature strands, and follows accumulation of lipid intermediates by extraction into butanol-pyridinium acetate.
Lipid II formation assay.
The assay was performed as described by Branstrom et al. (2000b ). Reaction components consisted of bacterial membranes (25 µg protein), 0·5 µM UDP-[14C]GlcNAc [
20000 d.p.m. (333 Bq)], 10 µM biotinylated UDP-MurNAc-pentapeptide and 0·1% (v/v) Triton X-100, all in 50 µl assay buffer (50 mM Tris/HCl, pH 8·0, 42 mM Mg(Ac)2 and 208 mM KCl). Reactions were preincubated without radiolabelled UDP-GlcNAc for 10 min at room temperature to allow formation of lipid I and then lipid II formation was initiated by addition of UDP-GlcNAc. Lipid II synthesis was allowed to proceed for 15 min at room temperature before being terminated with 25 µl 1% (w/v) SDS. Incorporation of labelled GlcNAc continued for 4050 min in control reactions. Lipid II was identified by mobility using paper chromatography (van Heijenoort et al., 1992
) and specific capture with Softlink avidin resin (Promega) (see below).
Streptavidin-bead capture.
Capture of biotinylated lipid II was as previously described by Branstrom et al. (2000b ). Binding buffer (10 mM Tris/HCl, pH 8·0, 150 mM NaCl, 0·2% v/v Triton X-100) either 500 µl or 100 µl, was added to Eppendorf tubes or 96-well filter plates, respectively, containing lipid II reaction mixtures. Tetralink (Promega) tetrameric avidin resin (35 µl as supplied) was added to each reaction to allow for capture of product. For the tube assay, samples were gently mixed for 1 h at room temperature, centrifuged for 3 min at 1500 g, and then washed four times with 500 µl binding buffer. Reactions run in filter plates were vacuum filtered and washed five times with 200 µl binding buffer. The washed beads from Eppendorf tubes were resuspended in buffer, mixed with scintillation cocktail, and counted in a scintillation counter to determine the amount of lipid II product made. Softlink monomeric avidin resin (Promega) was substituted for Tetralink resin when the captured product needed to be released for further analysis by paper chromatography (Anderson et al., 1966
).
Polymerization of lipid II into peptidoglycan.
Polymerization of lipid II into peptidoglycan was monitored in situ (Branstrom et al., 2000a ). The first step of this assay allowed synthesis and accumulation of lipid II in a reaction mixture containing Esc. coli membranes (0·5 mg ml-1), UDP-MurNAc pentapeptide (20 µM) and radiolabelled UDP-GlcNAc (0·5 µM) in reaction buffer (50 mM Tris; pH 8·0; 42 mM Mg(Ac)2; 208 mM KCl; 0·1% v/v Triton X-100; and 10% v/v DMSO). Reactions were incubated for 2 h at room temperature to accumulate endogenous lipid II. Inhibitors were added prior to removal of Triton X-100 by the addition of Detergent-Out resin (Geno-Technology). Reactions proceeded for an additional 2 h at room temperature to allow conversion of lipid II into peptidoglycan, which was monitored by ascending paper chromatography (Anderson et al., 1966
).
Effect on Candida albicans growth and glucan synthesis.
Candida albicans strain CCH442 was grown in BHI/CAA to OD600 0·1, and drugs added. Growth and viable counts were monitored. Microsomes were prepared as previously described for assay of 1,3-ß-glucan synthesis (Frost et al., 1994 ), monitoring incorporation of [14C]glucose from UDP-glucose into glucan.
In vitro cytotoxicity.
LD50 values for cytotoxicity of moenomycin and the synthetic disaccharide analogues were determined in three mammalian cell lines NIH3T3, HL-60 and HBL-100. Test compounds diluted in OptiMEM (Gibco-BRL), or medium alone were incubated for 6 h at 37 °C. Medium was removed and cells were incubated with fresh medium for 18 h. A Cytolux (EG & G Wallac) luminescent assay kit and Wallac Trilux plate reader were used to screen for cell viability as described by the manufacturer. At least eight concentrations of test compound were used to determine the LD50 computed by a four-parameter logistic fit of percentage cell viability versus drug concentration.
Antibiotics.
Moenomycin A was isolated and purified by standard preparative HPLC from commercial sources (Flavomycin; Hoechst). Reference standard antibiotics were purchased from Sigma. All synthetic disaccharide analogues were prepared at Incara Research Laboratories as described by Sofia et al. (1999) .
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RESULTS |
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In contrast, moenomycin showed only slight bactericidal activity on Gram-positive bacteria. Moenomycin gradually inhibited growth of Ent. faecalis at concentrations ranging from 4 to 600 times MIC, and the optical density gradually increased threefold by 4 h after drug addition (data not shown). Sta. aureus and Sta. epidermidis showed slightly faster growth shut-off occurring 1 h after treatment with moenomycin at 10 or 100 times the MIC, and the optical density declined slightly between 1 and 4 h post-drug addition (Fig. 3). The timing of growth shut-off was similar at 100 times MIC, but the decline in optical density was slightly faster (data not shown).
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Lipid II and peptidoglycan synthesis using Esc. coli membranes
Neither moenomycin, TS30153, TS30663, nor TS30888 inhibited formation of lipid II from UDP-MurNAc-pentapeptide and UDP-GlcNAc in an assay designed to measure only lipid II synthesis (see Branstrom et al., 2000b ; data not shown). By contrast, in the same assay, tunicamycin (an inhibitor of MraY) inhibited lipid II formation by 50% at 1 µg ml-1, as expected. We confirmed that peptidoglycan was in fact formed by Esc. coli membranes from biotinylated UDP-MurNAc-pentapeptide during a 2 h incubation (Fig. 5a
). As expected, moenomycin, at 400 times its IC50, inhibited peptidoglycan formation and caused accumulation of lipid II (Fig. 5b
). TS30153 also inhibited peptidoglycan formation at six times its IC50 (highest concentration testable), but not formation of lipid II (Fig. 5c
), and no accumulation of lipid II relative to controls was observed. Similar results were obtained for TS30888 and TS30663 (data not shown). Lipid II was identified by mobility, incorporation of labelled GlcNAc and the presence of biotin (from UDP-MurNAc-pentapeptide) as assessed by capture with Softlink avidin resin (not shown).
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Analysis of potential peptidoglycan degradation
The disaccharide analogues inhibited incorporation into peptidoglycan of radiolabelled [14C]GlcNAc from UDP-GlcNAc (Table 3). This could be due to either inhibition of peptidoglycan synthesis, or stimulation of peptidoglycan degradation. We thus examined the stability of newly synthesized peptidoglycan made during various time periods over the course of the 2 h incubation. Peptidoglycan synthesis was allowed to occur for various times, and then disaccharide inhibitors were added. Peptidoglycan made prior to disaccharide addition, even during the first 30 min, was stable during the remainder of the 2 h incubation (Fig. 6
). These data show that the disaccharide analogues inhibit formation of peptidoglycan, but do not stimulate its degradation.
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In vitro cell viability
The LD50 for cytotoxicity in three mammalian cell lines (NIH3T3, HL-60 and HBL-100) averaged 15 µg ml-1 for TS30663 and >50 µg ml-1 for both TS30888 and TS30153. A concentration of 100 µg moenomycin ml-1 failed to exhibit significant effects on viability in any of these cell lines (data not shown). The differential antimicrobial effects that were observed at MIC levels for moenomycin versus novel disaccharide analogues reported in the present studies are, therefore, unlikely to be due to non-specific cytotoxicity.
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DISCUSSION |
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A programme was initiated based on synthesis of analogues of the moenomycin disaccharide structure (Kakarla et al., 1999 ; Sofia et al., 1999
). Our rationale was that creating new sites of interaction with amino acid residues adjacent to the active site on a target enzyme could enhance even the weak activity reported for disaccharide analogues related to the moenomycin core structure (El-Abadla et al., 1999
). Simplified lipids were also included in the synthetic scheme in place of the C25 moenocinol. Combinatorial chemistry, using synthetic strategies developed in our laboratory (Sofia et al., 1999
; Kakarla et al., 1999
) was used to explore chemical modification of the disaccharide core. Several active compounds were identified and three (TS30153, TS30663 and TS30888) were investigated in detail.
The three disaccharide analogues studied all possess antibacterial activity against Gram-positive bacteria, including strains resistant to vancomycin, with MIC values ranging from 3 to 12 µg ml-1. Since the moenomycin disaccharide degradation product (TS0514) is inactive as an antibacterial agent, our data demonstrate that appropriate derivatization of the E and F units can lead to antibacterial agents active on both sensitive and resistant bacteria. These disaccharide analogues are active on vancomycin-resistant strains and are nearly as potent as vancomycin on vancomycin-sensitive strains.
Almost all the data in the literature pertaining to inhibition of transglycosylase activity by moenomycin and its physiological consequences on bacteria, comes from the study of Esc. coli (Hara & Suzuki, 1984 ; Kohlrausch & Holtje, 1991a
; Tamura et al., 1980
; van Heijenoort et al., 1978
, 1987
; van Heijenoort & van Heijenoort, 1980
). Moenomycin is rapidly bactericidal to growing Esc. coli, resulting in bacterial lysis and cell death, although lysis is not required for loss of viability. In contrast, our data reveal that the bactericidal activity of moenomycin is limited for Gram-positive bacteria. Only 12 log units of killing occurred when Gram-positive bacteria were treated with 510 times the MIC value of moenomycin. Killing was increased to 3 log units when the most sensitive species, Sta. epidermidis, was treated with 100 times the MIC of moenomycin. In contrast, the disaccharide analogues killed 36 log units when Gram-positive bacteria were treated with four to eight times the MIC. Thus, modifications to the disaccharide core resulted in the discovery of novel bactericidal analogues contrasting with the parent compound that had only bacteriostatic effects in Gram-positive bacteria.
Our data revealed that disaccharide analogues blocked peptidoglycan synthesis in ether-permeabilized Esc. coli at some point between the synthesis of lipid II and its polymerization into peptidoglycan by transglycosylation. This in vitro system requires the concerted action of MraY, MurG, lipid II translocation and recycling, and polymerization of lipid II into peptidoglycan. Evidence supporting inhibition of the transglycosylation stage by the synthetic disaccharide analogues is as follows. Neither the disaccharide analogues nor moenomycin inhibited lipid II synthesis in a system that depended on the functioning of MraY and MurG. However, both moenomycin and the disaccharide analogues did inhibit conversion of lipid II into peptidoglycan. Disaccharide analogues and moenomycin inhibited synthesis of both mature and immature peptidoglycan, data consistent with inhibition at the transglycosylase stage. However, the disaccharide analogues caused less accumulation of lipid II than moenomycin. In addition, stimulation of peptidoglycan degradation was ruled out, since peptidoglycan made during various time periods in ether permeabilized Esc. coli was stable following addition of the disaccharide inhibitors. Thus, one may conclude that the site of inhibition is between lipid II formation and its polymerization into peptidoglycan via the transglycosylation process.
The synthetic disaccharide analogues target cell-wall synthesis in vivo, in intact Gram-positive bacteria, as assessed by both direct and indirect experimental results. They all inhibited incorporation of lysine into cell-wall material in intact Ent. faecalis with IC50 values within twofold of their respective MIC values on the same strain. In addition, the disaccharide analogues were bactericidal only to actively growing cells. Stationary-phase cells and cells pretreated with protein synthesis inhibitors or moenomycin were resistant to the bactericidal effects of the disaccharide analogues. Such differential killing is indicative of cell-wall synthesis inhibitors. Although little is known about the stringent response in Gram-positive bacteria, our data indicate that protein synthesis may be required for killing caused by the disaccharide analogues. Disaccharide analogues did not inhibit growth of C. albicans CCH442, or synthesis of 1,3-ß-glucan from microsomes prepared from this strain (data not shown). The glucan-synthesis complex is known to be sensitive to agents which non-specifically perturb membrane structure (Goldman et al., 1995 ; Ko et al., 1994
). Testing for overt cytotoxicity at concentrations well above the MIC in several mammalian cell lines suggests that these novel compounds (and moenomycin itself) are unlikely to induce non-specific bacterial cell killing.
Our data are consistent with the following hypotheses and current base of knowledge regarding bacterial transglycosylases and their inhibition by moenomycin. Moenomycin is not a universal transglycosylase inhibitor, since it does not inhibit the monofunctional glycosyltransferase from Esc. coli (Di Berardino et al., 1996 ; Hara & Suzuki, 1984
), or the transglycosylase activity from Micrococcus luteus (Park & Matsuhashi, 1984
). Thus one would expect differential effects on bacteria depending on (a) the repertoire of transglycosylases inhibited, (b) the degree of inhibition and (c) the physiological consequences of inhibition. The pronounced bactericidal activity and subsequent rapid lysis of Esc. coli following treatment with moenomycin might result from inhibition of the transglycosylase activity of PBP1A and PBP1B, but it may also inhibit other transglycosylase enzymes such as PBP1C (Schiffer & Holtje, 1999
). Our data are consistent with the hypothesis that these synthetic disaccharide analogues of moenomycin target specific components of the transglycosylation process in Gram-positive bacteria in a unique manner not shared by the parent compound, moenomycin. Perhaps moenomycin inhibits an essential transglycosylase activity in Gram-positive bacteria, but one that accounts for only a minor fraction of the total peptidoglycan. Given the complexity of the interactions between the biosynthetic components involved in peptidoglycan synthesis (Holtje, 1996a
, b
, 1998
; Koch, 1998
; Vollmer et al., 1999
; von Rechenberg et al., 1996
), further work will be required to understand the physiological consequences of inhibition of the transglycosylation process by different inhibitors. We are currently using these and other synthetic disaccharide analogues to clarify some of these processes.
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ACKNOWLEDGEMENTS |
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Received 23 May 2000;
revised 8 August 2000;
accepted 13 September 2000.