Download PDF

FEMS MicrobiologyLetters94 (1992) 195-200
© 19q2Federation of European MicrobiologicalSocieties0378-1097/92/$05.00
Publishedby Elsevier
195
FEMSLE04952
Modified peptidoglycan precursors produced
by glycopeptide-resistant enterococci
Janet Messer and Peter E. Reynolds
Departmentof Biochemistry.Unitetsityof Cambridge.Cambridge,UK
Reeei,,'ed24 April Iqq2
Accepted 28 April 1992
Key words: Vancomycin resistance; Peptidoglycan synthesis; Peptidoglyean precursors
1. SUMMARY
Cytoplasmic precursors of the peptidoglycan
biosynthetic pathway were purified from vancomycin-treated, glycopeptide-sensitive and -resistant strains of Enterococcus faecium. Resis.
tance was due to production of a modified precursor, UDP-MurNAc-L-Ala-D-Glu-L-Lys-o-AlaD-lactate, where lactate was identified on the
basis of mass of the precursor and on its ability to
act as a substrate for o-lactate dehydrogenase
after release from the precursor. The presence of
the D-lactate residue instead of o-alanine in the
terminal position would hinder formation of a
vancomycin-precursor complex, without preventing incorporation of the precursor into mature
peptidoglycan.
2. INTRODUCTION
High-level resistance to glycopeptide antibiotics in Enterococcus faecium is at least partially
Correspondenceto: P.E. Reynolds,Department of Biochemistry, Universityof Cambridge, Tennis Court Road, Cambridge CB2 IQW, UK.
mediated by a 39-kDa protein VanA, whose
amino acid .,,equence is related to those of
Gram-negative o-Ala-o-Aia ligases [1]. These enzymes catalyse synthesis of the D-AIa-D-Ala
dipeptide which forms the C-terminal portion of
the peptide chain of peptidoglycan precursors.
VanA was p,lrified and shown to have a substantially modified substrate specificity in vitro, compared with that of Gram-negative D-AIa-D-AIa
ligases [21: it catalysed the synthesis of mixed
dipeptides, D-AIa-X, in which X was o-norvaline,
o-norleucine, o-methionine or o-phenylalanine.
In an e:aension of this research it was reported
that a protein encoded by a gene upstream of
vanA on the resistance plasmid from E. faecium
BM 4147, designated vanH, had extensive amino
acid homology with a 2-hydroxycarboxylic acid
dehydrogenase [3]. The authors proposed that
VanH synthesized a 2-h: droxycarboxylic acid that
would condense with o-Ala by formation of an
ester linkage in a reactio,n catalysed by VanA.
The ability of crude extracts from vancomycin-resistant and -sensitive E. faecium and of purified
preparations of VanH and VanA to synthesise
peptides and depsipeptides from o-amino acids
and D-hydroxycarboxylic acids in vitro was
196
demonstrated [4]. On the basis of the substrate
specificities and kinetic parameters it was proposed that the identity of the hydroxycarboxylic
acid was o-2-hydroxybutyrate.
Early work on the mode of action of glycopeptides showed that incubation in the presence of
these antibiotics resulted in the accumulation of
cytoplasmic peptidoglycan precursors in certain
bacteria, e.g. Staphylococcus aureus, Bacillus
megaterium [5,6]. in this investigation, the ability
of E. faecium to accumulate peptidoglycan precursors when cell wall growth was inhibited was
studied and their structure determined in order
to demonstrate unequivocally the pathway of
peptidoglycan synthesis in high-level, glycopeptide-resistant strains.
3. MATERIALS AND METHODS
3.1. Bacterial strains
E. faecium STR 211 (glycopeptide sensitive)
and E. faecium STR 207 (a high level glycopeptide-resistant clinical isolate) were obtained from
PHLS, Colindale, UK. Their MICs for vancomycin were 2/~g/ml and 1024 p.g/ml, respectively.
3.2. Accumulation of cell wall precursors in vancomycin-treated bacteria
3-1 cultures of E. faecium STR 211 and 207
were grown in brain heart infusion broth containing 0.5% (w/v) g!ucose. A sub-inhibitory concentration of vancomycin (4 ~g/ml) was added to
the culture of E. faecium STR 207 to induce
resistance [7]. Bacteria were grown to the exponential phase, an inhibitory concentration of vancomyein was added and incubation continued for
30 min at 37°C. Bacteria were harvested and
washed in 10 mM phosphate buffer (pH 7.0). The
pellet was extracted with 5% (w/v) trichloroacetic
acid for 10 min on ice and centrifuged at 39000
× g for 30 s. The supernatant was extracted three
times with an equal volume of ether and the
content of N-acetylhexosamine (indicative of cell
wall precursors) was determined by the method
of Strominger [8].
3.3. Purification and analysis of hexosamine-containing nucleotides from E. faecium
Peptidoglycan precursors were isolated and
purified essentially as described by Reynolds [6]
using a linear chloride gradient (0-0.2 M NaCI in
0.01 M HCI), followed by desalting on a G 10
Sephadex column, Fractions giving a 1 : 1 ratio for
UDP (based on uv absorption) and N-acetylhexosamine were combined and the amino acid
composition was determined after hydrolysis with
6 M HCi for 4 h at 105°C, using an automatic
amino acid analyser.
3.4. Electrophoretic mobility of peptidoglycan precursors
Samples containing 100 nmol UDP-MurNAcpeptides were subjected to flat-bed paper electrophoresis in 50 mM ammonium bicarbonate
(pH 7.9) at 3 kV for 45 min. Nucleotides were
located using a UV transilluminator (254 nm).
3.5. Analysis of peptidoglycan orecursors by mass
spectrometiy
Samples of purified cell wall precursors w e r e
analysed by negative Fast Ion Bombardment Mass
Spectrometry using a Kratos MS50 mass spectrometer. The matrix used was glycerol or dithioglycerol. The spectra obtained were compared
with those of authentic peptidoglycan precursors
isolated from S. aureus.
3.6. Identification of terminal residue of cell wall
precursors
The terminal residue of o,o-peptides, depsipeptides or cell wall precursors was released by
incubation of 20 nmol substrate with 5 ~g purified o,o-peptidase from Actinomadura R 39 in
50 mM Tris.CI (pH 7.5), 4 mM MgCt 2 (final
volume 30/zl) for 30 rain at 37°C [9]. o-alanine
was assayed using the o-amino acid oxidase/
peroxidase procedure [10]. o-Lactate was assayed
197
by incubation with 2 p,g o-lactate dehydrogenase
in 0.42 M glycine/0.33 M hydrazine buffer (pH
9) containing NAD + (4 mg/ml). The absorption
was determined at 340 nm after 30 min incubation at 30°C [11].
4. RESULTS
hexosamine-containing material was eluted from
the ion-exchange column by applying 0.5 M NaCI
in 0.01 M HCI after the initial chloride gradient
had finished. This peak contained UDP, muramic
acid, glucosamine and alanine suggesting that the
precursors UDP-N-acetylmuramic acid, UDP-Nacetylmuramyl-L-Ala and UDP-N-acetylglucosamine had also accumulated in small amounts.
4.1. Purification of cytoplasmic peptidoglycan precursors from E. faecium STR 211 and E. faecium
STR 207
4.Z Identification of hexosamine.containing nucleotides
In small scale tests it was found that maximal
accumulation of hexosamine-containing nucleotides was obtained by incubation of E. faecium STR 211 with 4 / z g / m l vancomycin and of
E. faecium STR 207 with 1500/zg/ml. Extraction
of 3-1 cultures of 17,.faecium STR 211 and STR
207 incubated in the presence of these concentrations of vancomycin resulted in yields of 17 and
22 gmol hexosamine-containing compounds/g
dry weight bacteria, respectively. Ion-exchange
chromatography of the extract from E. faecium
STR 211 resulted in one UV-absorbing peak containing UDP-N-acetylhexosamine ( A ) w h i c h
eluted at a salt concentration of approximately
0.1 M. Two peaks (B and C) containing UDP-Nacetylhexosamine were eluted close together at a
similar salt concentration during chromatography
of the extract from E. faecium STR 207. Further
faecium STR 211 (A) was identified as UDP-
The hexosamine-containing nucleotide from E.
MurNAc-r-Ala-v-Glu-L-Lys-o-Ala-o-A!a on the
basis of its absorption spectrum, content'of Nacetylhexosamine, identification of the hexosamine as muramic acid (after hydrolysis), and
the 3:1 : 1 ratio of alanine: glutamic acid: lysine
(Table 1). However, the nucleotides present in
both peaks B and C purified from E. faecium
STR 207 contained only four amino acid residues
instead of five. The ratio of alanine:glutamic
acid:lysine in these compounds was 2:1:1 (Table 1).
The nucleotide present in peak A and the
'tetrapeptide' sample from peak B from E. faecium STR 207 migrated to the same positions as
the standards of UDP-MurMAc-pentapeptide
and -tetrapeptide respectively. The 'tetrapeptide'
sample from peak C migrated more slowly than
Table 1
Characterisationof cell walt precursorsfromglycopeptide.sensitiveand -resistant strains of E [aecium
UDP a,
Muramicacidb,
Alanineb,
Giutamicacidb,
Lysineb,
Electrophoreticmobility(cm)¢
Mass
E. faeciura STR 211
(glycopeptide-sensitive)
Peak A
0.9
0.9
3.0
1.0
[1.0]
16
1149
E. faeciura STR 207
(glycopcptide-rcsistant)
Peak B
Peak C
1.0
0.9
1.0
0.9
2.0
2.0
!.1
1.0
[!.01
[1.0]
16
17
1150
i 078
a Basedon absorbanceat 262.5 nm. Identificationbased on 250/260 nm and 280/260 nm ratios.
b DeterminedaRer hydrolysisin 6 M HCI at 105°Cfor 4 h.
¢ Flat-I~delectropboresisat pH 7.9 for 45 rain at 3 kV.
* Relative to lysine.
198
the tetrapeptide standard, in a position similar to
that of UDP-MurNAe-pentapeptide, suggesting
that it was slightly larger than the tetrapeptide
precursor but carried the same charge at pH 7.9.
4.3. Analysis of wall precursors by mass spectrometry
The spectra obtained contained peaks corresponding to the masses of the molecular ion and
the molecular ion containing one or two sodium
ions. The masses of the nucleotides from peaks A
and B corresponded to the standards of UDPMurNAc-pentapeptide and -tetrapeptide respectively, a result consistent with their electrophoretic mobilities. The mass of the nucleotide from peak C was one greater than that of
authentic UDP-MurNAc-pentapeptide: this increase would be accounted for by the replacement of the terminal amide-Iinked alanine by
ester-linked lactate.
4.4. Determination of C-terminal residue of nucleotide
The o,D-peptidase from Actinomadura R 39
catalyses the hydrolysis of D,D-peptides, esters
and thioesters [12]. Incubation of the nucleotides
from peaks A, B and C and of standard cell wall
precursors with the D,D-peptidase, followed by
specific assays for a D-amino acid or D-lactate
confirmed the tentative identification based on
mass spectrometry that the cell wall precursor
from vancomycin-resistant E. faecium was UDPMurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-laetate (Table 2).
5. DISCUSSION
In the light of these results it is proposed that
VanA catalyses formation of the depsipeptide
D-Ala-D-lactate which is then added to UDPMurNAc-L-Ala-D-Glu-L-Lys: the modified precursor would then be incorporated into lipid intermediates and nascent peptidoglycan, the presumed targets of glycopeptide antibiotics. The
presence of the altered cell wall precursor (requiring both the activities of VanA and VanH) in
high-level, glycopeptide-resistant strains cf E.
faecium indicates that such organisms have acquired substantial amounts of genetic information to ensure that the different precursor is
synthesised and utilised in competition with the
normal peptidoglycan precursor. In our investigations there was no accumulation of UDPMurNAc-pentapeptide when vancomycin-resistant E. faecium was incubated in the presence
of ",ancomycin. Consequently the controls operate
at an early stage in the biosynthetic sequence.
This could be achieved by repression of the normal D-AIa-D-AIa ligase, control of its activity or
hydrolysis of any D-AIa-D-AIa produced. Gut-
Table 2
Identificationof C-terminalresidue present in cell wall precursorsfrom vancomycin-sensitiveand -resistant E, faecium
Substrate
UDP-MurNAc-pentapeptidefrom S. aureus
Ac2-L-Lys-D-AIa-D-AIa
Ac2-L-Lys-n-Ala-n-lactate
E. faecium STR 211 (vancomycin-sensitive)
Peak A
E. faecium STR 207 (vancomycin-resistant)
Peak B
Peak C
Residue hydrolysedby treatmentwith
D,t>-peptidasefrom ActinomaduraR 39
D-alaninea
D-lactate a
(nmol)
(nmol)
18
0
19
0
0
18
19
0
0
0
0
17
a D-alanineand D-lactatewere assayedenzymicallyas in MATERIALSAND METHODS.
199
mann et al. [13] demonstrated that vancomycin
induced a o,a-carboxypeptidase in glycopeptideresistant strains of enterocoeei that catalysed removal of o-alanine from UDP-MurNAc-pentapeptide or from D-AIa-D-AIa; however, this enzyme activity was not tested against molecules
terminating in acyl-o-Ala-D-lactate which are hydrolysed by penicillin-sensitive o,o-peptidases
[11,14,15]. The small amount of UDP-MurNActetrapeptide purified in this investigation probably arose as the result of chemical breakdown of
the modified precursor during the extraction and
purification procedures rather than by specific
enzymic hydrolysis.
Studies with model cell wall substrates have
demonstrated that Ae2-L-Lys-o-Ala-D-iactate and
other esters and thioesters can interact with o,opeptidases and transpeptidases [12,14], which
suggests that the modified precursor, once incorporated into linear peptidoglycan, could take part
in the final stage of peptidoglycan synthesis. As
this reaction involves removal of the C-terminal
residue of the modified precursor the final structure of the mature peptidoglycan would be virtually unchanged from that of a glycopeptide-sensitive Enterococcus, though some o-lactate residues
may remain as cross-linking of peptidoglycan
strands does not involve all peptide sidechains.
Formation of a vancomycin-precursor complex involves the formation of five hydrogen bonds
between the peptide backbone of the glycopeptide molecule and acyl-o-Ala-o-Ala [16]. Replacement of the amide nitrogen between the two
o-alanine residues by the oxygen of an ester link
would seriously disrupt the binding process. Depsipeptides terminating in D-2-hydroxybutyrate or
o-lactate showed poor affinity for vancomycin,
indicating the difficulty of forming a stable modified precursor-vancomycin complex [4].
It is surprising that some modified glycopeptide compounds that have been acylated or alkylated retain their activity against the vancomycin-resistant enterococci [17]. It is possible that
the activity of these modified compounds results
from a different mechanism of action, perhaps
involving disruption of membrane function, in
view of their increased hydrophobicity compared
with that of the vancomycin molecule.
The direct demonstration that o-lactate i~ present in the C-terminal position of the peptidoglycan precursor of the glycopeptide-resistant but
not the isogenic glycopeptide-sensitive strain of
E. faecium supports the less direct evidence that
addition of D-lactate to the growth medium can
compensate for the insertional inactivation of the
t'anH gene at a concentration five-fold lower than
that of D-2-hydroxybutyrate, which also restores
the resistance phenotype [18].
ACKNOWLEDGEMENTS
This study was funded by a Medical Research
Council Research Studentship to J.M. We thank
L. Packman and Z.J. Jacoby for amino acid analysis, P. Skelton for mass spectrometry, J.-M. Fr~re
for the gift of purified enzyme from Actinomadura R 39, R.C. George for the strains of
Enterococcus faecium and Marion Merrell Dow
for generous research support.
REFERENCES
[11 Dutka-Malen, S., Molinas, C, Arthur, M. and Courvalin,
P. (1990) Mol. Gen. Genet. 224, 364-372.
[2] Bugg, T.D.H., Dutka-Malen, S,, Arthur, M,, Courvalin,
P. and Walsh, C.T. (1991) Biochemistry 30, 20|7-2021~
[3] Arthur, M., Molinas, C., Dutka-Malen, S. and Courvalin,
P. (1991) Gene 103, 133-134.
[4] Bugg, T.D,H., Wright, G.D., Dutka-Malen, S., Arthur,
M., Courvalin, P. and Walsh, C.T. (1991) Biochemistry
30, 10408-10415,
[5] Reynolds, P.E. (1961) Biochim. Biophys. Acta 52, 403405.
[6] Reynolds, P.E, (1971) Biochim. Biophys. Acta 237, 239254.
[7] Leclercq, R., Derlot, E., Weber, M., Dural, J. and Courvalin, P. (1989)Antimierob. Agents Chemother. 33, 1915.
[8] Strominger, J.L. (1957) J. Biol. Chem. 224, 509-523.
[9] Fr~re, J.-M., Moreno, R., Ghuysen, J.-M., Perkins, H.R.,
Dieriekx. L. and Delcambe, L. (1974) Bioehem. J. 143,
233-240~
[10l Johnson~ K., Duez, C., Fr~:re, J.-M. and Ghuysen, J.-M.
(1975) Methods Enzymol. 43, 687-698.
[Ill Nguyen-Disteche, M., Leyh-Bouille, M., Pirlot, S., Fr~re,
J.-M. and Ghuysen, J.-M. (I986) Biochem. J. 235, 167176.
200
[t2] Adam, M., Damblon, C., Plaitin, B., Christiaens, L. and
Fr~re, J.-M. (1990) Biochem. J. 270, 525-529.
[13] Gutmann, L., Billot-Klein, D., AI-Obeid, S., Klare, 1.,
Francoual, S., Collatz, E. and van Heijenoort, J. (1992)
Antimicrob. Agents Chemother. 36, 77-80.
[14] Rasmussen, J.R. and Strominger, J.L. (1978) Proc. Natl.
Aead. Sei. USA 75, 84-88.
[15] Faraci, W,S, and Pratt, R,F, (1986) Biochem. J. 238,
309-312.
[16] Barna, J.C.J. and Williams, D,H. (1984) Ann, Rev. Microbiol. 38, 339-357.
[17] Nicas, T.I., Cole, C.T., Preston, D.A., Schabel, A.A. and
Nagarajan, R. (1989) Antimicrob. Agents Chemother. 33,
1477-1481.
[18] Arthur, M., Molinas, C., Bugg, T,D,H., Wright, G,D,,
Watsh, C.T. and Courvalin, P. (1992) Antimicrob. Agents
Chemother. 36, 867-869.