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High-Level ß-Lactam Resistance and Cell Wall Synthesis Catalyzed by the mecA Homologue of Staphylococcus sciuri Introduced into Staphylococcus aureus

Anatoly Severin,^1, Shang Wei Wu,¹ Keiko Tabei,² and Alexander Tomasz^1*

Laboratory of Microbiology, The Rockefeller University, New York, New York 10021,¹ Wyeth Research, 401 N. Middletown Rd., Pearl River, New York 10965²

Received 5 May 2005/ Accepted 5 July 2005

	ABSTRACT

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A close homologue of mecA, the determinant of broad-spectrum ß-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of DD- and LD-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.

	INTRODUCTION

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The genetic determinant of broad-spectrum ß-lactam antibiotic resistance mecA is an acquired gene in Staphylococcus aureus (8, 10). Efforts to identify the possible extra species source of this important drug resistance determinant have led to the detection of a close gene homologue in Staphylococcus sciuri (15), a staphylococcal species that is taxonomically distant from S. aureus and that is most frequently recovered from the skin of rodents and primitive mammals (3). Similarly to mecA in S. aureus, the mecA homologue carried by S. sciuri encodes a protein with a transpeptidase domain that has the linear structure and conserved residues typical of high-molecular-weight penicillin-binding proteins (PBPs) (7, 15). However, in contrast to S. aureus mecA, which is only present in resistant strains, the mecA homologue of S. sciuri was invariably present in all genetically and epidemiologically unrelated isolates (3), and it may represent one of the native PBP genes involved with cell wall synthesis in S. sciuri, most isolates of which are fully susceptible to all ß-lactam antibiotics (4).

Introduction of the mecA homologue from the antibiotic-susceptible S. sciuri strain K1 into a methicillin-susceptible S. aureus had no effect on ß-lactam resistance. However, if the source of the mecA homologue was a methicillin-resistant S. sciuri, the same genetic cross produced S. aureus transductants with significantly increased methicillin resistance (16). The mecA homologue of such laboratory mutants was shown to carry a single point mutation in the mecA promoter. S. aureus transductants that received this mecA homologue began to produce large amounts of a PBP that reacted with monoclonal antibodies prepared against the S. aureus mecA gene product PBP2A. Curing of the cells of the plasmid carrying the S. sciuri gene homologue resulted in complete loss of antibiotic resistance (16).

Methicillin-resistant S. aureus is known to produce a cell wall of unique muropeptide composition when grown in the presence of ß-lactam antibiotics (5). This cell wall is composed primarily of monomeric, dimeric, and trimeric muropeptides. It was proposed that this abnormal peptidoglycan is the product of PBP2A, the protein encoded by the resistance gene mecA. In S. aureus exposed to ß-lactam antibiotics the four native PBPs become inactivated and their transpeptidase function is taken over by PBP2A, which has very low affinity for most members of this family of antimicrobial agents (5).

The purpose of the present study was to determine the nature of the cell wall produced in S. aureus cells in which growth and cell wall synthesis in antibiotic-containing medium has an absolute dependence on the S. sciuri mecA gene homologue introduced into the cells on a plasmid vector (16). If the S. sciuri gene homologue were indeed an evolutionary relative or precursor of the S. aureus-resistant determinant mecA, then one would expect that the S. sciuri gene homologue introduced into the S. aureus background would produce cell wall characteristic of the host bacteria.

In order to answer this question, we determined the structure of the cell wall of antibiotic-susceptible and antibiotic-resistant S. sciuri and also the structure of the cell wall produced in S. aureus carrying the S. sciuri mecA homologue and growing in methicillin-containing medium.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, media, and growth conditions. The bacterial strains and plasmids used in the present study are described in Table 1. The S. aureus strain RU4 is a methicillin-susceptible Tn551 mutant of the methicillin-resistant strain COL in which the resident mecA was inactivated by the transposon insert (9). Introduction into RU4 of pSTSW8, a plasmid carrying the mecA homologue from the methicillin-resistant S. sciuri strain K1M200, produced transductants with moderately increased methicillin resistance and a heterogeneous phenotype (16). It was possible to select from the highly resistant subpopulation of such heterogeneous cultures bacteria that expressed high and homogeneous methicillin resistance. One of these isolates, SS1, was used in most of the studies described here. Curing SS1 of the mecA carrying plasmid caused a complete loss of antibiotic resistance, and reintroduction of the same plasmid into the cured SS1 strain (named SS*1) resulted in highly and homogeneously resistant transductants. Thus, the high-level methicillin resistance of the S. aureus strain SS1 had an absolute dependence on the presence of the mecA homologue from S. sciuri (S. W. Wu, H. de Lancastre, and A. Tomasz, unpublished data).

(i) Plasmid curing. Strains carrying plasmid constructs on the vector pSPT181C were grown in tryptic soy broth (TSB) at 37°C, and then the strains were restreaked on duplicate tryptic soy agar plates containing 10 µg/ml of chloramphenicol (a marker for the plasmid vector) or 10 µg/ml of erythromycin (a marker for the Tn551 insert in RU4), respectively. Isolates that could grow on the plate with erythromycin but not on the plate with chloramphenicol were selected, and plasmid preparation was performed to test for the absence of plasmid.

TSB (Difco Laboratories, Detroit, MI) was used to grow staphylococcal isolates, and chloramphenicol (10 µg/ml) was added for maintenance of the plasmids in staphylococci. Bacterial growth was monitored by measuring the optical density of the cultures at 600 nm.

(ii) Population analysis profile. A population analysis profile was determined by a previously described method (6, 13).

Preparation and analysis of peptidoglycan. Preparation and purification of the S. sciuri cell wall and peptidoglycan was performed essentially according to procedures established for S. aureus (2, 5, 11). The muropeptides solubilized by the enzymatic hydrolysis with muramidase M1 (Sigma, St. Louis, MO) were reduced by using borohydride and separated by reversed-phase high-performance liquid chromatography (HPLC) on a C₁₈ column (ODS-Hypersil [3 µm, 4.6 by 250 mm]; Thermo Electron, Bellefonte, PA). The column was eluted at a flow rate of 0.5 ml/min with a linear gradient starting immediately after injection of 5% (vol/vol) methanol in 100 mM NaH₂PO₄ (pH 2.5) to 30% methanol in 100 mM NaH₂PO₄ (pH 2.5) for 150 min as described previously (5). The relative abundance of muropeptides was estimated from the percentage of the integrate area of peaks detected by determining the absorbance at 206 nm. The peaks of interest were isolated, desalted by HPLC, and analyzed by mass spectroscopy.

Mass spectroscopic analysis. Samples of muropeptides were isolated by HPLC, lyophilized, and dissolved in H₂O:CH₃CN (50:50 [vol/vol]). A sample was injected at a flow rate of 50 µl/min into a Micromass quadrupole time-of-flight electrospray mass spectrometer operating in the positive ion mode.

Edman degradation. Approximately 200 pmol of muropeptides 7k and 11k were sequenced as recommended by the manufacturer's program in a Hewlett-Packard G-1000A protein sequencer at Sequencing Facilities of The Rockefeller University.

	RESULTS

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Cell wall composition of S. sciuri strain K1 and its methicillin-resistant laboratory mutant derivative S. sciuri K1M200. S. sciuri K1M200 was isolated in the laboratory by step selection against increasing concentrations of methicillin until the methicillin MIC increased to 200 µg/ml (16). The methicillin MIC of the parental strain K1 was 1 µg/ml.

Cultures of strain K1 and K1M200 were grown in TSB from small inocula, harvested in the middle of the exponential phase of growth, and used for the preparation of cell wall. After purification of the cell wall, isolation of peptidoglycan, and hydrolysis by the M1 murein hydrolase (mutanolysin), the family of muropeptides was separated by HPLC.

Figure 1 shows the muropeptide profiles of strains K1 and K1M200 and of these two strains grown in the presence of methicillin at drug concentrations corresponding to a fraction of their respective MICs. Individual muropeptide peaks were labeled with a number and a "k" suffix.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Muropeptide profiles of the peptidoglycans of S. sciuri strain K1 and its methicillin-resistant mutant K1M200. Strains S. sciuri K1 and its methicillin-resistant laboratory mutant derivative K1M200 were each grown in TSB and in TSB containing methicillin at sub-MIC concentrations: 0.5 µg/ml in K1 and 20 µg/ml in K1M200. Cell wall peptidoglycan was prepared and hydrolyzed by the M1 muramidase, and the family of muropeptides was separated by HPLC as described in Materials and Methods.

Molecular mass and amino acid composition of S. sciuri muropeptides. Individual muropeptide peaks separated by HPLC were isolated, desalted, and analyzed by mass spectrometry. Table 2 shows the molecular masses and suggested amino acid compositions for 24 of the major muropeptide species identified in strain K1.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Proposed structures for the main muropeptide components of the S. sciuri cell wall peptidoglycan.

High-level methicillin resistance in S. aureus transductants carrying the S. sciuri mecA homologue. S. aureus strain SS1 depended for its high level and homogeneous methicillin resistance (MIC 400 µg/ml) on the presence of the plasmid-borne mecA derived from the antibiotic-resistant S. sciuri strain K1M200. It was shown earlier that the promoter region of mecA in this resistant mutant carried a single T-to-A point mutation that was responsible for the high rate of transcription of the gene and also for the successful expression of high-level methicillin resistance when introduced into a methicillin-susceptible S. aureus strain with the appropriate genetic background (16; Wu et al., unpublished). When the temperature of cultivation of SS1 was shifted from 30 to 42°C, a temperature nonpermissive for plasmid replication, the methicillin MIC of SS1 (400 µg/ml) dropped to the MIC of the recipient strain (3 µg/ml) accompanied by the loss of the plasmid. The antibiotic-susceptible strain SS*1 cured of the plasmid was able to produce again highly and homogeneously methicillin-resistant transductants upon the reintroduction of the original pSTSW8. Exactly parallel phenomena were observed when SS*1 was used as a recipient for pSTSW2C, a plasmid carrying the mecA of S. aureus (Fig. 3).

View larger version (23K): [in this window] [in a new window]   FIG. 3. Dependence of the methicillin-resistant phenotype on the presence of the mecA in the bacteria. S. aureus mutant RU4 was transduced to high-level methicillin resistance either by the introduction of the S. sciuri mecA on plasmid pSTSW8 to generate transductant SS1 (•) or by the introduction of the S. aureus mecA on plasmid pSTSW2C to generate transductant SS2 (). Loss of the plasmid-born mecA constructs in the cured cells (SS*1 [] and SS*2 []) resulted in loss of resistance.

Cultures were harvested in the middle of the exponential phase of growth, cell walls and peptidoglycan were isolated, and enzymatic hydrolysates of the peptidoglycan muropeptides were analyzed by HPLC. The HPLC profiles of strains SS1, SS2, and COL and the S. sciuri strain K1M200, each grown in the presence of methicillin, are shown in Fig. 4. The HPLC profiles of SS1, SS2, and COL were identical and were quite different from the HPLC profile of strain K1M200. The quantitative representation of various muropeptides in these strains is shown in Table 3.

View larger version (20K): [in this window] [in a new window]   FIG. 4. S. sciuri mecA catalyzes the production of S. aureus-type peptidoglycan in methicillin-resistant transductants of S. aureus. Strains were grown from small inocula in the presence of the following concentrations of methicillin: S. sciuri K1M200 (20 µg/ml), S. aureus strain COL (20 µg/ml), and S. aureus transductants SS1 (5 µg/ml) and SS2 (20 µg/ml). Muropeptide hydrolysates were analyzed by HPLC as described in Materials and Methods.

	DISCUSSION

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The purpose of the investigations described here was to determine the chemical nature of the cell wall produced in such S. aureus transductants in which methicillin resistance is dependent on the expression of the S. sciuri mecA homologue.

In order to do this, it was necessary first to determine the structure of the S. sciuri cell wall in both antibiotic-susceptible and -resistant strains. A comparison of the muropeptide compositions of S. aureus and S. sciuri revealed several striking differences. In S. aureus, the majority of peptide branches and cross-links are composed of five glycine units, while in S. sciuri these are composed of four glycine and one alanine residues out of which the alanine is the one directly attached to the epsilon amino group of the stem peptide lysine. A second major difference is the frequent occurrence in the S. sciuri cell wall of muropeptide monomers carrying tetrapeptide chains such as in muropeptides 2k or 3k and the occurrence of muropeptide oligomers carrying a tetrapeptide (for instance, 8k and 13k) or a tripeptide unit (for instance, muropeptides 4k, 9k, 14k, 17k, 19k, and 23k) on the original acceptor components of muropeptide oligomers. These observations imply the presence of DD-carboxypeptidase and LD-carboxypeptidase activity in S. sciuri, which is in contrast to the lack of these enzyme activties in S. aureus.

Another interesting and contrasting feature of the S. sciuri was the apparent resistance of the hypothetical carboxypeptidases to ß-lactam antibiotics. Growth of strain K1 or the resistant strain K1M200 in methicillin-containing medium did not decrease the proportion of tetra- and tripeptide components in the cell wall (see Fig. 1 and Table 3). In several bacterial species such as Streptococcus pneumoniae the DD-carboxypeptidase is highly sensitive to ß-lactam antibiotics, and therefore cells grown in the presence of these agents cause drastic shifts in muropeptide composition, specifically, the appearance of muropeptides terminating in D-alanyl-D-alanine (12, 14). In fact, it seems that the sole effect of methicillin on wall composition in S. sciuri was the reduction in the proportion of the hump containing highly cross-linked muropeptides (from 41 to 13% in the case of K1 and from 61 to 24% in the case of K1M200) and the parallel increase in the representation of monomers and short oligomers (see Table 3).

Comparison of the muropeptide patterns of strain SS1 and COL in Fig. 4 allows one additional related conclusion. Growth of SS1 in the presence of methicillin depends on the participation of the PBP2A-like protein of S. sciuri in cell wall synthesis of the S. aureus host. One of the major muropeptides produced under these conditions is S. aureus muropeptide 5, a monomeric component that carries intact D-alanyl-D-alanine termini. If the S. sciuri mecA protein is involved with the synthesis of this peptidoglycan, then, clearly, the mecA protein of S. sciuri cannot have DD-carboxypeptidase activity. Thus, the DD-carboxypeptidase and LD-carboxypeptidase enzymes involved with the production of the tetra- and tripeptide components in the S. sciuri cell wall remain to be identified.

With this information in hand we proceeded to determine the structure of cell wall peptidoglycan produced in S. aureus transductant SS1 in which growth of the bacteria and cell wall synthesis in methicillin-containing medium had an absolute dependence on the S. sciuri gene, since removal of the plasmid-born gene caused a complete loss of drug resistance (see Fig. 3).

Analysis of the cell wall peptidoglycan produced in such cells clearly indicated that the cell wall was of the S. aureus and not the S. sciuri type: the HPLC profile of the SS1 cell wall was clearly different from that of the muropeptide profile of S. sciuri K1M200 and was indistinguishable from the profiles obtained in transductant SS2 or in the S. aureus strain COL growing under comparable conditions.

The simplest interpretation of these findings is that the PBP2A-like product of the S. sciuri mecA homologue is capable of using cell wall precursors produced by the host bacterium S. aureus.

These findings are reminiscent of the recent report in which the S. aureus mecA was introduced into Enterococcus faecalis (1). The introduction of mecA caused an increase in ß-lactam resistance, but the cell wall produced in such bacteria grown in antibiotic-containing medium has retained the composition of the bacterial host (1).

It seems that the mecA homologue from S. sciuri residing in an S. aureus cell utilizes cell wall precursors of the host bacterium with their pentaglycine branches for the production of a peptidoglycan, the cross-linking of which is catalyzed by the S. sciuri protein but the composition of which reflects the S. aureus host. The observations described here are consistent with the proposal that the S. sciuri mecA homologue may be an evolutionary precursor of the resistant mecA in S. aureus.

	ACKNOWLEDGMENTS

 
This study was supported in part by grant RO1 AI37275 from the U.S. Public Health Service.

	FOOTNOTES

 
* Corresponding author. Mailing address: Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8278. Fax: (212) 327-8688. E-mail: tomasz{at}rockefeller.edu.

Present address: Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965.

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