The Carboxyl Terminus of Peptidoglycan Stem Peptides Is a Determinant for Methicillin Resistance in Staphylococcus aureus

A mecA-containing Staphylococcus aureus strain was grown inthe presence of high concentrations of D-serine, D-threonine,and D-phenylalanine. These growth conditions resulted in thereplacement of the carboxyl-terminal (fifth) D-alanine residueof peptidoglycan stem peptides with the D-amino acid presentin the growth medium and a reduced ability to grow in the presenceof methicillin. The most dramatic effect was seen with D-serine.With 32 mM D-serine, strains that had been able to grow in thepresence of 800 µg of methicillin per ml were only ableto grow in the presence of less than 50 µg/ml. The resultsalso suggest that in S. aureus vancomycin resistance mediatedthrough the incorporation of precursors not terminating in D-alanyl-D-alaninewould be mutually exclusive with expression of mecA-mediatedmethicillin resistance.

INTRODUCTION

Top

Introduction

Expression of methicillin resistance in Staphylococcus aureusis mediated by the mecA gene product, penicillin-binding protein(PBP) 2A (PBP 2A) (15). PBP 2A is assumed to act as a peptidoglycan-synthesizingenzyme (8, 12), and because it has much lower affinities forß-lactam antibiotics compared to those of the setof regular staphylococcal peptidoglycan-synthesizing enzymes(4), PBP 2A could allow continued peptidoglycan biosynthesisin the presence of concentrations of ß-lactam antibioticsin the medium that inhibit the other peptidoglycan-synthesizingenzymes. Therefore, mecA-containing S. aureus strains are ableto grow in the presence of high concentrations of ß-lactamantibiotics. Although mecA expression is required, mecA expressionitself is not sufficient for expression of methicillin resistancein certain backgrounds. Several studies have identified otherimportant genes (2, 11). Some of those genes, called fem, havebeen recognized as determinants in the biosynthesis of peptidoglycanprecursors (6, 7, 14, 18, 19, 20).

Environmental conditions that cause changes in peptidoglycanprecursor biosynthesis may also alter expression of methicillinresistance in S. aureus. Growth of a mecA-containing S. aureusstrain in the presence of high concentrations of glycine producedpeptidoglycan in which the carboxyl-terminal D-alanine residueof the stem peptides was replaced with glycine, and this substitutionwas paralleled by heterogeneous expression and a reduction inthe level of methicillin resistance (9). The replacement ofthe carboxyl-terminal residue of peptidoglycan stem peptidesapparently resulted from the synthesis of peptidoglycan precursorsterminating in glycine rather than D-alanine. This situationis analogous to that in the fem mutants, in which altered precursorsalso led to a reduced susceptibility to methicillin (6, 7, 14,18, 19, 20).

In order to determine whether modifications in peptidoglycanprecursors are always accompanied by a change in the level ofmethicillin resistance, a mecA-containing S. aureus strain wasgrown in the presence of high concentrations of a variety ofD-amino acids. Such conditions are known to lead in many bacteriato a peptidoglycan in which the carboxyl-terminal D-alanineof the stem peptides is replaced by the D-amino acid presentin the growth medium (17, 21). Provided that this also occursin S. aureus, these growth conditions would be expected to rendera mecA-containing strain more susceptible to methicillin.

MATERIALS AND METHODS

Top

Materials and Methods

Growth conditions and growth in the presence of methicillin. A total of 10⁷ cells of an overnight culture of the mecA-containingS. aureus strain COL (11) grown in tryptic soy broth (TSB; Difco)was transferred to 2 ml of TSB supplemented with 0.25 M glycine,L-serine, D-serine, D-phenylalanine, and D-threonine (Sigma)and twofold increasing concentrations of methicillin. Growthwas measured after 24 and 48 h of incubation at 37°C. Inanother set of experiments, the concentration of methicillinwas kept constant at 5 or 50 µg/ml and the amount of D-aminoacid was varied.

Peptidoglycan and muropeptide analysis. Cells were grown in TSB with the addition of D-amino acids.Peptidoglycan was isolated from exponentially grown cells, andits muropeptide composition was analyzed by reversed-phasedhigh-performance liquid chromatography (HPLC) as described before(5). The structures of the muropeptides were determined by acombination of amino acid analysis and mass spectrometric (MS)techniques (23).

RESULTS AND DISCUSSION

Top

Results and Discussion

Peptidoglycan composition and muropeptide structures. Growth in the presence of 0.25 M D-serine and D-threonine alteredthe peptidoglycan composition of S. aureus dramatically (Fig.1). The D-alanine-terminating pentapeptide-containing muropeptides(unsubstituted monomeric species and pentaglycine-substitutedmono-, di-, tri-, tetra-, penta-, and hexameric species [peaks1, 5, 11, 15, 16, 17, and 18, respectively]) present in peptidoglycanin the absence of the D-amino acids (Fig. 1A) virtually disappearedand were replaced by peaks with different retention times (Thr1,Thr3, Thr7 to Thr10 [Fig. 1B], and Ser1 to Ser11 [Fig. 1C]).Amino acid analysis showed that when D-threonine was presentin the medium, muropeptides of peptidoglycan contained a threonineresidue, and when cells were grown in the presence of 0.25 MD-serine, at least one serine residue was incorporated intothe peptidoglycan (Table 1). MS-MS analysis revealed the aminoacid sequence and showed threonine and serine to be at the carboxyl-terminusposition of the muropeptide stem peptides (data not shown).

View larger version (16K):
[in this window]
[in a new window]
FIG. 1. HPLC elution profiles of muropeptides isolated from peptidoglycan of S. aureus COL grown in TSB (A), TSB supplemented with 0.25 M D-threonine (B), and TSB supplemented with 0.25 M D-serine (C). For structures of peaks, see Table 1 and reference 5.

View this table:
[in this window]
[in a new window]
TABLE 1. Amino acid compositions, molecular masses and proposed structures of muropeptides present in peptidoglycan of S. aureus strain COL grown in the presence of D-serine or D-threonine

The elution times and mass spectrometric analyses of Thr3, Thr7,Thr8, Thr9, and Thr10 indicated that these peaks are pentaglycine-substitutedthreonine-terminating mono-, di-, tri-, tetra-, and pentamericspecies, respectively (Table 1). Similarly, in the presenceof D-serine, monomeric (Ser1 to Ser4), dimeric (Ser6 to Ser9),and trimeric (Ser10 and Ser11) serine-terminating pentapeptidespecies could be detected (Table 1). The proposed structuresof the peaks were confirmed by MS-MS analysis (data not shown).

Carboxyl-terminal D-threonine and D-serine in peptidoglycanstem peptides have been reported before in other bacteria grownunder similar conditions (3, 21), as has the presence of twoserine residues in stem peptides of peptidoglycan (22).

The relative abundance of the peaks depended on the concentrationof D-amino acids in the medium, as shown in Fig. 2 for D-serine.The presence of D-amino acids other than D-alanine in the peptidoglycanmust be the result of the incorporation of D-alanyl--D-aminoacid-terminating peptidoglycan precursors synthesized in thecytoplasm. Such D-alanyl-D-amino acid-terminating precursorsare likely the result of the addition of the D-alanyl--D-aminoacid rather than D-alanyl--D-alanine to the UDP-linked tripeptideat the cytoplasmic stage of peptidoglycan precursor synthesis(21). Information on the sequence of the S. aureus genome didnot reveal the presence of more than one ligase in this organism(13), and therefore, synthesis of the altered dipeptide is likelyto occur by the D-alanyl--D-alanine ligase. Such proposed broadsubstrate specificities for the D-alanyl--D-alanine ligaseshave been demonstrated for the enzymes of Escherichia coli (24).

View larger version (17K):
[in this window]
[in a new window]
FIG. 2. HPLC elution profiles of muropeptides isolated from peptidoglycan of S. aureus COL grown in TSB supplemented with 0.01 M D-serine (A), TSB supplemented with 0.1 M D-serine (B), and TSB supplemented with 0.25 M D-serine (C). For structures of peaks see Table 1 and reference 5.

Equimolar concentrations of L-serine in the growth medium didnot affect the muropeptide profile of peptidoglycan (data notshown).

D-Amino acids in growth medium and ability to grow in the presence of methicillin. Growth in the presence of D-amino acids also had a profoundeffect on the ability to grow in the presence of methicillin.Addition of 0.25 M D-serine, D-threonine, or D-phenylalanineto the growth medium allowed growth in the presence of methicillinonly at concentrations below 10 µg/ml, whereas growthoccurred in the presence of methicillin at concentrations of800 µg/ml when the medium was not supplemented with D-aminoacids (Table 2). The degree of growth reduction in the presenceof methicillin differed for each D-amino acid. Addition of 0.25M L-serine to the growth medium did not affect the ability togrow in the presence of methicillin (Table 2).

View this table:
[in this window]
[in a new window]
TABLE 2. Growth of S. aureus strain COL in the presence of 0.25 M amino and methicillin

The extent of growth reduction in the presence of methicillindepended on the concentration of the D-amino acid in the medium.Addition of 0.125 M D-threonine allowed cell growth in the presenceof methicillin at concentrations above 50 µg/ml, whereas0.25 M D-threonine allowed growth in the presence of methicillinonly at concentrations below 5 µg/ml (Table 3). D-Serinewas more effective in reducing cell growth in the presence ofmethicillin, and it was effective at even lower concentrationsof the amino acid than those of D-threonine. Addition of 0.125M serine did not allow cell growth in the presence of methicillinat concentrations above 5 µg/ml in the medium, and additionof 0.032 M D-serine halted cell growth in the presence of between5 and 50 µg of methicillin per ml in the growth medium(Table 3). It was observed that growth did occur upon prolongedincubation (Tables 2 and 3), and this may have been caused bya heterogeneous expression of resistance (16). Population analysisprofiles showed that this was indeed the case (data not shown).

View this table:
[in this window]
[in a new window]
TABLE 3. Growth of S. aureus strain COL in the presence of 5 and 50 µg of methicillin per ml and D-serine or D-threonine

The presence of these high concentrations of D-amino acids hadlittle effect on cell growth in the presence of vancomycin (datanot shown).

Cell growth in the presence of methicillin and peptidoglycan composition. This study extends the observation that there is a correlationbetween the presence of a D-amino acid other than D-alanineat the fifth (terminal) position of peptidoglycan stem peptidesand the expression of methicillin resistance in S. aureus (9).The presence of D-amino acids other than D-alanine at the carboxylterminus of these stem peptides must have resulted from theincorporation of D-alanyl-D-amino acid-terminating peptidoglycanprecursors synthesized in the cytoplasm. In the absence of methicillin,synthesis and incorporation of these precursors had little effecton cell growth, indicating that these precursors are suitablesubstrates for the enzymes that usually process D-alanyl-D-alanine-terminatingspecies, i.e., the staphylococcal PBPs. It should be noted,though, that the peptidoglycan of these cells contained a reducedamount of oligopeptides, as evidenced by the disappearance ofthe hump at the end of the chromatogram (Fig. 1B and C). Thereduction in cross-linking itself could make the cells moresusceptible to methicillin (and ß-lactams in general)and thus explain the correlation between the reduced methicillinsusceptibility and the altered peptidoglycan composition. Alternatively,it is possible that D-alanyl--D-serine and D-alanyl--D-threonineare poor substrates for PBP 2A, and biosynthesis of these precursorswould therefore fail to support growth under conditions in whichPBP 2A is the only active PBP, i.e., when methicillin is presentin the growth medium and all the staphylococcal PBPs with theexception of the low-affinity PBP 2A are inactivated (4, 10).

This study presents another example of how alterations in peptidoglycanprecursor biosynthesis in S. aureus affect susceptibility tomethicillin (6, 7, 9, 10, 14, 18, 19). It demonstrated thatsubstitution of the carboxyl-terminal D-alanine residue resultedin decreased methicillin susceptibility. It therefore seemsmost likely that expression of vancomycin resistance throughincorporation of D-alanyl--D-lactate-terminating peptidoglycanprecursors, as seen in enterococci (1), is mutually exclusivewith the expression of methicillin resistance mediated throughPBP 2A.

ACKNOWLEDGMENTS

We thank A. Tomasz and A. Severin for helpful and stimulatingdiscussions.

Amino acid determination was provided by The Rockefeller UniversityProtein Sequence Facility, which is supported in part by NIHshared instrumentation grants and by funds provided by the U.S.Army and Navy for purchase of equipment. MS analysis was performedat the MSU MS facility, which is in part supported by NIH grantRR00480 (to D.G.).

FOOTNOTES

* Corresponding author. Present address: AstraZeneca R&D Boston, 35 Gatehouse Dr., Waltham, MA 02451. Phone: (781) 839-4535. Fax: (781) 839-4500. E-mail: boudewijn.dejonge{at}astrazeneca.com.

Present address: Pfizer Global R&D, Ann Arbor, MI 48105.

Present address: Abbott Laboratories, Abbott Park, IL 60064.

REFERENCES

Top