Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci

Messer, Janet; Reynolds, Peter E.

doi:10.1016/0378-1097(92)90608-q

Cited by 66 publications

(79 citation statements)

References 17 publications

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“…When acquired, as in Enterococcus faecalis or Enterococcus faecium, it is due to the presence of a new set of genes (2, 15), leading to the phenotype VanA or VanB, both of which are characterized by the synthesis of a cytoplasmic precursor containing a Cterminal D-lactate instead of the normal C-terminal D-alanine (1,7,14,19,25). The level of glycopeptide resistance of these organisms is associated with two main parameters: the lower affinity of the C-terminal D-alanyl-D-lactate of the new cytoplasmic precursors for the glycopeptides (3, 23) and the residual cell pool of the normal UDP-MurNAc-pentapeptide precursor, the D-alanyl-D-alanine terminus of which has a high affinity for glycopeptides (4, 6, 26).…”

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Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics

et al. 1997

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The structure of the peptidoglycan of Lactobacillus casei ATCC 393, a species highly resistant to glycopeptide antibiotics, was examined. After digestion, 23 muropeptides were identified; monomers represented 44.7% of all muropeptides, with monomer tetrapeptides being the major ones. Fifty-nine percent of the peptidoglycan was O-acetylated. The cross-bridge between D-alanine and L-lysine consisted of one asparagine, although aspartate could be found in minor quantities. Since UDP-MurNAc-tetrapeptide-D-lactate is the normal cytoplasmic precursor found in this species, monomer tetrapeptide-lactate was expected to be found. However, such a monomer was found only after exposure to penicillin, suggesting that penicillin-sensitive D,D-carboxypeptidases were very active in normal growing cells.Resistance to glycopeptide antibiotics among gram-positive bacteria is either acquired or naturally expressed (3, 7). When acquired, as in Enterococcus faecalis or Enterococcus faecium, it is due to the presence of a new set of genes (2, 15), leading to the phenotype VanA or VanB, both of which are characterized by the synthesis of a cytoplasmic precursor containing a Cterminal D-lactate instead of the normal C-terminal D-alanine (1,7,14,19,25). The level of glycopeptide resistance of these organisms is associated with two main parameters: the lower affinity of the C-terminal D-alanyl-D-lactate of the new cytoplasmic precursors for the glycopeptides (3, 23) and the residual cell pool of the normal UDP-MurNAc-pentapeptide precursor, the D-alanyl-D-alanine terminus of which has a high affinity for glycopeptides (4, 6, 26). The natural high-level resistance of Leuconostoc mesenteroides, Pediococcus sp., and Lactobacillus casei to glycopeptides can be explained by the presence of a cytoplasmic precursor with a C-terminal D-lactate and the total absence of one with a C-terminal D-alanine (7,20). The primary structure of the peptidoglycan of L. casei, like that of E. faecium, belongs to subgroup A4 (27) and has a common monomer structure, GlcNAc-MurNAc-L-Ala-␥-DGlu-L-Lys-D-Ala, with an asparagine attached to the ε-amino group of lysine (21, 27). Thus, we were interested in determining if any difference in the overall composition of the peptidoglycan would exist between L. casei ATCC 393 and the VanB-type E. faecium D366 that we had previously described (8), both of which synthesize a lactate precursor.Muropeptide composition of L. casei ATCC 393 in the absence of penicillin G. L. casei ATCC 393 was obtained from the Institut Pasteur collection and grown in MRS broth (Diagnostic Pasteur). The MIC of vancomycin was Ͼ512 g/ml, and that of teicoplanin was 512 g/ml (7). Peptidoglycan was extracted as previously described (8). Briefly, a 500-ml exponential-phase culture was grown to an A 650 of 0.4 in the absence of vancomycin and quickly chilled in an ice-ethanol bath. After concentration by centrifugation, cells were boiled in 4% (wt/vol) sodium dodecyl sulfate (SDS) and cell walls were purified by using pronase followed by trypsin, each a...

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Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics

et al. 1997

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“…In particular, the presence of unusual nucleotide precursors such as UDPMurNAc-pentadepsipeptide and UDP-MurNAc-tetrapeptide-D-Ser involved in the resistance of various gram-positive bacteria against glycopeptide antibiotics has been investigated by this method (1,4,5,12,13,21). More recently, qualitative and quantitative analyses of complex mixtures were greatly facilitated by coupling HPLC with mass spectrometry (MS).…”

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Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin

et al. 1997

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Analyses of the peptidoglycan nucleotide precursor contents of enterococci and staphylococci treated with ramoplanin, tunicamycin, or vancomycin were carried out by high-pressure liquid chromatography coupled with mass spectrometry (MS). In all cases, a sharp increase in the UDP-N-actetylmuramoyl-pentapeptide or -pentadepsipeptide pool was observed. Concomitantly, new peptidoglycan nucleotide peptides of higher molecular masses with hexa-or heptapeptide moieties were identified: UDP-MurNAc-pentapeptide-Asp or pentadepsipeptide-Asp in enterococci and UDP-MurNAc-pentapeptide-Gly or -Ala and UDP-MurNAc-pentapeptide-Gly-Gly or -Ala-Gly in staphylococci. These new compounds are derivatives of normal UDP-MurNAcpentapeptide or -pentadepsipeptide precursors with the extra amino acid(s) linked to the lysine -amino group as established by various analytical procedures (MS, MS-MS fragmentation, chemical analysis, and digestion with R39 D,D carboxypeptidase). Except for tunicamycin-treated cells, it was not possible to ascertain whether these unusual nucleotides were formed by direct addition of the amino acids to UDP-MurNAc-pentapeptide (or -pentadepsipeptide) or whether they arose by reverse reactions from lipid I intermediates to which the amino acids had been added.Analysis of the pools of peptidoglycan nucleotide precursors by high-pressure liquid chromatography (HPLC) is now a wellestablished procedure which was initially developed for E. coli (19,20) and which has led to a better understanding of the metabolism of bacterial peptidoglycan (31). In particular, the presence of unusual nucleotide precursors such as UDPMurNAc-pentadepsipeptide and UDP-MurNAc-tetrapeptide-D-Ser involved in the resistance of various gram-positive bacteria against glycopeptide antibiotics has been investigated by this method (1,4,5,12,13,21). More recently, qualitative and quantitative analyses of complex mixtures were greatly facilitated by coupling HPLC with mass spectrometry (MS). This approach has just been used successfully to analyze both nucleotide precursors (12, 13) and fragments (6, 7) of bacterial peptidoglycan. Here it was extended to the analysis of the nucleotide precursor contents of extracts from enterococci and staphylococci treated with ramoplanin, tunicamycin, or vancomycin. As observed previously in many cases (25), the inhibition of peptidoglycan polymerization or of the formation of the lipid precursors led to sharp increases in the UDP-MurNAcpentapeptide or -pentadepsipeptide pool. Moreover, concomitantly new peptidoglycan nucleotide precursors of higher molecular masses with hexa-or heptapeptide moieties were identified in this study. Their plausible origins are also discussed. MATERIALS AND METHODSStrains and growth conditions. All strains used in this study are listed in Table 1. Enterococcus faecium MT9 expresses glycopeptide resistance constitutively and was derived from E. faecium D366, a clinical isolate expressing an inducible VanB-type resistance (2), grown on teicoplanin. E. faecium MT10R is a vancomycin-sen...

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“…Alaz = 40 mM) (Dutka-Malen et al, 1990;Bugg et al, 1991a,b). A predicted D-Ala-D-lactate terminus, resistant to vancomycin recognition, has been detected in these glycopeptide-resistant bacteria (Handwerger et al, 1992;Messer & Reynolds, 1992). Given the similarities in primary structure and the alteration in substrate specificity among Ddl and VanA ligases, we have sought to identify regions of the enzymes that are important to function.…”

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Identification of a common protease‐sensitive region in d‐alanyl‐d‐alanine and d‐alanyl‐d‐lactate ligases and photoaffinity labeling with 8‐azido ATP

Wright

Walsh

1993

Protein Science

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Modified peptidoglycan precursors produced by glycopeptide-resistant enterococci

Cited by 66 publications

References 17 publications

Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics

Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics

Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin

Identification of a common protease‐sensitive region in d‐alanyl‐d‐alanine and d‐alanyl‐d‐lactate ligases and photoaffinity labeling with 8‐azido ATP

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