FEMS Microbiology Letters 98 (1992) 109-116 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
109
FEMSLE 05119
Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium N.E. Allen, J.N. Hobbs Jr., J.M. Richardson and R.M. Riggin Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA Received 5 August 1992 Accepted 6 August 1992
Key words: Vancomycin resistance; Peptidoglycan precursor; UDP-MurNAc-pentapeptide; UDP-MurNAc-tetrapeptide; UDP-MurNAc-depsipentapeptide; Enterococcus faecium
1. SUMMARY In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (r,anA phenotype) accumulate UDP-N-acetylmuramyl(UDP-MurNAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-I)-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.
2. INTRODUCTION
Enterococcus faecium strains of the vanA phenotype are inducibly resistant to high levels of vancomycin and teicoplanin [1]. Recent reports [2,3] indicate that two of the genes required for
Correspondence to: N.E. Allen, Infectious Disease Research, Eli Lilly and Company, Indianapolis, IN 46285, USA
expression of resistance, ~'anA and vanH, encode enzyme activities which may be involved in the biosynthesis of a modified form of the nucleotidelinked precursor, UDP-N-acetylmuramyl-Lalanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine (U D P-MurNAc-L-ala-D-glu-L-lys-D-ala-D-ala) which functions in the polymerization cycle of peptidoglycan biosynthesis. The VANA protein has ligase activity similar to bacterial D-ala-D-ala ligases but has a preference for condensing D-ala with a I)-2-hydroxycarboxylic acid in the C-terminal position via an ester bond [2,3]. VANH has D-2-hydroxycarboxylic acid dehydrogenase activity with specificity for reducing D-2-ketobutyric acid and i)-pyruvic acid to the corresponding hydroxy acids [3]. The activity and specificities of these gene products suggest that glycopeptide-resistant enterococci synthesize a modified depsipentapeptide-containing precursor to which vancomycin does not bind. We report here on the isolation and identification of nucleotide-linked precursors synthesized by vancomycin-resistant enterococci which confer resistance to vancomycin in an in vitro assay for peptidoglycan polymerization.
110
3. M A T E R I A L S AND M E T H O D S
3.1. Bacteria and growth conditions The following strains were used: E. faecium 180 (inducible vancomycin resistance [4,5]); E. faecium 180-1 (vancomycin-susceptible, cured derivative of E. faecium 180; [4]); Staphylococcus aureus FDA 209P; Bacillus cereus X254. Bacteria were routinely grown in brain heart infusion broth at 35°C. The medium was supplemented with 20 # g / m l vancomycin to induce expression of glycopeptide resistance. 3.2. Isolation of UDP-MurNAc-peptides and -depsipeptide Bacitracin was added to mid-log phase cultures of E. faecium to give a final concentration of 100 p~g/ml and incubation was continued for another 60 min. Following harvesting and washing of cells in 0.9% saline, UDP-MurNAcpeptides were isolated and purified according to the methods of Gorecki et al. [6] using anion exchange (Bio-Rad A G 1-2x) chromatography. Samples were desalted on Sephadex G25 prior to amino acid analysis. UDP-MurNAc-pentapeptide was isolated and purified from S. aureus 209P using the same methodology except cells were grown in L broth and vancomycin (20 /zg/ml) was used to induce accumulation of the precursor. Isolation of the UDP-MurNAc-pentapeptide from Bacillus cereus X254 was reported previously [7]. 3.3. Amino acid analyses Amino acid composition of UDP-MurNAcpeptides and -depsipeptides was determined using a model 6300 Beckman amino acid analyser. 3.4. Analytical HPLC Samples were analysed by HPLC using a /xbondapak C18 column (2 x 300 mm). Samples were subjected to isocratic elution at room temperature as described [8] using 0.01 M ammonium acetate, pH 5 substituted for 0.05 M ammonium phosphate.
3.5. EIectrospray ionization mass-spectrometry Electrospray ionization mass spectrometry was conducted using a Sciex API III mass spectrometer equipped with a pneumatically assisted electrospray (Ionspray) interface, which has been described previously [9]. Spectra were obtained by continuously infusing the sample into the interface at a rate of 2-5 ~ l / m i n u t e using a Harvard infusion pump. Negative ion mass spectra were obtained using aqueous solutions adjusted to pH 8-9 with ammonium hydroxide. Positive ion spectra were obtained using 5 0 / 5 0 acetonitrile/water solutions acidified with glacial acetic acid (5% final concentration). Chromatographic peaks collected from the analytical HPLC system were lyophilized on a Speedvac vacuum centrifuge and redissolved in 0.1% ammonium hydroxide solution just prior to negative ion mass spectral analysis. M S / M S spectra were obtained using a collision gas thickness of approximately 5 × 1014 a t o m s / c m 2. Most of the data were obtained using an inlet orifice potential of - 2 0 V (for negative ion mode) or + 20 V (for positive ion mode) relative to the R0 potential. In order to enhance fragmentation, and allow collision-induced dissociation (CID) of major fragment ions, some of the M S / M S data were obtained using an inlet orifice potential of +70 V relative to the R0 potential. Unless otherwise indicated, mass spectra were obtained using a step size of 0.1 ainu (0.2 amu for M S / M S experiments), a scan range of 350 to 1400 ainu, and a scan time of 5 s/scan. Multiple scans (10-40 total) were acquired per sample to provide an averaged final mass spectrum.
3.6. Peptidoglycan polymerization assay Bacteria were permeabilized to nucleotide-linked precursors by ether treatment according to the procedure of Mirelman et al. [10]. Polymerization assays contained (100 p~l): 50 mM Tris • HCI (pH 8.3), 50 mM NH4C1, 20 mM MgSO 4 • 7 H 2 0 , 10 mM ATP, 0.5 mM /3-mercaptoethanol, 0.15 m M D-aspartic acid, 0.05 m M UDP-Nacetyl[14C]glucosamine, 0.15 mM UDP-MurNacpeptide or -depsipeptide, 10 /~g tetracycline and 50 /~g ETB (ether-treated bacteria) protein. Reactions were incubated for 120 min at 27°C and
111 trichloroacetic acid-insoluble m e a s u r e d as d e s c r i b e d [7].
radioactivity was
0
4. R E S U L T S
4.1. Identification and characterization of UDPMurNAc-peptides and -depsipeptides
0
T-
T--
E
E
0
0
0
X
a.
By p a p e r c h r o m a t o g r a p h y , p r e c u r s o r s isolated from v a n c o m y c i n - s u s c e p t i b l e a n d -resistant E. faecium w e r e i n d i s t i n g u i s h a b l e f r o m e a c h o t h e r and from the p r e c u r s o r synthesized by S. aureus (Fig. 1). T h e d i a m i n o p i m e l i c a c i d - c o n t a i n i n g U D P - M u r N A c - p e n t a p e p t i d e from Bacillus cereus was easily d i s t i n g u i s h e d in this system. E x c e p t for U D P , the c o m p o u n d s d e p i c t e d in Fig. 1 w e r e also ninhydrin-positive. C o m p a r i s o n of t h e s a m e p r e p a r a t i o n s by analytical H P L C (Fig. 2) r e v e a l e d that the p r e c u r s o r from E. faecium 180-1 (cured; Fig. 2a) c o n t a i n e d a m a j o r p e a k (I) which was i n d i s t i n g u i s h a b l e f r o m the p r e c u r s o r p r o d u c e d by S. aureus (Fig. 2c). In contrast, the p r e c u r s o r p r e p a r a t i o n f r o m E. faecium 180 i n d u c e d for v a n c o m y c i n r e s i s t a n c e s h o w e d the p r e s e n c e of two p e a k s (II a n d III; Fig. 2b) n e i t h e r of which c o r r e s p o n d e d to the m a j o r p e a k in Fig. 2a. T h e a m i n o acid c o m p o s i t i o n s of the p r e p a r a tions d e p i c t e d in Fig. 2 a r e given in T a b l e 1. T h e c o m p o s i t i o n of p r e c u r s o r s i s o l a t e d from S. aureus 209P a n d E. faecium 180-1 ( c u r e d ) w e r e identical. B o t h p r e c u r s o r s c o n t a i n e d ala : glu : lys in a 3 : 1 : 1 ratio. T h e p e p t i d o g l y c a n o f E. faecium is classiA
0
i i*
, ¢
Fig. 1. Comparison of UDP-MurNAc-peptides by paper chromatography. Precursors were isolated as described in MATERIALS AND METHODS.Samples were spotted on Whatman 3MM paper and subjected to descending chromatography in isobutyric acid-1 M NH4OH (5:3, v/v). Lanes A-D contained precursors isolated from strains as indicated in the figure. Lane A, 22 nmol; lane B, 62 nmol; lane C, 21 nmol; lane D, 20 nmol. Concentrations were determined by quantitative amino acid analysis. Lane E contained 50 nmol of UDP. Spots were visualized by UV light, marked and traced for this illustration. fied as an A 4 type a c c o r d i n g to the s c h e m e o f Schleifer a n d K a n d l e r [11]. T h e a m i n o acid comp o s i t i o n of t h e p r e c u r s o r from E. faecium n e e d e d
B
C
c
0
!
or .
£ III
,,Q
0
I
I
I
I
I
I
10
20
30
10
20
30
10
I
I
20
30
Minutes
Fig. 2. Comparison of UDP-MurNAc-peptides and -depsipeptides by HPLC. Samples (1 nmol in 20 tzl) were analysed as described in MATERIALSAND METHODS.Precursors prepared from: (a) E. faecium 180-1 (cured); (b) E. faecium 1-80 (induced); (e) S. aureus 209P.
112
to synthesize an A4 type peptidoglycan is L-ala-oglu-L-lys-D-ala-D-ala in a ratio of 3 a l a : l g l u : l lys. The composition of the precursor from S. aureus is the same [11]. The amino acid composition of the precursor preparation from E. faecium 180 induced for vancomycin resistance revealed only 2 mol of alanine for each lysine (i.e., 2 a l a : l glu: 1 lys). This is consistent with either a tetrapeptide structure, a depsipentapeptide containing a nonamino-bearing moiety, or a combination of the two. Lesser amounts of glycine and aspartic acid (or asparagine) were found in each preparation (see Table 1). Muramic acid was detected in each sample, but molar ratios were not calculated due to its sensitivity to acid hydrolysis. Alkaline hydro[ysis followed by thin-layer silica gel chromatography (in water-saturated diethyl ether-formic acid [7: 1]; [12]) of the precursor preparation from E. faecium 180 (induced) revealed the presence of an ammonium molybdatestaining material having the same mobility as lactic acid. The material was readily separated from 2-hydroxybutyrate and was not detected in the extract prepared from E. faecium 180-1 (cured). Chromatographically purified precursors were ana[ysed using negative ion electrospray mass spectrometry, with the results shown in Fig. 3. Spectra for the precursor from E. faecium 180-1 (cured; peak I, Fig. 2a) indicated a compound with a molecular mass of 1149.2 (calculated from the M - H - ion at 1148.2 m / z and the M-2H 2
573.6
100
Im - 2 H 2 - )
A
75 50 25
I
0
1148.2 . . . . . . .
•. . . . . ".
........
v
B
--
50
"
25
1077.3 ,. (m- H-)
~lluJLlJt]..,H m
C ,r/a~...~.,~._~
. . . . . . . . . . ~............... :...................... .. . . . . . . . .
L....-..-~.. ,,..:........
574.1
100
(m - 2 H 2- )
75
C
50 25
1149.1
0~
~
500
600
........ ~.......................................,.......................... ~"!~:.H)
Z00
800
900 mlz
1000
1100 1200
Fig. 3. Negative ion ¢]¢ctrospray mass spectra for isolated precursors: (a) E. faecium 180-1 (cured; peak I, Fig. 2a); (b) E. faecium 180 (induced; peak II, Fig. 2b); and (c) E. faecium 180 (induced; peak IIL Fig. 2B).
ion at 573.6 m,/z shown in Fig. 3A). This result corresponds closely with the expected molecular mass (1149.3 Da) for UDP-MurNAc-ala-glu-lys-
Amino acid composition of UDP-MurNAc-peptides from S. aureus and E. faecium S. aureus 209P
-
(m - 2 H 2 - )
"->'751,~
Table l
Compound
-
o~ 100 538.2
E. f a e c i u m 180-1 (cured) a
E. faecium 180 (induced)
nmol
Molar ratio
nmol
Molar ratio
nmol
Molar ratio
Alanine Glutamic acid Lysine
306 105 103
3.0 0.98 1.0
177 81 60
3.0 1.4 1.0
239 142 123
1.9 1.2 1.0
Aspartic acid Glycine Muramic acid
1.9 6.4 103
0.02 0.06 nc b
20 21 31
18 20 54
0.15 0.16 nc b
" 7.7 nmol serine and 12 nmol histidine were also detected in this sample. b Not calculated.
0.33 0.35 nc b
113
ala-ala. Spectra of the two chromatographic peaks from E. faecium 180 (induced; peaks II and III, Fig. 2b) indicated the presence of two compounds. Peak II had a molecular mass of 1078.4 (Fig. 3B), corresponding to a tetrapeptide (UDPMurNAc-ala-glu-lys-ala; expected molecular mass of 1078.3) and Peak III had a molecular mass of 1150.2 (Fig. 3C), corresponding to a lactate-containing depsipentapeptide (UDP-MurNAc-alaglu-lys-ala-lactate; expected molecular mass of 1150.3). Both positive and negative ion mass spectra of the samples before fractionation were consistent with the results obtained for the chromatographically purified peaks. Additional evidence supporting the proposed structures for the two components from E. faecium 180 (induced) was obtained using tandem mass spectrometry (MS/MS). MS/MS fragmentation of Peak II (the putative UDP-MurNAcala-glu-lys-ala peptide) demonstrated the presence of fragments corresponding to -MurNAcala-glu-lys-ala (m/z 675), -O-lactyl-ala-glu-lys-ala (m/z 490), and -lys-ala (m/z 218). Peak III provided fragments at m / z 747, 562, and 290, corresponding to the same fragmentation points as in Peak II, except for the addition of a lactic acid residue to each fragment. Free lysine was observed from both Peaks II and III.
4.3. Vancomycin sensitir,ity of in L,itro peptidoglycan polymerization employing the isolated precursors Peptidoglycan polymerization was measured in vitro in ether-treated bacteria (ETB) using UDPN-acetyl-[14C]glucosamine plus the precursor preparations from E. faecium 180-1 (cured) or E. faecium 180 (resistant and induced) depicted in Figs. 2a and b, respectively. Both preparations supported peptidoglycan polymerization (Table 2), and each was functional in homologous (precursor and ETB prepared from same cells) as well as heterologous (precursor and ETB prepared from different cells) assays. Polymerization was noticeably reduced when the precursor preparation from E. faecium 180 (induced) was used. Peptidoglycan polymerization by ETB prepared from either E. faecium 180-1 (cured) or E. faecium 180 (induced) was inhibited more than 90% by vancomycin (300 /xg/ml) when the precursor from E. faecium 180-1 (cured) was used (Table 2). In contrast, polymerization was resistant (< 25% inhibition) to vancomycin when the precursor preparation from E. faecium 180 (induced) was used. In a separate experiment (data not shown) 100 /xg/ml of vancomycin inhibited polymerization in ETB prepared from E. faecium
Table 2 Effects of vancomycin on in vitro peptidoglycan polymerization Vancomycin
Peptidoglycan polymerization c
ETB b
Precursor source
(~zg/ml)
dpm
nmol/mgP
% Inhibition
Cured
Cured
0 300 0 30O 0 300 0 3OO
40239 1487 8536 6447 10284 653 5416 4436
18.1 0.67 3.8 2.9 4.6 0.29 2.4 2.0
96 25 94 18
Bacteria ~
Resistant Resistant
Cured Resistant
a Cured: E. faecium 180-1; resistant: E. faecium 180 (induced). b Ether-treated bacteria. c See M A T E R I A L S AND METHODS,
114
180 (induced) and E. faecium 180 (non-induced) using the precursor from E. faecium 180-1 (cured), but polymerization was completely resistant to vancomycin when the precursor preparation from E. faecium 180 (induced) was used. It should be noted that resistance to vancomycin was conferred by the precursors isolated from E. faecium 180 (induced) regardless of the source of ETB (see Table 2).
5. DISCUSSION Vancomycin inhibits peptidoglycan biosynthesis in susceptible bacteria by binding to D-ala-D-ala residues of peptidoglycan precursors [13]. The lethal effect of vancomycin likely results from binding to the D-ala-o-ala terminus of the undecaprenyl-pyrophosphoryl-disaccharide-pentapeptide intermediate in the cytoplasmic membrane preventing polymerization via the transglycosylation reaction. The process blocks regeneration of the undecaprenyl lipid carrier shutting down the polymerization cycle and causing an accumulation of the UDP-MurNAc-pentapeptide precursor. Exposure of growing bacteria to vancomcyin has provided a convenient way to block the polymerization cycle and generate increased quantities of the UDP-MurNAc-pentapeptide precursor [14]. Isolation of precursors from vancomycin-resistant E. faecium in this study required the use of bacitracin (a non-glycopeptide inhibitor of peptidoglycan polymerization [15]). Genetic [16,17] and biochemical [3,4] studies of vanA and uanH and their gene products in E. faecium have suggested that vancomycin-resistant enterococci may synthesize a modified pentapeptide, containing a depsidipeptide terminus (D-alaX) in place of D-ala-D-ala. A study by Rasmussen and Strominger [18] predicted that a modification of the peptidoglycan biosynthetic process of this sort could be tolerated without compromising the overall structure of peptidoglycan. We have shown in this study that bacitracin effected the accumulation of modified precursors in E. faecium induced for resistance to vancomycin. A combination of paper and thin-layer chromatography, amino acid analyses and electrospray ionization
mass-spectrometry indicate the presence of both depsipentapeptide and tetrapeptide precursors. The presence of the latter is consistent with the recent demonstration of a unique DD-carboxypeptidase in vanA strains [19]. Although our methodology does not allow assignment of chirality, our results coupled with previous biochemical studies [2,3] indicate that the structure of the depsipentapeptide is L-ala-D-glu-L-lys-D-ala-D-lac. The precusor isolated from vancomycin-susceptible cells and the mixture of precursors isolated from cells induced for vancomycin resistance were functional in an in vitro peptidoglycan polymerization assay. Polymerization was significantly inhibited by vancomycin only when the precursor from susceptible cells was used. Polymerization in the presence of the tetrapeptide and depsipentapeptide precursors was resistant to vancomycin. Our observation that the modified precursors confer resistance to vancomycin in ETB derived from vancomycin-sensitive strains (E. faecium 180-1 ( c u r e d ) a n d E. faecium 180 (non-induced)) supports the notion that precursor modification is necessary and sufficient to confer vancomycin-resistant peptidoglycan formation in vanA strains.
ADDENDUM Following the completion of this work, we learned of two other studies (Messer, J. and Reynolds, P.E. (1992) FEMS Microbiol. Lett. 94, 195-200; Handwerger, S., et al. (1992) J. Bacteriol., in press) reporting the isolation of a lactate-containing depsipentapeptide precursor from glycopeptide-resistant Enterococcus.
ACKNOWLEDGEMENT We thank Robert M. Ellis for the amino acid analyses.
REFERENCES [1] Courvalin, P. (1990) Antimicrob. Agents Chemother. 34, 2291-2296.
115 [2] Bugg, T.D.H., Dutka-Malen, S., Arthur, M., Courvalin, P. and Walsh, C.T. (1991) Biochemistry 30, 2017-2021. [3] Bugg, T.D.H., Wright, G.D., Dutka-Malen, S., Arthur, M., Courvalin, P. and Walsh, C.T. (1991) Biochemistry 30, 10408-10415 [4] Nicas, T.I., Wu, C.Y.E., Hobbs, J.N. Jr., Preston, D.A. and Allen, N.E. (1989) Antimicrob. Agents Chemother. 33, 1121-1124. [5] Uttley, A.H.C., Collins, C.H., Naidoo, J. and GeOrge, R.C. (1988) Lancet i, 57-58. [6] Gorecki, M., Bar-Eli, A., Burstein, Y., Patchornik, A. and Chain, E.B. (1975) Biochem. J. 147, 131-137. [7] Allen, N.E., Hobbs, J.N. Jr. and Alborn, W.E. Jr. (1987) Antimicrob. Agents Chemother. 31, 1093-1099. [8] Flouret, B., Mengin-Lecreulx, D. and van Heijenoort, J. (1981) Anal. Biochem. 114, 59 63. [9] Bruins, A.P., Covey, T.R. and Henion, J.D. (1987) Anal. Chem. 59, 2642-2646. [10] Mirelman, D., Yashouv-Gan, Y. and Schwarz, U. (1976) Biochemistry 15, 1781-1790. [11] Schleifer, K.H. and Kandler, O. (1972) Bacteriol. Rev. 36, 407-477.
[12] Zweig, G. and Sherma, J. (1972) In: Handbook of Chromatography, Vol. I, p. 475, CRC Press, Cleveland, OH. [13] Reynolds, P.E. (1989) Eur. J. Clin. Microbiol. Infect. Dis. 8, 943-950. [14] Kohlrausch, U. and Holtje, J.-V. (1991) FEMS Microbiol. Lett. 78, 253-258. [15] Gale, E.F., Cundliffe, E., Reynolds. P.E., Richmond, M.H. and Waring, M.J. (1981) In: The Molecular Basis of Antibiotic Action, 2nd edn., pp. 137-143. John Wiley, New York, NY. [16] Dutka-Malen, S., Molinas, C., Arthur, M. and Courvalin, P. (1990) Mol. Gen. Genet. 224, 364-372. [17] Arthur, M., Molinas, C., Dutka-Malen, S. and Courvalin, P. (1991) Gene 103, 133-134. [18] Rasmussen, J.R. and Strominger, J.L. (1978) Proc. Natl. Acad. Sci. USA 75, 84-88. [19] Wright, G.D., Molinas, C., Arthur, M., Corvalin, P. and Walsh, C.T. (1992) Antimicrob. Agents Chemother. 36, 1514 1518.
109
FEMSLE 05119
Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium N.E. Allen, J.N. Hobbs Jr., J.M. Richardson and R.M. Riggin Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA Received 5 August 1992 Accepted 6 August 1992
Key words: Vancomycin resistance; Peptidoglycan precursor; UDP-MurNAc-pentapeptide; UDP-MurNAc-tetrapeptide; UDP-MurNAc-depsipentapeptide; Enterococcus faecium
1. SUMMARY In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (r,anA phenotype) accumulate UDP-N-acetylmuramyl(UDP-MurNAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-I)-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.
2. INTRODUCTION
Enterococcus faecium strains of the vanA phenotype are inducibly resistant to high levels of vancomycin and teicoplanin [1]. Recent reports [2,3] indicate that two of the genes required for
Correspondence to: N.E. Allen, Infectious Disease Research, Eli Lilly and Company, Indianapolis, IN 46285, USA
expression of resistance, ~'anA and vanH, encode enzyme activities which may be involved in the biosynthesis of a modified form of the nucleotidelinked precursor, UDP-N-acetylmuramyl-Lalanyl-D-glutamyl-L-lysyl-D-alanyl-D-alanine (U D P-MurNAc-L-ala-D-glu-L-lys-D-ala-D-ala) which functions in the polymerization cycle of peptidoglycan biosynthesis. The VANA protein has ligase activity similar to bacterial D-ala-D-ala ligases but has a preference for condensing D-ala with a I)-2-hydroxycarboxylic acid in the C-terminal position via an ester bond [2,3]. VANH has D-2-hydroxycarboxylic acid dehydrogenase activity with specificity for reducing D-2-ketobutyric acid and i)-pyruvic acid to the corresponding hydroxy acids [3]. The activity and specificities of these gene products suggest that glycopeptide-resistant enterococci synthesize a modified depsipentapeptide-containing precursor to which vancomycin does not bind. We report here on the isolation and identification of nucleotide-linked precursors synthesized by vancomycin-resistant enterococci which confer resistance to vancomycin in an in vitro assay for peptidoglycan polymerization.
110
3. M A T E R I A L S AND M E T H O D S
3.1. Bacteria and growth conditions The following strains were used: E. faecium 180 (inducible vancomycin resistance [4,5]); E. faecium 180-1 (vancomycin-susceptible, cured derivative of E. faecium 180; [4]); Staphylococcus aureus FDA 209P; Bacillus cereus X254. Bacteria were routinely grown in brain heart infusion broth at 35°C. The medium was supplemented with 20 # g / m l vancomycin to induce expression of glycopeptide resistance. 3.2. Isolation of UDP-MurNAc-peptides and -depsipeptide Bacitracin was added to mid-log phase cultures of E. faecium to give a final concentration of 100 p~g/ml and incubation was continued for another 60 min. Following harvesting and washing of cells in 0.9% saline, UDP-MurNAcpeptides were isolated and purified according to the methods of Gorecki et al. [6] using anion exchange (Bio-Rad A G 1-2x) chromatography. Samples were desalted on Sephadex G25 prior to amino acid analysis. UDP-MurNAc-pentapeptide was isolated and purified from S. aureus 209P using the same methodology except cells were grown in L broth and vancomycin (20 /zg/ml) was used to induce accumulation of the precursor. Isolation of the UDP-MurNAc-pentapeptide from Bacillus cereus X254 was reported previously [7]. 3.3. Amino acid analyses Amino acid composition of UDP-MurNAcpeptides and -depsipeptides was determined using a model 6300 Beckman amino acid analyser. 3.4. Analytical HPLC Samples were analysed by HPLC using a /xbondapak C18 column (2 x 300 mm). Samples were subjected to isocratic elution at room temperature as described [8] using 0.01 M ammonium acetate, pH 5 substituted for 0.05 M ammonium phosphate.
3.5. EIectrospray ionization mass-spectrometry Electrospray ionization mass spectrometry was conducted using a Sciex API III mass spectrometer equipped with a pneumatically assisted electrospray (Ionspray) interface, which has been described previously [9]. Spectra were obtained by continuously infusing the sample into the interface at a rate of 2-5 ~ l / m i n u t e using a Harvard infusion pump. Negative ion mass spectra were obtained using aqueous solutions adjusted to pH 8-9 with ammonium hydroxide. Positive ion spectra were obtained using 5 0 / 5 0 acetonitrile/water solutions acidified with glacial acetic acid (5% final concentration). Chromatographic peaks collected from the analytical HPLC system were lyophilized on a Speedvac vacuum centrifuge and redissolved in 0.1% ammonium hydroxide solution just prior to negative ion mass spectral analysis. M S / M S spectra were obtained using a collision gas thickness of approximately 5 × 1014 a t o m s / c m 2. Most of the data were obtained using an inlet orifice potential of - 2 0 V (for negative ion mode) or + 20 V (for positive ion mode) relative to the R0 potential. In order to enhance fragmentation, and allow collision-induced dissociation (CID) of major fragment ions, some of the M S / M S data were obtained using an inlet orifice potential of +70 V relative to the R0 potential. Unless otherwise indicated, mass spectra were obtained using a step size of 0.1 ainu (0.2 amu for M S / M S experiments), a scan range of 350 to 1400 ainu, and a scan time of 5 s/scan. Multiple scans (10-40 total) were acquired per sample to provide an averaged final mass spectrum.
3.6. Peptidoglycan polymerization assay Bacteria were permeabilized to nucleotide-linked precursors by ether treatment according to the procedure of Mirelman et al. [10]. Polymerization assays contained (100 p~l): 50 mM Tris • HCI (pH 8.3), 50 mM NH4C1, 20 mM MgSO 4 • 7 H 2 0 , 10 mM ATP, 0.5 mM /3-mercaptoethanol, 0.15 m M D-aspartic acid, 0.05 m M UDP-Nacetyl[14C]glucosamine, 0.15 mM UDP-MurNacpeptide or -depsipeptide, 10 /~g tetracycline and 50 /~g ETB (ether-treated bacteria) protein. Reactions were incubated for 120 min at 27°C and
111 trichloroacetic acid-insoluble m e a s u r e d as d e s c r i b e d [7].
radioactivity was
0
4. R E S U L T S
4.1. Identification and characterization of UDPMurNAc-peptides and -depsipeptides
0
T-
T--
E
E
0
0
0
X
a.
By p a p e r c h r o m a t o g r a p h y , p r e c u r s o r s isolated from v a n c o m y c i n - s u s c e p t i b l e a n d -resistant E. faecium w e r e i n d i s t i n g u i s h a b l e f r o m e a c h o t h e r and from the p r e c u r s o r synthesized by S. aureus (Fig. 1). T h e d i a m i n o p i m e l i c a c i d - c o n t a i n i n g U D P - M u r N A c - p e n t a p e p t i d e from Bacillus cereus was easily d i s t i n g u i s h e d in this system. E x c e p t for U D P , the c o m p o u n d s d e p i c t e d in Fig. 1 w e r e also ninhydrin-positive. C o m p a r i s o n of t h e s a m e p r e p a r a t i o n s by analytical H P L C (Fig. 2) r e v e a l e d that the p r e c u r s o r from E. faecium 180-1 (cured; Fig. 2a) c o n t a i n e d a m a j o r p e a k (I) which was i n d i s t i n g u i s h a b l e f r o m the p r e c u r s o r p r o d u c e d by S. aureus (Fig. 2c). In contrast, the p r e c u r s o r p r e p a r a t i o n f r o m E. faecium 180 i n d u c e d for v a n c o m y c i n r e s i s t a n c e s h o w e d the p r e s e n c e of two p e a k s (II a n d III; Fig. 2b) n e i t h e r of which c o r r e s p o n d e d to the m a j o r p e a k in Fig. 2a. T h e a m i n o acid c o m p o s i t i o n s of the p r e p a r a tions d e p i c t e d in Fig. 2 a r e given in T a b l e 1. T h e c o m p o s i t i o n of p r e c u r s o r s i s o l a t e d from S. aureus 209P a n d E. faecium 180-1 ( c u r e d ) w e r e identical. B o t h p r e c u r s o r s c o n t a i n e d ala : glu : lys in a 3 : 1 : 1 ratio. T h e p e p t i d o g l y c a n o f E. faecium is classiA
0
i i*
, ¢
Fig. 1. Comparison of UDP-MurNAc-peptides by paper chromatography. Precursors were isolated as described in MATERIALS AND METHODS.Samples were spotted on Whatman 3MM paper and subjected to descending chromatography in isobutyric acid-1 M NH4OH (5:3, v/v). Lanes A-D contained precursors isolated from strains as indicated in the figure. Lane A, 22 nmol; lane B, 62 nmol; lane C, 21 nmol; lane D, 20 nmol. Concentrations were determined by quantitative amino acid analysis. Lane E contained 50 nmol of UDP. Spots were visualized by UV light, marked and traced for this illustration. fied as an A 4 type a c c o r d i n g to the s c h e m e o f Schleifer a n d K a n d l e r [11]. T h e a m i n o acid comp o s i t i o n of t h e p r e c u r s o r from E. faecium n e e d e d
B
C
c
0
!
or .
£ III
,,Q
0
I
I
I
I
I
I
10
20
30
10
20
30
10
I
I
20
30
Minutes
Fig. 2. Comparison of UDP-MurNAc-peptides and -depsipeptides by HPLC. Samples (1 nmol in 20 tzl) were analysed as described in MATERIALSAND METHODS.Precursors prepared from: (a) E. faecium 180-1 (cured); (b) E. faecium 1-80 (induced); (e) S. aureus 209P.
112
to synthesize an A4 type peptidoglycan is L-ala-oglu-L-lys-D-ala-D-ala in a ratio of 3 a l a : l g l u : l lys. The composition of the precursor from S. aureus is the same [11]. The amino acid composition of the precursor preparation from E. faecium 180 induced for vancomycin resistance revealed only 2 mol of alanine for each lysine (i.e., 2 a l a : l glu: 1 lys). This is consistent with either a tetrapeptide structure, a depsipentapeptide containing a nonamino-bearing moiety, or a combination of the two. Lesser amounts of glycine and aspartic acid (or asparagine) were found in each preparation (see Table 1). Muramic acid was detected in each sample, but molar ratios were not calculated due to its sensitivity to acid hydrolysis. Alkaline hydro[ysis followed by thin-layer silica gel chromatography (in water-saturated diethyl ether-formic acid [7: 1]; [12]) of the precursor preparation from E. faecium 180 (induced) revealed the presence of an ammonium molybdatestaining material having the same mobility as lactic acid. The material was readily separated from 2-hydroxybutyrate and was not detected in the extract prepared from E. faecium 180-1 (cured). Chromatographically purified precursors were ana[ysed using negative ion electrospray mass spectrometry, with the results shown in Fig. 3. Spectra for the precursor from E. faecium 180-1 (cured; peak I, Fig. 2a) indicated a compound with a molecular mass of 1149.2 (calculated from the M - H - ion at 1148.2 m / z and the M-2H 2
573.6
100
Im - 2 H 2 - )
A
75 50 25
I
0
1148.2 . . . . . . .
•. . . . . ".
........
v
B
--
50
"
25
1077.3 ,. (m- H-)
~lluJLlJt]..,H m
C ,r/a~...~.,~._~
. . . . . . . . . . ~............... :...................... .. . . . . . . . .
L....-..-~.. ,,..:........
574.1
100
(m - 2 H 2- )
75
C
50 25
1149.1
0~
~
500
600
........ ~.......................................,.......................... ~"!~:.H)
Z00
800
900 mlz
1000
1100 1200
Fig. 3. Negative ion ¢]¢ctrospray mass spectra for isolated precursors: (a) E. faecium 180-1 (cured; peak I, Fig. 2a); (b) E. faecium 180 (induced; peak II, Fig. 2b); and (c) E. faecium 180 (induced; peak IIL Fig. 2B).
ion at 573.6 m,/z shown in Fig. 3A). This result corresponds closely with the expected molecular mass (1149.3 Da) for UDP-MurNAc-ala-glu-lys-
Amino acid composition of UDP-MurNAc-peptides from S. aureus and E. faecium S. aureus 209P
-
(m - 2 H 2 - )
"->'751,~
Table l
Compound
-
o~ 100 538.2
E. f a e c i u m 180-1 (cured) a
E. faecium 180 (induced)
nmol
Molar ratio
nmol
Molar ratio
nmol
Molar ratio
Alanine Glutamic acid Lysine
306 105 103
3.0 0.98 1.0
177 81 60
3.0 1.4 1.0
239 142 123
1.9 1.2 1.0
Aspartic acid Glycine Muramic acid
1.9 6.4 103
0.02 0.06 nc b
20 21 31
18 20 54
0.15 0.16 nc b
" 7.7 nmol serine and 12 nmol histidine were also detected in this sample. b Not calculated.
0.33 0.35 nc b
113
ala-ala. Spectra of the two chromatographic peaks from E. faecium 180 (induced; peaks II and III, Fig. 2b) indicated the presence of two compounds. Peak II had a molecular mass of 1078.4 (Fig. 3B), corresponding to a tetrapeptide (UDPMurNAc-ala-glu-lys-ala; expected molecular mass of 1078.3) and Peak III had a molecular mass of 1150.2 (Fig. 3C), corresponding to a lactate-containing depsipentapeptide (UDP-MurNAc-alaglu-lys-ala-lactate; expected molecular mass of 1150.3). Both positive and negative ion mass spectra of the samples before fractionation were consistent with the results obtained for the chromatographically purified peaks. Additional evidence supporting the proposed structures for the two components from E. faecium 180 (induced) was obtained using tandem mass spectrometry (MS/MS). MS/MS fragmentation of Peak II (the putative UDP-MurNAcala-glu-lys-ala peptide) demonstrated the presence of fragments corresponding to -MurNAcala-glu-lys-ala (m/z 675), -O-lactyl-ala-glu-lys-ala (m/z 490), and -lys-ala (m/z 218). Peak III provided fragments at m / z 747, 562, and 290, corresponding to the same fragmentation points as in Peak II, except for the addition of a lactic acid residue to each fragment. Free lysine was observed from both Peaks II and III.
4.3. Vancomycin sensitir,ity of in L,itro peptidoglycan polymerization employing the isolated precursors Peptidoglycan polymerization was measured in vitro in ether-treated bacteria (ETB) using UDPN-acetyl-[14C]glucosamine plus the precursor preparations from E. faecium 180-1 (cured) or E. faecium 180 (resistant and induced) depicted in Figs. 2a and b, respectively. Both preparations supported peptidoglycan polymerization (Table 2), and each was functional in homologous (precursor and ETB prepared from same cells) as well as heterologous (precursor and ETB prepared from different cells) assays. Polymerization was noticeably reduced when the precursor preparation from E. faecium 180 (induced) was used. Peptidoglycan polymerization by ETB prepared from either E. faecium 180-1 (cured) or E. faecium 180 (induced) was inhibited more than 90% by vancomycin (300 /xg/ml) when the precursor from E. faecium 180-1 (cured) was used (Table 2). In contrast, polymerization was resistant (< 25% inhibition) to vancomycin when the precursor preparation from E. faecium 180 (induced) was used. In a separate experiment (data not shown) 100 /xg/ml of vancomycin inhibited polymerization in ETB prepared from E. faecium
Table 2 Effects of vancomycin on in vitro peptidoglycan polymerization Vancomycin
Peptidoglycan polymerization c
ETB b
Precursor source
(~zg/ml)
dpm
nmol/mgP
% Inhibition
Cured
Cured
0 300 0 30O 0 300 0 3OO
40239 1487 8536 6447 10284 653 5416 4436
18.1 0.67 3.8 2.9 4.6 0.29 2.4 2.0
96 25 94 18
Bacteria ~
Resistant Resistant
Cured Resistant
a Cured: E. faecium 180-1; resistant: E. faecium 180 (induced). b Ether-treated bacteria. c See M A T E R I A L S AND METHODS,
114
180 (induced) and E. faecium 180 (non-induced) using the precursor from E. faecium 180-1 (cured), but polymerization was completely resistant to vancomycin when the precursor preparation from E. faecium 180 (induced) was used. It should be noted that resistance to vancomycin was conferred by the precursors isolated from E. faecium 180 (induced) regardless of the source of ETB (see Table 2).
5. DISCUSSION Vancomycin inhibits peptidoglycan biosynthesis in susceptible bacteria by binding to D-ala-D-ala residues of peptidoglycan precursors [13]. The lethal effect of vancomycin likely results from binding to the D-ala-o-ala terminus of the undecaprenyl-pyrophosphoryl-disaccharide-pentapeptide intermediate in the cytoplasmic membrane preventing polymerization via the transglycosylation reaction. The process blocks regeneration of the undecaprenyl lipid carrier shutting down the polymerization cycle and causing an accumulation of the UDP-MurNAc-pentapeptide precursor. Exposure of growing bacteria to vancomcyin has provided a convenient way to block the polymerization cycle and generate increased quantities of the UDP-MurNAc-pentapeptide precursor [14]. Isolation of precursors from vancomycin-resistant E. faecium in this study required the use of bacitracin (a non-glycopeptide inhibitor of peptidoglycan polymerization [15]). Genetic [16,17] and biochemical [3,4] studies of vanA and uanH and their gene products in E. faecium have suggested that vancomycin-resistant enterococci may synthesize a modified pentapeptide, containing a depsidipeptide terminus (D-alaX) in place of D-ala-D-ala. A study by Rasmussen and Strominger [18] predicted that a modification of the peptidoglycan biosynthetic process of this sort could be tolerated without compromising the overall structure of peptidoglycan. We have shown in this study that bacitracin effected the accumulation of modified precursors in E. faecium induced for resistance to vancomycin. A combination of paper and thin-layer chromatography, amino acid analyses and electrospray ionization
mass-spectrometry indicate the presence of both depsipentapeptide and tetrapeptide precursors. The presence of the latter is consistent with the recent demonstration of a unique DD-carboxypeptidase in vanA strains [19]. Although our methodology does not allow assignment of chirality, our results coupled with previous biochemical studies [2,3] indicate that the structure of the depsipentapeptide is L-ala-D-glu-L-lys-D-ala-D-lac. The precusor isolated from vancomycin-susceptible cells and the mixture of precursors isolated from cells induced for vancomycin resistance were functional in an in vitro peptidoglycan polymerization assay. Polymerization was significantly inhibited by vancomycin only when the precursor from susceptible cells was used. Polymerization in the presence of the tetrapeptide and depsipentapeptide precursors was resistant to vancomycin. Our observation that the modified precursors confer resistance to vancomycin in ETB derived from vancomycin-sensitive strains (E. faecium 180-1 ( c u r e d ) a n d E. faecium 180 (non-induced)) supports the notion that precursor modification is necessary and sufficient to confer vancomycin-resistant peptidoglycan formation in vanA strains.
ADDENDUM Following the completion of this work, we learned of two other studies (Messer, J. and Reynolds, P.E. (1992) FEMS Microbiol. Lett. 94, 195-200; Handwerger, S., et al. (1992) J. Bacteriol., in press) reporting the isolation of a lactate-containing depsipentapeptide precursor from glycopeptide-resistant Enterococcus.
ACKNOWLEDGEMENT We thank Robert M. Ellis for the amino acid analyses.
REFERENCES [1] Courvalin, P. (1990) Antimicrob. Agents Chemother. 34, 2291-2296.
115 [2] Bugg, T.D.H., Dutka-Malen, S., Arthur, M., Courvalin, P. and Walsh, C.T. (1991) Biochemistry 30, 2017-2021. [3] Bugg, T.D.H., Wright, G.D., Dutka-Malen, S., Arthur, M., Courvalin, P. and Walsh, C.T. (1991) Biochemistry 30, 10408-10415 [4] Nicas, T.I., Wu, C.Y.E., Hobbs, J.N. Jr., Preston, D.A. and Allen, N.E. (1989) Antimicrob. Agents Chemother. 33, 1121-1124. [5] Uttley, A.H.C., Collins, C.H., Naidoo, J. and GeOrge, R.C. (1988) Lancet i, 57-58. [6] Gorecki, M., Bar-Eli, A., Burstein, Y., Patchornik, A. and Chain, E.B. (1975) Biochem. J. 147, 131-137. [7] Allen, N.E., Hobbs, J.N. Jr. and Alborn, W.E. Jr. (1987) Antimicrob. Agents Chemother. 31, 1093-1099. [8] Flouret, B., Mengin-Lecreulx, D. and van Heijenoort, J. (1981) Anal. Biochem. 114, 59 63. [9] Bruins, A.P., Covey, T.R. and Henion, J.D. (1987) Anal. Chem. 59, 2642-2646. [10] Mirelman, D., Yashouv-Gan, Y. and Schwarz, U. (1976) Biochemistry 15, 1781-1790. [11] Schleifer, K.H. and Kandler, O. (1972) Bacteriol. Rev. 36, 407-477.
[12] Zweig, G. and Sherma, J. (1972) In: Handbook of Chromatography, Vol. I, p. 475, CRC Press, Cleveland, OH. [13] Reynolds, P.E. (1989) Eur. J. Clin. Microbiol. Infect. Dis. 8, 943-950. [14] Kohlrausch, U. and Holtje, J.-V. (1991) FEMS Microbiol. Lett. 78, 253-258. [15] Gale, E.F., Cundliffe, E., Reynolds. P.E., Richmond, M.H. and Waring, M.J. (1981) In: The Molecular Basis of Antibiotic Action, 2nd edn., pp. 137-143. John Wiley, New York, NY. [16] Dutka-Malen, S., Molinas, C., Arthur, M. and Courvalin, P. (1990) Mol. Gen. Genet. 224, 364-372. [17] Arthur, M., Molinas, C., Dutka-Malen, S. and Courvalin, P. (1991) Gene 103, 133-134. [18] Rasmussen, J.R. and Strominger, J.L. (1978) Proc. Natl. Acad. Sci. USA 75, 84-88. [19] Wright, G.D., Molinas, C., Arthur, M., Corvalin, P. and Walsh, C.T. (1992) Antimicrob. Agents Chemother. 36, 1514 1518.
