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Antimicrobial Agents and Chemotherapy, February 2009, p. 435-441, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01099-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Reconstruction of the Phenotypes of Methicillin-Resistant Staphylococcus aureus by Replacement of the Staphylococcal Cassette Chromosome mec with a Plasmid-Borne Copy of Staphylococcus sciuri pbpD Gene {triangledown}

Aude Antignac and Alexander Tomasz*

Laboratory of Microbiology, The Rockefeller University, New York, New York 10021

Received 15 August 2008/ Returned for modification 29 September 2008/ Accepted 6 November 2008


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ABSTRACT
 
The mecA gene, the central determinant of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), is not native to this bacterial species but may have originated in the animal commensal species Staphylococcus sciuri. All S. sciuri strains carry a close homologue of mecA in the form of pbpD, the genetic determinant of penicillin binding protein 4 (PBP 4) of S. sciuri. Here we describe an experimental system that could be used for additional tests for this proposition. The S. sciuri pbpD gene was cloned into a shuttle plasmid and introduced into methicillin-susceptible S. aureus strain COL-S derived from parental MRSA strain COL from which the resistance cassette staphylococcal cassette chromosome mec was excised. The S. sciuri pbpD determinant was transcribed and translated in the S. aureus transductants producing large amounts of the 84-kDa S. sciuri PBP 4 and was then deposited in the plasma membrane of the host bacterium. Transductants carrying the heterologous S. sciuri pbpD gene exhibited properties typical of those of parental MRSA strain COL, including broad-spectrum, high-level, and homogeneous resistance to structurally different β-lactams. Antibiotic resistance was dependent on the functioning of S. aureus PBP 2 and was suppressed by the specific regulatory genes mecI and mecR and by inhibitors of an early step in cell wall biosynthesis. S. sciuri PBP 4 was also able to replace the essential physiological function(s) of the native PBP 2 of S. aureus and produce peptidoglycan typical of that of parental MRSA strain COL. Our results provide further support for the proposition that the resistance determinant mecA of MRSA strains has evolved from S. sciuri pbpD.


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INTRODUCTION
 
Staphylococcus aureus is a major human pathogen responsible for several life-threatening infections, including septicemia, endocarditis, and toxic shock syndrome. Methicillin (meticillin)-resistant S. aureus (MRSA) strains were first reported in 1961, shortly after the introduction of methicillin in clinical practice. Since then, MRSA had become an increasing critical threat in hospital and community environments worldwide. The genetic determinant of β-lactam resistance, the mecA gene, is carried on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec), and encodes a low-affinity penicillin binding protein (PBP), PBP 2A (8). Several lines of evidence suggest that the heterologous β-lactam resistance gene, mecA, which is resident in all MRSA strains, may have its evolutionary origin in a close homologue of this gene that is ubiquitous in both β-lactam-susceptible and -resistant isolates of the animal commensal species Staphylococcus sciuri (2, 3). The S. sciuri pbpD gene (the S. aureus mecA homologue) is the genetic determinant of PBP 4, one of the six PBPs recently identified in this species (20). Previous studies have shown that an upregulated form of the S. sciuri pbpD gene transduced into a susceptible S. aureus strain was able to produce a moderate but significant (twofold) increase in the oxacillin MIC of the transductants (19, 20). In that experiment, the source of the upregulated pbpD gene was the laboratory mutant K1M200, obtained by stepwise exposure of antibiotic-susceptible S. sciuri strain K1 to gradually increasing concentrations of methicillin. The recipient strain was S. aureus COL, in which the mecA resistance determinant was inactivated by a transposon insert.

The purpose of the studies described here was to construct a new experimental system that would allow one to further test the validity of the proposition that the resistance determinant of MRSA strains may have originated from the S. sciuri pbpD gene. The chromosomal resistance determinant SCCmec was excised from highly and homogeneously methicillin-resistant S. aureus strain COL to provide a methicillin-susceptible S. aureus (MSSA) strain, COL-S. In some of the new experiments, the essential S. aureus pbpB gene was also put under the control of an inducible promoter producing strain, COL-Sspac::pbpB. Both strains were then used as the recipient for plasmid-borne copies of the upregulated S. sciuri pbpD gene recovered from oxacillin-resistant clinical isolate SS37 of S. sciuri (3, 4). Transductants carrying the S. aureus mecA determinant on the same plasmid were used as controls. The physiological, genetic, and biochemical properties of the S. aureus COL-S transductants carrying the heterologous S. sciuri pbpD gene were then compared to the properties of original MRSA strain COL to determine to what extent the drug resistance-related phenotypes of the original MRSA strain were reconstructed in the transductants.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. The characteristics of the bacterial strains and plasmids used in this study are described in Table 1. Bacterial cultures were grown in tryptic soy broth (Difco Laboratories) or on tryptic soy agar (Difco Laboratories) at 30°C with aeration.


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  		TABLE 1. Bacterial strains and plasmidsa

Antibiotic susceptibility testing. The susceptibilities of the S. aureus strains to β-lactam antibiotics were determined by population analysis, as described previously (17).

Peptidoglycan purification and analysis by HPLC. Peptidoglycan was purified from 1-liter cultures of bacteria grown at 30°C to mid-exponential phase, as described previously (5). Purified peptidoglycan was digested with mutanolysin (Sigma-Aldrich); and muropeptides were reduced with sodium borohydride (Sigma-Aldrich), separated by high-performance liquid chromatography (HPLC) on a C18 column (3 µm, 4.6 by 250 mm; ODS-Hypersil; Thermo Electron Corporation), and detected by measurement of the absorbance at 206 nm, as described previously (5).

Membrane purification and PBP assay. Membrane preparations were purified from cultures grown to the late exponential phase, as described previously (15). The detection of PBPs was performed by incubating the membrane preparations (150 µg of proteins) with a saturating concentration (20 µg/ml) of [14C]benzylpenicillin (59 mCi/mmol, 158 µCi/mg; GE Healthcare) at 30°C for 10 min. The reactions were stopped by adding an excess of unlabeled penicillin G. The proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) in an 8% (wt/vol) acrylamide-0.06% (wt/vol) bisacrylamide gel. The labeled PBPs were detected with a tritium storage phosphor screen (GE Healthcare).

Detection of S. aureus PBP 2A and S. sciuri PBP 4 by Western blotting. Membrane preparations (80 µg of proteins) were separated by SDS-PAGE as described above. Transfer and blotting were done according to the instructions accompanying the ECL Western blotting analysis system (GE Healthcare). The ChromPure human immunoglobulin G Fc fragment (Jackson ImmunoResearch Laboratories) was used at 3 µg/ml to eliminate nonspecific hybridization with protein A. The primary antibody was a monoclonal antibody against S. aureus PBP 2A (dilution, 1:20,000) obtained by injecting a rabbit with the synthetic peptide sequence CDKNFKQVYKDSSYISKSDNG conjugated to keyhole limpet hemocyanin (a gift from JoAnn Hoskins, Eli Lilly, Indianapolis, IN), and the secondary antibody was the anti-rabbit antibody included in the kit (dilution, 1:5,000). S. sciuri PBP 4 has previously been shown to react also with the monoclonal antibody raised against S. aureus PBP 2A (4, 19).

RNA isolation and Northern blot analysis. Total RNA was extracted from cultures grown up to an optical density at 620 nm (OD620) of 0.7. RNA (5 µg) was resolved by electrophoresis on 1.2% agarose-0.66 M formaldehyde gels in morpholinepropanesulfonic acid running buffer. RNA was blotted onto Hybond-N+ membranes (GE Healthcare) with a turbo blotter alkaline transfer system (Schleicher & Schuell) with 20x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The PCR-amplified DNA probes were labeled with [{alpha}-32P]dCTP (GE Healthcare) by using a Ready-To-Go labeling kit (GE Healthcare) and hybridized under high-stringency conditions. The blots were subsequently washed and autoradiographed.

Introduction of a plasmid-borne S. aureus COL mecA gene and S. sciuri SS37 pbpD gene into S. aureus strain COL-S and into a pbpB conditional mutant of COL-S. Plasmid pPBP2i (13) was transduced into oxacillin-susceptible S. aureus strain COL-S (11) to produce strain COL-Sspac::pbpB, in which the expression of the chromosomal pbpB gene is under the control of the isopropyl-β-D-1-thiogalactopyranoside (IPTG)-inducible spac promoter. A PCR-amplified sequence of the 3,737-bp region of S. aureus COL mecA was ligated into shuttle plasmid pSPT181C to form pSTSW2C (19). A 4,130-bp fragment that included the promoter region of pbpD gene with a complete copy of IS256 inserted 109 bp upstream of the start codon and the entire pbpD gene was amplified from S. sciuri SS37 by PCR and was cloned into shuttle plasmid pSPT181C to form pSS37MA (20). Recombinant plasmids pSPSW2C and pSS37MA were subsequently introduced into S. aureus strain RN4220 by electroporation and were then transduced by phage 80{alpha} into S. aureus strain COL-S to yield the transductants COL-SSAmecA and COL-SSSpbpD, respectively, and into COL-Sspac::pbpB to yield the transductants COL-Sspac::pbpB/SAmecA and COL-Sspac::pbpB/SSpbpD, respectively.

Cloning of mecI and mecR1 regulatory genes and introduction into S. aureus. The 2,466-bp mecI-mecR1 region from S. aureus strain N315 was amplified with primers mecIP5 (5'-AGAGGGGATCCTCAACGACTTGATTGTTTCC-3') and mecRP9 (5'-GTTCGAATTCTTCTACTTCACCATTATCGC-3') and was ligated into the BamHI and EcoRI sites of high-copy-number plasmid pGC2 (D. C. Oliveira, unpublished data). The resulting recombinant plasmid, pGC2::mecI-mecR1, was electroporated into RN4220 and then transduced into COL, COL-SSAmecA, and COL-SSSpbpD to produce the transductants COLmecI-mecR1, COL-SSAmecA/mecI-mecR1, and COL-SSSpbpD/mecI-mecR1, respectively.


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RESULTS
 
The experimental system: construction of MSSA strain COL-S and introduction of plasmid-borne copies of S. sciuri pbpD and S. aureus mecA genes. The chromosomal SCCmec type I cassette was removed from MRSA strain COL by precise excision, producing strain COL-S, which was fully susceptible to all beta-lactam antibiotics tested (Table 2) (11). A pbpB conditional mutant of strain COL-S (COL-Sspac::pbpB) was constructed by placing the genetic determinant of PBP 2 under the control of the IPTG-inducible spac promoter (13).


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  		TABLE 2. Susceptibility to β-lactam antibiotics

The source of the S. sciuri pbpD gene was clinical isolate S. sciuri SS37, which was recovered from the nasopharynx of a patient (3). Strain SS37 exhibited heterogeneous resistance to oxacillin and carried a pbpD gene that was overexpressed due to the insertion of IS256 upstream of the gene (3, 4). The upregulated pbpD gene was cloned into a shuttle plasmid (20) and introduced into S. aureus COL-S and COL-Sspac::pbpB to produce transductants COL-SSSpbpD and COL-Sspac::pbpB/SSpbpD, respectively. As a control, the same shuttle plasmid carrying the mecA gene from MRSA strain COL (19) was also introduced into the same S. aureus backgrounds, yielding transductants COL-SSAmecA and COL-Sspac::pbpB/SAmecA, respectively.

Transcription and translation of plasmid-borne S. aureus mecA and S. sciuri pbpD in S. aureus. Figure 1 shows the results of Northern blot analysis, performed to estimate the degree of expression of S. aureus mecA and pbpB and S. sciuri pbpD in the different S. aureus backgrounds. No mecA transcript was detected in COL-S or its pbpB conditional mutant, COL-Sspac::pbpB. S. aureus mecA and S. sciuri pbpD were both effectively and highly expressed in transductants COL-Sspac::pbpB/SAmecA and COL-Sspac::pbpB/SSpbpD. The inhibition of pbpB transcription was confirmed in cultures of strains COL-Sspac::pbpB/SAmecA and COL-Sspac::pbpB/SSpbpD grown in the absence of the IPTG inducer.


Figure 1
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  		FIG. 1. Transcription of S. aureus mecA and pbpB genes and S. sciuri pbpD gene. RNA was purified from cultures grown at 30°C to an OD620 of 0.7. RNA (5 µg) was resolved by electrophoresis on agarose-formaldehyde gels. After transfer, the membranes were hybridized with 32P-labeled S. aureus mecA and pbpB and S. sciuri pbpD DNA probes.

The PBP patterns were determined by incubating membrane preparations in the presence of radioactive penicillin (Fig. 2A), and a Western blot analysis was performed with an antibody raised against S. aureus PBP 2A (Fig. 2B), which has previously been shown to also react with S. sciuri PBP 4 (4, 19, 20). In accordance with the results of the Northern blot analysis, these experiments confirmed that PBP 2 was absent from the membrane preparation of COL-Sspac::pbpB/SSpbpD grown in the absence of IPTG and that PBP 2A was absent from COL-S and its derivative, COL-Sspac::pbpB. Large amounts of PBP 2A were detected in the membrane preparations of COL-Sspac::pbpB/SAmecA. The foreign S. sciuri PBP 4 was also correctly translocated and deposited in large amounts in the plasma membrane of S. aureus host cells. As shown in Fig. 2C, S. aureus PBP 2A (78 kDa) and S. sciuri PBP 4 (84 kDa) were already evident from the SDS-polyacrylamide gel stained with Coomassie blue.


Figure 2
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  		FIG. 2. PBP patterns and detection of protein products of S. aureus mecA (PBP 2A) and S. sciuri pbpD (PBP 4). (A) Membrane preparations (150 µg of proteins) were incubated with a single saturating concentration of [14C]benzylpenicillin. After SDS-PAGE, the gel was exposed to a tritium storage phosphor screen for 2 weeks. (B) Membrane preparations (80 µg of proteins) were tested by Western blot analysis for the production of proteins that react with a monoclonal antibody raised against S. aureus PBP 2A. The results for S. aureus COL and S. sciuri SS37 are provided as positive controls and size markers for PBP 2A and PBP 4, respectively. (C) Protein patterns on an SDS-polyacrylamide gel stained with Coomassie blue.

Similarly, high levels of transcription of S. aureus mecA and S. sciuri pbpD and large amounts of PBP 2 and PBP 4 were also observed for transductants COL-SSAmecA and COL-SSSpbpD, respectively (data not shown).

Phenotypic expression of β-lactam resistance. Table 2 shows that the introduction of both the S. sciuri pbpD gene and the S. aureus mecA gene into MSSA strain COL-S was able to provide virtually identical levels of resistance to a group of β-lactam antibiotics which have considerably different degrees of selective binding to the four S. aureus PBPs. Transductants COL-SSSpbpD and COL-SSAmecA produced high-level and homogeneous resistance to ceftizoxime and cefotaxime (selective affinity for PBP 2), cephradine (PBP 3), cefoxitin (PBP 4), and oxacillin.

Inhibition of β-lactam resistance in transductants COL-SSSpbpD and COL-SSAmecA by subinhibitory concentrations of D-cycloserine and by the mecI-mecR1 regulatory genes. It has previously been shown that inhibitors of the early steps of cell wall synthesis can reduce the level of methicillin resistance and change the homogeneous resistance phenotype of strain COL to a heterogeneous one (16). Susceptibility to oxacillin in the presence of subinhibitory concentrations (0.25x MIC) of the cell wall synthesis inhibitor D-cycloserine was determined by population analysis. While the oxacillin MIC of strain COL-S remained unchanged, the resistance levels were drastically reduced in the presence of D-cycloserine, from MICs of 800 to 400 µg/ml for strains COL, COL-SSAmecA, and COL-SSSpbpD to MICs as low as 1, 8, and 0.75 µg/ml, respectively. In addition, the homogeneous expression of oxacillin resistance in COL-SSAmecA and COL-SSSpbpD was converted to heterogeneous expression (data not shown), as previously described for COL (16).

The S. aureus mecA gene has been shown to be transcriptionally regulated in some clinical isolates by mecR1 and mecI, cotranscribed chromosomal genes that encode a signal transducer and a repressor, respectively (9). The mecI-mecR1 region from S. aureus strain N315 was cloned in a high-copy-number plasmid and introduced into COL, COL-SSAmecA, and COL-SSSpbpD. The mecI and mecR1 regulatory genes repressed the phenotypic expression of β-lactam resistance in COL, as described previously (10), and well as in COL-SSAmecA (Table 2). Most interestingly, the level of β-lactam resistance was also extensively repressed in strain COL-SSSpbpD, which expressed the plasmid-borne S. sciuri pbpD gene (Table 2), suggesting that the MecI repressor can bind to the promoter region of S. sciuri pbpD similar to the way in which it binds to the promoter region of S. aureus mecA (14).

Replacement of the normal (essential) physiological function(s) of the S. aureus host PBP 2 by S. sciuri PBP 4. Previous studies have shown that S. aureus PBP 2, the protein product of the pbpB gene, is essential for growth in β-lactam-susceptible S. aureus strains. However, the additional PBP 2A present in MRSA strains can replace the essential function(s) of PBP 2 (7, 13). In order to test if S. sciuri PBP 4 was also able to support growth in PBP 2-deprived S. aureus cells, the plasmid-borne S. sciuri pbpD gene was introduced into S. aureus strain COL-Sspac::pbpB, in which the pbpB gene was put under the control of an IPTG-inducible promoter. As a control, the plasmid-borne S. aureus mecA gene was also introduced in the same background. As previously described for a β-lactam-susceptible S. aureus strain, the absence of IPTG prevented the growth of COL-Sspac::pbpB but only slowed the growth of the pbpB conditional mutant of MRSA strain COL, COLspac::pbpB (Fig. 3A). The introduction of the plasmid-borne S. sciuri pbpD gene into COL-Sspac::pbpB allowed the growth of strain COL-Sspac::pbpB/SSpbpD in the absence of IPTG, and the growth rate was comparable to that of COL-Sspac::pbpB/SAmecA carrying the plasmid-borne S. aureus mecA gene (Fig. 3B) indicating that, similarly to S. aureus PBP 2A, S. sciuri PBP 4 can replace the essential function(s) of the native PBP 2 of S. aureus.


Figure 3
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  		FIG. 3. Effect of suppression of pbpB transcription and introduction of the plasmid-borne S. aureus mecA or S. sciuri pbpD gene on growth in S. aureus. Overnight cultures supplemented with IPTG were centrifuged, washed twice, and suspended in fresh medium to an initial OD620 of 0.01 in the presence of 500 µM IPTG (closed symbols and solid lines) or in the absence of IPTG (open symbols and dashed lines). The turbidities of the cultures were monitored at 30°C for 12 h. (A) COL-Sspac::pbpB ({blacksquare} and {square}) and COLspac::pbpB ({blacktriangleup} and {triangleup}); (B) COL-Sspac::pbpB/SAmecA (• and {circ}) and COL-Sspac::pbpB/SSpbpD ({diamondsuit} and {diamond}).

Effect of suppression of pbpB transcription on peptidoglycan composition of transductants carrying the plasmid-borne S. sciuri pbpD or S. aureus mecA gene. Peptidoglycans were purified from MRSA strain COL and from the two transductants COL-Sspac::pbpB/SAmecA and COL-Sspac::pbpB/SSpbpD carrying the plasmid-borne S. aureus mecA and S. sciuri pbpD genes, respectively, in the background of the pbpB conditional mutant of COL-S grown in the absence of IPTG. Under these conditions of growth (the absence of IPTG), the plasmid-borne S. aureus mecA and S. sciuri pbpD genes provide the normal (essential) function(s) of the native PBP 2 of S. aureus. The muropeptide compositions of the peptidoglycans were analyzed by HPLC (Fig. 4). The suppression of pbpB transcription in cultures of COL-Sspac::pbpB/SAmecA (Fig. 4B) resulted in the production of peptidoglycan with a moderate decrease in the relative proportion of the highly cross-linked muropeptides with retention times of over 100 min and a parallel slight increase in the proportion of the monomeric muropeptide 5 compared to the peptidoglycan composition of COL as shown in Fig. 4A. Similar variations in the peptidoglycan composition were observed in cultures of strain COL in which pbpB transcription was suppressed (7). The HPLC elution profile of COL-Sspac::pbpB/SSpbpD grown in the absence of IPTG (Fig. 4C) clearly differed from that of S. sciuri strain SS37 (Fig. 4D) but was identical to that of COL-Sspac::pbpB/SAmecA. These results indicate that S. sciuri PBP 4 was able to replace the essential enzymatic activity of S. aureus PBP 2 and catalyze the biosynthesis of peptidoglycan to produce an S. aureus type of cell wall with the cell wall precursors of the S. aureus host.


Figure 4
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  		FIG. 4. HPLC elution profiles of purified peptidoglycans. Peptidoglycans were purified from 1-liter cultures grown at 30°C to an OD620 of 0.4, digested with mutanolysin, and separated by HPLC. The peptidoglycan compositions of S. aureus COL (A), transductants COL-Sspac::pbpB/SAmecA (B) and COL-Sspac::pbpB/SSpbpD (C) grown in the absence of IPTG, and S. sciuri SS37 (D) were determined. Muropeptides were detected by measurement of the absorbance at 206 nm.

Effect of suppression of pbpB transcription on the oxacillin resistance level of transductants carrying the plasmid-borne S. sciuri pbpD or S. aureus mecA gene. High-level and homogeneous resistance to oxacillin in MRSA strain COL is known to depend on the level of transcription of the S. aureus pbpB gene (7). As shown in Fig. 5, the inhibition of pbpB transcription in the conditional mutant of strain COL (COLspac::pbpB) reduced the oxacillin resistance levels of the majority of the cells and converted the homogeneous phenotype to a heterogeneous one. In the presence of IPTG, the introduction of either the plasmid-borne S. sciuri pbpD gene or the S. aureus mecA gene into the pbpB conditional mutant COL-Sspac::pbpB produced high-level and homogeneous resistance that was close to that of S. aureus strain COL (Fig. 5). However, in the absence of IPTG, the oxacillin MICs for the majority of the cells decreased 16-fold and the population analysis profiles became heterogeneous (Fig. 5). These results indicate that even in the presence of large amounts of either S. sciuri PBP 4 or S. aureus PBP 2A in the transductants, the host PBP 2 was still required for the optimal expression of antibiotic resistance in S. aureus, as was already shown for MRSA strain COL (7, 12).


Figure 5
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  		FIG. 5. Effect of introduction of the plasmid-borne S. aureus mecA or S. sciuri pbpD genes and suppression of pbpB transcription on the expression of oxacillin resistance in S. aureus. Aliquots of overnight cultures were plated on tryptic soy agar containing increasing concentrations of oxacillin. The numbers of CFU were counted after incubation for 72 h at 30°C. The oxacillin susceptibility profiles were determined for COL ({diamondsuit}), COLspac::pbpB grown in the presence ({blacktriangleup}) and absence ({triangleup}) of IPTG, COL-Sspac::pbpB grown in the presence of IPTG ({blacktriangledown}), COL-Sspac::pbpB/SAmecA grown in the presence (•) and absence ({circ}) of IPTG, and COL-Sspac::pbpB/SSpbpD grown in the presence ({blacksquare}) and absence ({square}) of IPTG.


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DISCUSSION
 
The genetic determinant of β-lactam resistance, mecA, is not native to S. aureus but was acquired from an extraspecies source (1). The S. sciuri pbpD gene, which is uniformly present in both β-lactam-susceptible and -resistant isolates of this widely spread animal commensal species, was first identified on the basis of its high degree of structural similarity with the S. aureus mecA gene (2, 18). Previous studies have proposed that S. sciuri pbpD may represent the evolutionary precursor of the S. aureus mecA gene, mainly on the basis of epidemiological and genetic evidence. The protein product of pbpD was subsequently identified as PBP 4, one of the six PBPs detected in S. sciuri (20). Recombinant S. sciuri PBP 4 was purified and was shown to share several biochemical properties with S. aureus PBP 2A (6).

In this report, a new experimental system was designed to further test the validity of the proposition that the mecA resistance determinant present in all MRSA strains may have originated from the S. sciuri pbpD gene. The resistance cassette SCCmec was excised from extensively studied MRSA strain COL to generate MSSA strain COL-S, which was subsequently used as the recipient of a plasmid-borne copy of an upregulated form of S. sciuri pbpD recovered from resistant S. sciuri clinical isolate SS37.

The transductants carrying the S. sciuri pbpD gene exhibited most, if not all, of the resistance-related properties of the original MRSA strain COL.

(i) S. aureus cells were able to transcribe and translate this foreign genetic determinant producing large amounts of S. sciuri PBP 4, which was deposited in the plasma membrane of the host bacterium. (ii) S. sciuri pbpD could function as an effective component of the antibiotic resistance mechanism in the heterologous background of S. aureus. The transductants had extremely high-level, homogeneous, and broad-spectrum resistance to structurally diverse β-lactam antibiotics, similar to the properties of MRSA strain COL. Also, similarly to strain COL, (iii) the oxacillin resistance level of the transductants was dependent on the function of the host S. aureus PBP 2 (7) and (iv) oxacillin resistance was repressed by inhibitors of an early step in cell wall biosynthesis (16) and by the specific regulatory genes mecI and mecR (10), suggesting that the MecI repressor can bind to the promoter region of the S. sciuri pbpD gene. (v) S. sciuri PBP 4 was able to fully replace the essential physiological function(s) of the native S. aureus PBP 2 in a conditional mutant in which transcription of pbpB gene was inhibited (7, 13). (vi) The composition of the cell wall peptidoglycan produced under these conditions was typical of that of the S. aureus peptidoglycan (5), indicating that S. sciuri PBP 4 can function as a cell wall synthetic enzyme in S. aureus and cooperate with the other PBPs and monofunctional transglycosylases of the host to build cell wall peptidoglycan.

Thus, by replacing the S. aureus chromosomal determinant of resistance to β-lactams with a plasmid-borne copy of the heterologous upregulated S. sciuri pbpD gene, we were able to reconstruct a typical MRSA strain which was phenotypically indistinguishable from original MRSA strain COL. These results provide further support for the proposition that the wide-spectrum β-lactam resistance determinant mecA carried by all MRSA strains has evolved from the S. sciuri pbpD gene.


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ACKNOWLEDGMENTS
 
We are grateful to D. C. Oliveira (ITQB/UNL) for providing the plasmid carrying the construct pGC2::mecI-mecR1 and strain COLmecI-mecR1.

Partial support for these investigations was provided by grant 2 RO1AI045738 from the U.S. Public Health Service.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Microbiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Phone: (212) 327-8277. Fax: (212) 327-8688. E-mail: tomasz{at}rockefeller.edu Back

{triangledown} Published ahead of print on 17 November 2008. Back


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Antimicrobial Agents and Chemotherapy, February 2009, p. 435-441, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01099-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Llarrull, L. I., Fisher, J. F., Mobashery, S. (2009). Molecular Basis and Phenotype of Methicillin Resistance in Staphylococcus aureus and Insights into New {beta}-Lactams That Meet the Challenge. Antimicrob. Agents Chemother. 53: 4051-4063 [Full Text]  

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