Abstract
A close homologue of the acquired Staphylococcus aureus mecA gene is present as a native gene in Staphylococcus sciuri. We determined the patterns of penicillin-binding proteins (PBPs) and the peptidoglycan compositions of several S. sciuri strains to explore the functions of this mecA homologue, named pbpD, in its native S. sciuri environment. The protein product of pbpD was identified as PBP4 with a molecular mass of 84 kDa, one of the six PBPs present in representatives of each of three subspecies of S. sciuri examined. PBP4 had a low affinity for nafcillin, reacted with a monoclonal antibody raised against S. aureus PBP2A, and was greatly overproduced in oxacillin-resistant clinical isolate S. sciuri SS37 and to a lesser extent in resistant laboratory mutant K1M200. An additional PBP inducible by oxacillin and corresponding to S. aureus PBP2A was identified in another oxacillin-resistant clinical isolate, S. sciuri K3, which harbors an S. aureus copy of mecA. Oxacillin resistance depended on the overtranscribed S. sciuri pbpD gene in strains SS37 and K1M200, while the resistance of strain K3 depended on the S. aureus copy of mecA. Our data provide evidence that both S. aureus mecA and S. sciuri pbpD can function as resistance determinants in either an S. aureus or an S. sciuri background and that the protein products of these genes, S. aureus PBP2A and S. sciuri PBP4, can participate in the biosynthesis of peptidoglycan, the muropeptide composition of which depends on the bacterium “hosting” the resistance gene.
The important wide-spectrum β-lactam resistance gene mecA is present in all strains of methicillin-resistant Staphylococcus aureus (MRSA), as well as in coagulase-negative staphylococci. However, mecA is not native to these species. In an attempt to identify the potential evolutionary source of this genetic determinant, we recently tested multiple isolates of various species of the genus Staphylococcus for strains that would react with a DNA probe internal to mecA of S. aureus. This effort has led to the animal commensal species Staphylococcus sciuri, in which a close homologue of mecA was present in all of the epidemiologically unrelated isolates tested (1). The S. sciuri mecA homologue, referred to in this paper as S. sciuri pbpD, showed a linear structure and conserved motifs characteristic of genetic determinants of bacterial penicillin-binding proteins (PBPs) (16), and it was proposed that the S. sciuri mecA homologue may represent the evolutionary precursor of the resistance determinant mecA carried by all MRSA strains (17).
S. sciuri is a widely spread inhabitant of the skin of both domestic and wild animals and is considered one of the most abundant staphylococcal species on this planet (9, 10). The overwhelming majority of S. sciuri isolates examined were found to be fully susceptible to β-lactam antibiotics, including penicillin (1). However, recently S. sciuri has been repeatedly isolated from human clinical specimens also and the MICs of β-lactam antibiotics for several of these clinical isolates are increased (2, 14, 15). A closer examination of such drug-resistant clinical S. sciuri isolates indicated that they were of two different types. The first type contained the S. sciuri pbpD gene in combination with strong promoters, resulting in overexpression of the gene (3). The second type of resistant S. sciuri isolates had unaltered native pbpD but also carried a copy of a staphylococcal chromosome cassette mec (SCCmec), similar to strains of MRSA and coagulase-negative staphylococci (3).
One purpose of the studies described here was to identify the protein product of S. sciuri pbpD as a PBP and establish its molecular size and β-lactam affinity, together with those of the other PBPs present in S. sciuri. The second purpose of these studies was to provide experimental evidence for the capacity of the up-regulated forms of S. sciuri pbpD and/or S. aureus mecA to deliver antibiotic resistance and participate in the synthesis of cell walls in the genetic background of either S. sciuri or S. aureus.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
The bacterial strains used in this study are described in Table 1. Both the S. sciuri and the S. aureus strains were grown in tryptic soy broth or tryptic soy agar (TSA; Difco Laboratories, Detroit, MI) at 37°C with aeration. All but three of the S. sciuri strains were fully susceptible to β-lactam antibiotics.
TABLE 1.
S. sciuri and S. aureus strains used in this study
| Strain | Description | Source or reference |
|---|---|---|
| S. sciuri subsp. sciuri | ||
| K1 | Isolated from an eastern gray squirrel, methicillin susceptible | 1 |
| K56 | Isolated from a Norway rat, methicillin susceptible | 1 |
| S. sciuri subsp. rodentium | ||
| K3 | Clinical isolate recovered from a patient in a neonatal ward, forms pigmented (yellow) colonies on agar, expresses heterogeneous methicillin resistance, contains both S. aureus SCCmecIII and S. sciuri pbpD, segregates nonpigmented (white) methicillin-susceptible colonies (K3W) and pigmented (yellow) methicillin-resistant colonies (K3Y); pigmented colonies carry S. aureus SCCmecIII which is lost from the white segregants along with the loss of methicillin resistance | 1, 3 |
| K3W | Methicillin-susceptible, nonpigmented (white) segregant of K3 (see above) | 3 |
| K8 | Isolated from a Norway rat, expresses heterogeneous intermediate methicillin resistance, contains both S. aureus mecA and S. sciuri pbpD | 1 |
| S. sciuri subsp. carnaticum | ||
| K11 | Isolated from a veal leg, methicillin susceptible | 1 |
| K30 | Isolated from a Jersey cattle heifer, methicillin susceptible | 1 |
| Other S. sciuri strains | ||
| K1M200 | Highly methicillin-resistant laboratory mutant of strain K1, increased transcription of pbpD | 18 |
| SS37 | Methicillin-resistant clinical isolate recovered from the nasopharynx of a patient, increased transcription of pbpD | 3 |
| S. aureus control strains | ||
| COL | MRSA, homogeneous methicillin resistance | RUa collection |
| COL-S | COL with SCCmec excised, methicillin susceptible | 11 |
RU, The Rockefeller University.
Of the three resistant strains, S. sciuri K1M200 is an oxacillin-resistant, laboratory-derived mutant isolated from strain K1 (18), which is a wild-type S. sciuri strain susceptible to oxacillin and contains the pbpD gene native to this species (1). K1M200 was shown to carry a single point mutation in the promoter region of pbpD which was associated with the overexpression of the gene (18).
The heteroresistant clinical isolate S. sciuri K3 forms pigmented colonies and contains both S. sciuri pbpD and a copy of S. aureus mecA, which is primarily responsible for the heterogeneous oxacillin resistance phenotype of this strain (16). The oxacillin MIC for the majority of K3 cells was shown to be 12 μg/ml. Strain K3 is not stable but segregates white (K3W) and yellow (K3Y) colonies on TSA containing no antibiotics. Segregated strain K3W was shown to carry only the native pbpD gene and has reduced resistance to oxacillin (MIC of 1.5 μg/ml) (3).
The third oxacillin-resistant strain used in this study, S. sciuri SS37, was isolated from a patient with an infection (3). The strain was resistant to oxacillin, with an MIC of 25 μg/ml for the majority of the cells tested. Unlike K3, strain SS37 carried only pbpD native to S. sciuri but the gene was shown to be overexpressed, which appears to be related to the insertion of IS256 upstream of the gene (3).
MRSA strain COL and its isogenic methicillin-susceptible derivative COL-S, lacking the mecA gene and the associated SCCmec cassette (11), were also used in some experiments as controls.
PAPs.
Population analysis profiles (PAPs) were determined by spreading aliquots of overnight cultures at various dilutions onto TSA plates containing increasing concentrations of oxacillin. CFU were counted after 48 h of incubation at 37°C (5).
Membrane purification.
Membrane proteins were prepared from cultures that were grown until the optical density (OD) at 620 nm reached 0.7, as previously described (13). Oxacillin at a concentration of 3, 6, or 9 μg/ml was added to cultures of K3 in order to induce the expression of S. aureus mecA. The protein concentrations of membrane preparations were determined by the modified Lowry protein assay (Pierce, Rockford, IL) with bovine serum albumin as the standard.
Detection of staphylococcal PBP2A by Western blotting.
Membrane preparations (80 μg of proteins) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in 8% (wt/vol) acrylamide-0.06% (wt/vol) bisacrylamide gel as previously described (13). Transfer and blotting were done according to the ECL Western blotting analysis system (GE Healthcare, Little Chalfont, United Kingdom). The ChromPure human immunoglobulin G Fc fragment (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used at 3 μg/ml to eliminate nonspecific hybridization with protein A. The primary antibody was a monoclonal antibody against S. aureus PBP2A (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).
PBP analysis.
Membrane protein preparations (150 μg of proteins) were used to detect PBPs. In direct labeling, membrane proteins were labeled with [14C]benzylpenicillin (GE Healthcare, Little Chalfont, United Kingdom) at a final concentration of 20 μg/ml, followed by incubation at 30°C for 10 min. In competition assays, the membrane preparations were preincubated with nafcillin (20 μg/ml) at 30°C for 10 min prior to labeling with [14C]benzylpenicillin. The reaction with [14C]benzylpenicillin was terminated by the addition of an excess amount (1 mg/ml) of nonlabeled penicillin G. Electrophoresis was performed as described for the Western blot analysis. The gels were exposed to a tritium storage phosphor screen for 2 weeks, and the screen was scanned with a typhoon scanner (GE Healthcare, Little Chalfont, United Kingdom).
Peptidoglycan preparation and analysis by HPLC.
Peptidoglycan was purified from S. sciuri or S. aureus strains. One-liter batches of the cultures were collected by centrifugation and extracted with hot SDS, followed by mechanical rupture of the cells, removal of teichoic acids, and enzymatic digestion with mutanolysin, as previously described (4). The isolated muropeptides were reduced with sodium borohydride and separated by reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (ODS-Hypersil [3 μm, 4.6 by 250 mm]; Thermo Electron, Bellefonte, PA) at a flow rate of 0.5 ml/min for 150 min with a gradient from 5% (vol/vol) methanol in 100 mM NaH2PO4 (pH 2.5) to 30% methanol in 100 mM NaH2PO4 (pH 2.5). The abundance of muropeptides was estimated by calculating the percentage of the integrated area of the peaks detected by the absorbance at 206 nm.
Introduction of a plasmid-borne S. sciuri pbpD gene from S. sciuri strains SS37 and K1M200 into S. aureus strain COL-S.
Primers SS37MAF2 (5′-CGGGATCCCGGAATTTTCTGACTTCAGTGT-3′) and SS37MAR (5′-CGGGATCCCGTGGAAGGATAGTTGTGAGTG-3′) were used to amplify the 4,130-bp region of pbpD in strain SS37. The sequence obtained by PCR was ligated into shuttle plasmid pSPT181C to form pSS37MA. The recombinant plasmid was subsequently introduced into S. aureus strain RN4220 by electroporation and then transduced into S. aureus strain COL-S by phage 80α. To produce strain COL-S, SCCmecI of strain COL was removed by the method of precise excision as described by Katayama et al. (8, 11). Transductant TDSS37 was obtained after selection with tetracycline. The construction of another transductant, TDK1M200, was described in a previous communication (18), except that the recipient strain was COL-S.
RESULTS AND DISCUSSION
PBPs of S. sciuri.
14C-labeled penicillin was used to detect PBPs in a group of S. sciuri strains selected to represent various subspecies and sources of isolation, including some strains that showed resistance to oxacillin (Fig. 1 and Table 1). Lanes 1 and 2 of Fig. 1 show the PBP patterns of S. sciuri subsp. sciuri methicillin-susceptible strains K1 and K56 recovered from rodents (1). Of the two S. sciuri subsp. rodentium isolates shown in lanes 3 and 4, strain K3 was recovered from a patient and strain K8 was from a rodent (1). Both strains K3 and K8 showed heterogeneous resistance to oxacillin. Methicillin-susceptible strains K11 and K30 (lanes 5 and 6) belonged to S. sciuri subsp. carnaticum and were isolated from the skin of domestic animals (1). Strain K1M200 (lane 7) is an oxacillin-resistant laboratory mutant isolated through step selection from S. sciuri strain K1 (18). S. sciuri strain SS37 (lane 8) is a clinical isolate showing heterogeneous resistance to oxacillin (2, 3).
FIG. 1.
PBP patterns of oxacillin-susceptible and -resistant S. sciuri strains. Membrane proteins were purified from S. sciuri strains grown in antibiotic-free medium (lanes 1 to 9) and from strain K3 grown in the presence of 3, 6, or 9 μg/ml oxacillin (lanes 10 to 12). Membrane preparations (150 μg of proteins) were incubated with a single saturating concentration of [14C]benzylpenicillin. After SDS-polyacrylamide gel electrophoresis analysis, the gel was exposed to a storage phosphor screen for 2 weeks. PBPs and their corresponding molecular masses are indicated on the left. Protein standard size markers are indicated on the right.
Each of these eight S. sciuri isolates showed five clearly identifiable PBPs (PBP1, -2, -3, -5, and -6) that had molecular sizes that ranged from 98 kDa to 50 kDa and showed minor strain-to-strain variations in size. One additional PBP, named PBP4, with an approximate molecular mass of 84 kDa was detected at a high amount in strain SS37 (lane 8) and at a lower amount in strain K1M200 (lane 7). Only a faint band corresponding to PBP4 could be detected in methicillin-resistant strains K3 and K8 (lanes 3 and 4). However, PBP4 was not detected in methicillin-susceptible strains K1, K56, K11, and K30. The pbpD gene has been previously shown to be overexpressed in both oxacillin-resistant strains K1M200 and SS37 (3, 18), suggesting that PBP4 might correspond to the protein product of S. sciuri pbpD.
Previous studies (3, 16) have demonstrated that the oxacillin-resistant phenotype of S. sciuri K3 was associated with a copy of S. aureus-type SCCmecIII carried by this strain. It was also shown that in the absence of antibiotic selection, the resistance of strain K3 was unstable, producing oxacillin-susceptible progeny from which the S. aureus copy of mecA was lost (3). The PBP profile of such an antibiotic-susceptible segregant, K3W, showed the prominent bands of S. sciuri PBP1, -2, -3, and -5 but only a very faint PBP4 band (lane 9). Surprisingly, PBP6, one of the most prominent PBPs in all S. sciuri strains, including strain K3, was also missing in strain K3W. Lanes 10 through 12 of Fig. 1 show the PBP patterns of resistant isolate K3 grown in the presence of low concentrations of oxacillin (3, 6, and 9 μg/ml) sufficient to stabilize resistance and also needed to induce the expression of the S. aureus copy of mecA, which is under the control of mecI/mecR regulatory genes (1, 16). The PBP pattern in lane 10 shows only two strong bands (in addition to the faint band of PBP4), one band representing PBP6 of S. sciuri and a second, new, band with an approximate molecular mass of 78 kDa which was inducible by oxacillin and corresponded in molecular mass to that of S. aureus PBP2A. PBP1, -2, -3, and -5, prominent bands in all of the other S. sciuri strains, were only faintly visible in lane 10, suggesting that these PBPs had high enough affinity to bind oxacillin in the concentration range (3 to 9 μg/ml) used for the induction of S. aureus PBP2A.
Reactivity of S. sciuri PBPs with a monoclonal antibody raised against PBP2A of MRSA.
Previous studies have demonstrated that the protein product of pbpD (the mecA gene homologue native to all S. sciuri strains) reacts with a monoclonal antibody raised against PBP2A, the protein product of the S. aureus mecA determinant (3, 18). As shown in Fig. 2, membrane protein preparations from several S. sciuri strains were tested by Western blotting with the monoclonal antibody raised against PBP2A of S. aureus. MRSA strain COL is shown as a control to provide a size marker (78 kDa) for S. aureus PBP2A. Only single reactive bands corresponding to the molecular mass of S. sciuri PBP4 (84 kDa) were detected in the membrane preparations from S. sciuri strains K1M200, SS37, and K3W. Two reactive bands, one corresponding to the molecular mass of S. sciuri PBP4 and another to that of S. aureus PBP2A, were detected in oxacillin-resistant S. sciuri strains K3 and K8, which contain both S. sciuri pbpD and S. aureus mecA. When K3 was grown in the presence of 3 μg/ml oxacillin, PBP2A was present at a high amount and a very faint band corresponding to PBP4 was detected. Similar results were obtained when K8 was grown in the presence of oxacillin (data not shown). No reactive bands corresponding to PBP4 and/or PBP2A were detected in oxacillin-susceptible S. sciuri strains K1, K56, K11, and K30 and S. aureus strain COL-S.
FIG. 2.
Detection by Western blotting of protein products of S. aureus mecA and S. sciuri pbpD. 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 PBP2A. Membrane proteins were purified from oxacillin-susceptible S. sciuri strains K1, K56, K11, K30, and K3W, from oxacillin-resistant S. sciuri strains K3, K8, K1M200, SS37, and K3 grown in the presence of 3 μg/ml oxacillin, and from S. aureus strains COL and COL-S. The protein products of S. aureus mecA (PBP2A) and S. sciuri pbpD (PBP4) are indicated on the left.
These results allowed us to verify PBP4 (84 kDa) as the protein product of S. sciuri pbpD and confirmed that the additional PBP detected in K3 grown in the presence of oxacillin corresponded to the protein product of S. aureus mecA (PBP2A). Moreover, our data showed that PBP4 was not expressed in the oxacillin-susceptible S. sciuri strains whereas PBP4 was overexpressed in oxacillin-resistant S. sciuri strains K1M200 and SS37. In the other type of oxacillin-resistant S. sciuri represented by strains K3 and K8, which contain both S. sciuri pbpD and S. aureus mecA, PBP4 and PBP2A were produced at a low level. However, in the presence of oxacillin, the expression of PBP2A was strongly induced.
S. sciuri PBP4 detected in oxacillin-resistant strains has a low affinity for nafcillin.
Membrane preparations from oxacillin-resistant laboratory mutant K1M200 (Fig. 3, lanes 3 and 5) and from oxacillin-resistant clinical isolate SS37 (Fig. 3, lanes 4 and 6) were incubated in the presence of 14C-labeled penicillin with or without preincubation of the preparations with nonradioactive nafcillin at 20 μg/ml. Only PBP4 remained detectable in the preparations that were preincubated with nafcillin (lanes 5 and 6). As with PBP2A of S. aureus, PBP4 of S. sciuri is a low-affinity PBP. Lanes 1 and 2 of Fig. 3 provide a comparison for the S. sciuri PBP pattern to the more familiar PBP pattern of MRSA strain COL (lane 1) and its isogenic derivate COL-S (lane 2), from which the genetic determinant of PBP2A was removed by precise excision (11).
FIG. 3.
Determination of S. sciuri PBP affinity by competition assay. Membrane preparations (150 μg of proteins) purified from strains K1M200 and SS37 were incubated first with nafcillin (20 μg/ml) and then with a saturating concentration of [14C]benzylpenicillin (lanes 5 and 6). In parallel, the same preparations were incubated with [14C]benzylpenicillin without preincubation with nafcillin (lanes 3 and 4). PBP patterns of S. aureus strains COL (lane 1) and COL-S (lane 2) are also provided as a comparison. Protein standard size markers are indicated on the right.
Expression of oxacillin resistance in S. aureus transductants carrying a plasmid-borne copy of the upregulated pbpD gene from antibiotic-resistant S. sciuri strain K1M200 or SS37.
The oxacillin susceptibility profiles (PAPs) of susceptible parental strain S. sciuri K1, its resistant laboratory mutant K1M200, and resistant S. sciuri clinical isolate SS37 are shown in Fig. 4A. A plasmid-borne S. sciuri pbpD gene from strain K1M200 or SS37 was transduced into susceptible S. aureus recipient strain COL-S to obtain transductants TDK1M200 and TDSS37, respectively. Figure 4B shows that introduction of the upregulated pbpD gene from either strain K1M200 or SS37 caused a significant increase in the oxacillin MIC for susceptible S. aureus recipient COL-S. This increased resistance was clearly associated with the S. sciuri copy of pbpD since curing the transductants of the plasmid led to a complete loss of resistance (Fig. 4B). The degree of the increase in the oxacillin MIC differed substantially for the two transductants, TDSS37 expressing the more substantial increase, which seems to be correlated with the greater amount of PBP4 produced by strain SS37 compared to strain K1M200, as determined by Western blot analysis as described above (Fig. 2). Interestingly, the resistance profiles of S. aureus transductant TDSS37 and original S. sciuri strain SS37 were very close (compare Fig. 4A and B).
FIG. 4.
Oxacillin susceptibility profiles of S. sciuri strains and S. aureus transductants carrying a plasmid-borne copy of the upregulated pbpD gene from S. sciuri strains K1M200 and SS37. (A) S. sciuri strains K1 (open squares and dashed lines), K1M200 (closed squares and solid lines), SS37 (closed triangles and solid lines), K3 (solid circles and solid lines), and K3W (open circles and dashed lines). (B) Oxacillin-susceptible S. aureus recipient COL-S (closed diamonds and dashed lines) and transductants of COL-S carrying plasmid-borne mecA from S. aureus strain COL (TDCOL; closed circles and solid lines); transductants of COL-S carrying plasmid-borne pbpD from S. sciuri strain K1M200 (TDK1M200; closed squares and solid lines) and from S. sciuri strain SS37 (TDSS37; closed triangles and solid lines); transductants TDK1M200 (open squares) and TDSS37 (open triangles) cured of the plasmid carrying pbpD.
Also shown in Fig. 4B is the PAP of S. aureus transductant TDCOL, in which the same susceptible S. aureus strain, COL-S, received a copy of unregulated S. aureus mecA carried on a plasmid. In this case, the oxacillin MIC for the transductant was very high at more than 400 μg/ml and homogeneous, similar to the MIC for MRSA strain COL, which served both as a genetic background and also as a source of the mecA gene for these experiments. Thus, the genetic background of S. aureus strain COL was not optimal to generate a very high level and homogeneous resistance from S. sciuri pbpD (6).
S. sciuri PBP4, the protein product of pbpD, appears to be the only low-affinity PBP of S. sciuri, and the capacity of pbpD to deliver phenotypic resistance to S. aureus is clear from the transduction experiments illustrated in Fig. 4. Whether or not the overexpression of PBP4 is the only correlate of β-lactam resistance in S. sciuri is less obvious; oxacillin-resistant strain K1M200 also seemed to overproduce low-molecular-mass PBP6 compared to susceptible parental strain K1, and PBP6 was missing in strain K3W, along with oxacillin resistance compared to resistant parental strain K3 (Fig. 1).
Peptidoglycan composition and oxacillin resistance in S. sciuri.
Peptidoglycans were purified from oxacillin-susceptible S. sciuri strains K1 and K3W and from resistant strains K1M200, K3, and SS37 (Fig. 4A). Enzymatic hydrolysates of the peptidoglycans were analyzed for muropeptide composition by HPLC. A common feature of the HPLC profiles of the resistant strains was the increased representation of highly cross-linked muropeptides, which roughly paralleled the increasing oxacillin MIC for the strains (Fig. 5). PBP4 and/or PBP6 were shown to be overproduced in these resistant strains (Fig. 1 and 2), suggesting that PBP4 and PBP6 may contribute to the production of highly cross-linked peptidoglycans.
FIG. 5.
Comparison of the muropeptide compositions of peptidoglycans purified from oxacillin-susceptible and -resistant S. sciuri strains. Peptidoglycans were purified from 1-liter cultures grown at 37°C with aeration to an OD of 0.4, digested with mutanolysin, and separated by RP-HPLC. Oxacillin-susceptible S. sciuri strains K1 (A) and K3W (C); oxacillin-resistant S. sciuri strains K1M200 (B), K3 (D), and SS37 (F); and (E) relative amounts of monomeric, dimeric, and highly cross-linked muropeptides are shown.
Peptidoglycan composition of oxacillin-resistant S. sciuri strain K3 grown in the presence of sub-MICs of oxacillin.
MRSA strains that carry the mecA gene are known to produce a characteristically abnormal, distorted muropeptide profile when the bacteria are grown in oxacillin-containing media (compare panels A and B in Fig. 6). It has been suggested that the altered muropeptide pattern is the molecular “fingerprint” of the activity of resistance protein PBP2A (4). Therefore, it was of interest to determine the composition of peptidoglycan produced in S. sciuri strain K3, a strain whose oxacillin resistance was shown to be based on the activity of the S. aureus PBP2A-encoding gene carried by this S. sciuri isolate (3).
FIG. 6.
Comparison of the muropeptide compositions of peptidoglycans purified from S. sciuri and S. aureus strains grown in the presence of sub-MICs of oxacillin. Peptidoglycans were purified from 1-liter cultures grown at 37°C with aeration to an OD of 0.4, digested with mutanolysin, and separated by RP-HPLC. S. aureus strain COL grown in the absence (A) or in the presence (B) of 20 μg/ml oxacillin (oxa); S. sciuri strain K1M200 grown in the absence (C) or in the presence (D) of 20 μg/ml oxacillin; S. sciuri strain K3 grown in the absence (E) or in the presence (F) of 3 μg/ml oxacillin to induce the expression of S. aureus mecA are shown.
The muropeptide composition of peptidoglycan was analyzed from S. sciuri strain K3 grown in antibiotic-free medium (Fig. 6E) and in medium containing a sub-MIC (3 μg/ml) of oxacillin (Fig. 6F). The HPLC profile of the peptidoglycan from the bacteria grown in the presence of oxacillin showed an increase in the proportions of monomeric (peaks 4K and 7K) and dimeric (peaks 9K and 11K) muropeptides and a substantial reduction in the proportion of highly cross-linked oligomers (muropeptides with retention times of greater than 75 min on the HPLC column). Since oxacillin resistance in S. sciuri strain K3 is based on the functioning of S. aureus-derived PBP2A, one may have expected that strain K3 grown in the presence of a sub-MIC of oxacillin would produce a peptidoglycan with a muropeptide composition typical of an MRSA grown in the presence of β-lactam antibiotics (Fig. 6B). However, this was not the case. The muropeptide composition of S. sciuri strain K3 grown in the presence of 3 μg/ml oxacillin (Fig. 6F) was different from that of S. aureus strain COL (Fig. 6B) but similar to that of oxacillin-resistant S. sciuri strain K1M200 grown in the presence of a sub-MIC of oxacillin (Fig. 6D), whose resistance is based on altered PBPs native to this bacterium and not on an acquired heterologous determinant.
These observations indicate that the S. aureus mecA gene product produced an S. sciuri type of cell wall when S. sciuri strain K3 was grown in oxacillin-containing medium. The parallel, reverse, situation has already been documented recently in the case of S. aureus transductant SS1, in which the high level of oxacillin resistance depended upon the upregulated S. sciuri pbpD gene introduced into an S. aureus strain on a plasmid (19). The HPLC profile of peptidoglycan in transductant SS1 grown in the presence of a sub-MIC of oxacillin was typical of the peptidoglycan of S. aureus (12).
Our data provide further support for the similarity between the protein products of the S. sciuri pbpD (PBP4) and S. aureus mecA (PBP2A) genes (7). (i) Both the pbpD and mecA genes confer increased oxacillin resistance in either an S. sciuri or an S. aureus background, and (ii) both PBP4 and PBP2A are capable of catalyzing the biosynthesis of peptidoglycan in either an S. sciuri or an S. aureus background and produce peptidoglycans, the muropeptide composition of which is dictated by “blueprints” provided by the particular bacterial host.
Acknowledgments
Partial support for these studies was provided by a grant to Alexander Tomasz from the Irene Diamond Foundation and by the U.S. Public Health Service (2 RO1AI045738).
Footnotes
Published ahead of print on 16 November 2007.
REFERENCES
- 1.Couto, I., H. de Lencastre, E. Severina, W. Kloos, J. A. Webster, R. J. Hubner, I. S. Sanches, and A. Tomasz. 1996. Ubiquitous presence of a mecA homologue in natural isolates of Staphylococcus sciuri. Microb. Drug Resist. 2377-391. [DOI] [PubMed] [Google Scholar]
- 2.Couto, I., I. S. Sanches, R. Sa-Leao, and H. de Lencastre. 2000. Molecular characterization of Staphylococcus sciuri strains isolated from humans. J. Clin. Microbiol. 381136-1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Couto, I., S. W. Wu, A. Tomasz, and H. de Lencastre. 2003. Development of methicillin resistance in clinical isolates of Staphylococcus sciuri by transcriptional activation of the mecA homologue native to the species. J. Bacteriol. 185645-653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.de Jonge, B. L., Y. S. Chang, D. Gage, and A. Tomasz. 1992. Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A. J. Biol. Chem. 26711248-11254. [PubMed] [Google Scholar]
- 5.de Lencastre, H., A. M. Sa Figueiredo, C. Urban, J. Rahal, and A. Tomasz. 1991. Multiple mechanisms of methicillin resistance and improved methods for detection in clinical isolates of Staphylococcus aureus. Antimicrob. Agents Chemother. 35632-639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.de Lencastre, H., S. W. Wu, M. G. Pinho, A. M. Ludovice, S. Filipe, S. Gardete, R. Sobral, S. Gill, M. Chung, and A. Tomasz. 1999. Antibiotic resistance as a stress response: complete sequencing of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin. Microb. Drug Resist. 5163-175. [DOI] [PubMed] [Google Scholar]
- 7.Fuda, C., M. Suvorov, Q. Shi, D. Hesek, M. Lee, and S. Mobashery. 2007. Shared functional attributes between the mecA gene product of Staphylococcus sciuri and penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus. Biochemistry 468050-8057. [DOI] [PubMed] [Google Scholar]
- 8.Katayama, Y., T. Ito, and K. Hiramatsu. 2000. A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 441549-1555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kloos, W. E., D. N. Ballard, J. A. Webster, R. J. Hubner, A. Tomasz, I. Couto, G. L. Sloan, H. P. Dehart, F. Fiedler, K. Schubert, H. de Lencastre, I. S. Sanches, H. E. Heath, P. A. Leblanc, and A. Ljungh. 1997. Ribotype delineation and description of Staphylococcus sciuri subspecies and their potential as reservoirs of methicillin resistance and staphylolytic enzyme genes. Int. J. Syst. Bacteriol. 47313-323. [DOI] [PubMed] [Google Scholar]
- 10.Kloos, W. E., and T. L. Bannerman. 1994. Update on clinical significance of coagulase-negative staphylococci. Clin. Microbiol. Rev. 7117-140. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Pereira, S. F., A. O. Henriques, M. G. Pinho, H. de Lencastre, and A. Tomasz. 2007. Role of PBP1 in cell division of Staphylococcus aureus. J. Bacteriol. 1893525-3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Severin, A., S. W. Wu, K. Tabei, and A. Tomasz. 2005. High-level β-lactam resistance and cell wall synthesis catalyzed by the mecA homologue of Staphylococcus sciuri introduced into Staphylococcus aureus. J. Bacteriol. 1876651-6658. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Sieradzki, K., M. G. Pinho, and A. Tomasz. 1999. Inactivated pbp4 in highly glycopeptide-resistant laboratory mutants of Staphylococcus aureus. J. Biol. Chem. 27418942-18946. [DOI] [PubMed] [Google Scholar]
- 14.Stepanović, S., I. Dakic, D. Morrison, T. Hauschild, P. Jezek, P. Petras, A. Martel, D. Vukovic, A. Shittu, and L. A. Devriese. 2005. Identification and characterization of clinical isolates of members of the Staphylococcus sciuri group. J. Clin. Microbiol. 43956-958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Stepanović, S., P. Jezek, I. Dakic, D. Vukovic, and L. Seifert. 2005. Staphylococcus sciuri: an unusual cause of pelvic inflammatory disease. Int. J. STD AIDS 16452-453. [DOI] [PubMed] [Google Scholar]
- 16.Wu, S., H. de Lencastre, and A. Tomasz. 1998. Genetic organization of the mecA region in methicillin-susceptible and methicillin-resistant strains of Staphylococcus sciuri. J. Bacteriol. 180236-242. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wu, S., C. Piscitelli, H. de Lencastre, and A. Tomasz. 1996. Tracking the evolutionary origin of the methicillin resistance gene: cloning and sequencing of a homologue of mecA from a methicillin susceptible strain of Staphylococcus sciuri. Microb. Drug Resist. 2435-441. [DOI] [PubMed] [Google Scholar]
- 18.Wu, S. W., H. de Lencastre, and A. Tomasz. 2001. Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus. J. Bacteriol. 1832417-2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wu, S. W., H. D. Lencastre, and A. Tomasz. 2005. Expression of high-level methicillin resistance in Staphylococcus aureus from the Staphylococcus sciuri mecA homologue: role of mutation(s) in the genetic background and in the coding region of mecA. Microb. Drug Resist. 11215-224. [DOI] [PubMed] [Google Scholar]