Molecular Analysis of Resistance to Streptogramin A Compounds Conferred by the Vga Proteins of Staphylococci

Strains and growth conditions. Escherichia coli strains XL-2 Blue (Stratagene, Cedar Creek, Tex.) and PM9, which is a derivative of strain JM109 carrying a deletion of the malE gene (9), were used for the large-scale preparation of plasmids and expression of the MalE-fusion protein, respectively. Staphylococcus aureus RN4220 (22) and Staphylococcus epidermidis BM3302 (12), both of which are susceptible to MLS antibiotics, were used as recipient strains in the transformation experiments. Three clinical isolates were investigated: S. aureus BM3327 and S. epidermidis BM10385, which are resistant to SGA by the presence of the vga(A)v and vga(A) genes, respectively (16, 25), and S. epidermidis IPF69, which is resistant to ERY and SGB by the presence of the msr(A) gene.

E. coli strains and staphylococci were grown in Luria medium and brain heart infusion (BHI) broth or agar (Becton Dickinson, le Pont de Claix, France), respectively, supplemented as necessary with antibiotics. Bacterial growth was monitored by measuring the optical density of the cell suspension at 600 nm. Transformation of the staphylococcal strains was performed as described previously (2, 7).

Chemicals and reagents.Ampicillin, kanamycin, and chloramphenicol were purchased from Sigma (Lisle d'Abeau, Chesnes, France). The other antibiotics were kindly provided by their manufacturers: lincomycin (LC) and clindamycin (CLI) were provided by Pharmacia & Upjohn (Kalamazoo, Mich.); and the two components of pristinamycin (PT), with PTI as the SGB compound and PTII as the SGA compound, were provided by Aventis-Pharma (Vitry-sur Seine, France). Antibiotic disks came from Bio-Rad (Marnes-la-Coquette, France) or Eurobio (les Ulis, France). Disks of the SGA and SGB compounds, which contained 20 μg of PTII and 40 μg of PTI, respectively, were homemade. Isopropyl-β-d-thiogalactopyranoside (IPTG) was from Euromedex (Mundolsheim, France).

Restriction enzymes, T4 DNA ligase, alkaline phosphatase, and antiprotease cocktail were from Roche Diagnostics (Mannheim, Germany). Amylose resin was supplied by New England BioLabs (Beverly, Mass.), and enhanced chemiluminescence detection products, including polyvinylidene difluoride membranes, were from Amersham Biosciences (Little Chalfont, England). Size markers were a 1-kb DNA ladder (BioLabs) for agarose gel electrophoresis and Rainbow (Amersham) for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined with a bicinchoninic acid kit (Pierce, Rockford, Ill.) with bovine serum albumin (Sigma) as the standard. Lysostaphin was from Ambi UK (Trowbridge, England). Lysozyme and all the other chemical reagents were from Sigma.

DNA manipulations.All plasmids were constructed by common cloning techniques (36). Extraction of the DNA fragments separated by agarose gel electrophoresis was carried out with a Gene-Clean II kit (Bio 101, La Jolla, Calif.). Sequencing reactions and synthesis of the oligodeoxynucleotides (Table 1) were performed by Genome-Express (Meylan, France). The mutagenic primers were designed and used according to the guidelines supplied with the reagents of the QuikChange site-directed mutagenesis kit (Stratagene). High-fidelity DNA synthesis was done with Pfu DNA polymerase (Stratagene). The PCR assays were run on a Crocodile III apparatus (Appligène, Illkirch, France) with thermocycling conditions that varied according to the length of the DNA fragment amplified. Amplicons of the expected size were cloned into pCR4 (Invitrogen, Carlsbad, Calif.) and were sequenced to verify the integrity of the gene constructs. Inserts were subcloned into vectors of the pRB series, which enabled propagation of the plasmid constructs in staphylococci (7). Because transformation of S. epidermidis yielded more plasmid-less small-colony variants on kanamycin plates than that of S. aureus, pRB474 (with the cat gene as a resistance marker) was used instead of pRB374 (with the aadD gene as a resistance marker) throughout the study (Table 2).

The wild-type vga(A) gene was amplified from plasmid pIP1617 (4). The resulting BamHI-EcoRI-digested product was inserted into pRB374, leading to pIP1707 (Fig. 1A). Two rounds of site-directed mutagenesis were then performed to remove undesirable XbaI and BamHI sites and to add an EcoRI site between the ribosome-binding site (RBS) and the translation start of vga(A), leading to pIP1748 (Fig. 1B). Plasmid pIP1835, obtained by subcloning the HindIII-XbaI fragment of pIP1748 into pRB474, was the basis of all subsequent modifications. This plasmid codes for the N-terminal half of Vga(A) (Fig. 1C). Plasmids pIP1845, pIP1837, and pIP1867, which express the wild-type Vga(A) protein, the C-terminal half of Vga(A), and the Lmo0919 protein, respectively, resulted from the replacement of the EcoRI fragment of pIP1835 by the corresponding EcoRI-digested PCR products. Plasmid pIP1846, which directs the independent expression of the two halves of Vga(A), was obtained by subcloning a KpnI-digested PCR product encompassing the RBS and the distal half of vga(A) from pIP1837 into the unique KpnI site of pIP1835. Plasmids that encode chimeric proteins were constructed by using the internal XbaI site of the vga(A) gene as a frontier for domain shuffling (4). The proximal half of the msr(A) gene, which contains an additional XbaI site at the 3′ end of the sequence, was amplified and digested with EcoRI to replace the EcoRI fragment of pIP1835, leading to pIP1873 (Fig. 1D). XbaI-digested PCR products of the distal half of msr(A) were inserted into the unique XbaI sites of pIP1835 and pIP1873. The resulting plasmids, pIP1879 and pIP1880, respectively, encode the N-terminal ABC domain of Vga(A) fused to the C-terminal domain of Msr(A) and a full-length Msr(A) protein in which histidine 281 is replaced by leucine (H281L), respectively. This change in the amino acid sequence of Msr(A) was due to the creation of the XbaI site within the nucleotide sequence of the gene. The 900-bp XbaI fragment from plasmid pIP1739 (see below), which encodes the C-terminal half of Vga(A), was inserted into the unique XbaI site of pIP1873 to generate the fusion of the N-terminal ABC domain of Msr(A) with the C-terminal domain of Vga(A). Plasmids pIP1861 and pIP1863, which carry the vga(B) and vga(A)v genes, respectively, were constructed by subcloning HindIII-KpnI and BamHI-KpnI fragments from pIP1717 (3) and pIP1810 (15), respectively, into pRB474. All the gene constructs were therefore under the control of the Bacillus subtilis vegII promoter (7), and all but the last two constructions were placed 9 nucleotides downstream of the RBS of vga(A) (Fig. 1).

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FIG. 1.

Progeny of the plasmids used. Abbreviations: PvegII, constitutive promoter; RBS, ribosome-binding site of the vga(A) gene; B, BamHI; E, EcoRI; X, XbaI; H, HindIII, S, SmaI; K, KpnI.

Expression and purification of the MalE fusion protein.The MalE fusion pMal-c0 expression vector was a modified version of pMal-c2 (New England Biolabs). In pMal-c0, the polylinker at the 5′ end of the malE gene was replaced by the authentic malE sequence (J.-M. Clément, unpublished data). The EcoRI fragment of pIP1746 carrying the vga(A) gene was inserted into pMal-c0 at the EcoRI site. The orientation of the insert, in frame with the 3′ end of the malE gene, was confirmed by restriction mapping. The resulting plasmid, pIP1739, was transformed into E. coli PM9. Cultures of a transformant were induced with 0.2 mM IPTG, and the MalE-Vga(A) fusion protein was observed at 100 kDa in Coomassie blue-stained gels (data not shown). Cells were broken in a French press at 16,000 lb/in², and the fusion protein was purified from the lysate on amylose resin by standard protocols. A total of 6 mg of protein, which eluted from the column as a single peak, was obtained from 2 liters of culture. The purity of the eluate was checked by SDS-PAGE. The size of the purified protein, which reacted with an antibody elicited against MalE, was consistent with that expected for the hybrid protein. The final preparation was dialyzed against phosphate-buffered saline, concentrated to 1.5 ml, and injected into rabbits to raise antisera.

Production of antibodies directed against MalE-Vga(A).Two New Zealand rabbits were immunized by intradermal injection of 200 μg of antigen preparation in Freund's complete adjuvant. The immune response was boosted with the same dose of proteins in Freund's incomplete adjuvant given subcutaneously every 2 weeks for 1.5 months. Serum samples were then taken twice weekly. The animals were bled 3 weeks after the last immunization. Antibodies were purified on a protein A-Sepharose column.

Susceptibility tests.The MICs of lincosamides and streptogramins were determined in triplicate by the twofold dilution method with Mueller-Hinton agar and 10⁴ CFU per spot after 24 h of incubation at 37°C. The concentrations of antibiotics ranged from 0.064 to 512 μg/ml. Disk diffusion assays carried out with various antibiotics were used in parallel for each recombinant strain to check the purity of the bacterial culture.

Preparation and analysis of cell fractions.Cultures of staphylococci were grown in brain heart infusion medium at 37°C with vigorous shaking until the absorbance at 600 nm reached 0.7 units. Cells from 50-ml cultures were harvested by centrifugation at 5,000 × g for 10 min at 4°C, washed once in 50 mM Tris (pH 7.5), and resuspended in 1/10 of the same buffer with 30% (wt/vol) sucrose. Protoplasts were prepared by using 1 mg of lysostaphin per liter and 5 mg of lysozyme per liter at 37°C for 20 min. The protoplast pellets were lysed in 2 ml of buffer consisting of 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgCl₂, and 20 mM dithiothreitol, hereafter called the sample buffer, in the presence of 15 μg of protease inhibitors per ml and with the help of ultrasonic disruption for 3 min in 30-s pulses at 4°C. Unbroken cells and debris were removed by centrifugation at 16,000 × g for 4 min, and the lysates were ultracentrifuged in a Beckman TL-100 instrument. Membranes were isolated by centrifugation at 30,000 × g for 30 min, and ribosomes were separated from the cytoplasm by centrifugation at 150,000 × g for 1 h. Because the membranes and ribosomes were resuspended in 200 μl of sample buffer, the cytosolic fractions were 10 times as dilute as both particulate fractions. For SDS-PAGE analysis, each sample was mixed with an identical volume of 2× Laemmli buffer and boiled for 5 min. Gels (10% [wt/vol] acrylamide) in Tris-glycine buffer were loaded with 1 volume of membranes and ribosomes and 10 volumes of the cytosolic fractions. Runs were performed at room temperature under a constant current of 40 mA. Proteins were either stained with Coomassie brilliant blue R-250 (Sigma) or transferred to a polyvinylidene difluoride membrane (Amersham).

Immunoblotting was carried out with antibodies directed against MalE-Vga(A) and with two antiserum samples used as controls for cellular fractionation. The first was raised against the β subunit of the F1 protein of E. coli ATP synthase, and the second was raised against the L24 protein from B. subtilis. The working dilutions of these antibodies were 1/400 for those reacting against MalE and Vga(A), 1/15,000 for those reacting against anti-F1β, and 1/1,000 for those reacting against anti-L24. Anti-rabbit or anti-mouse immunoglobulin G-peroxidase conjugates were diluted 10,000-fold. Bound peroxidase activity was revealed with enhanced chemiluminescence substrates on Fuji HR-E30 films.

Phenotypes conferred by wild-type genes.The MICs of lincosamides (LC and CLI) and streptogramins (SGA, SGB, and PT) were determined for two staphylococcal hosts, S. aureus RN4220 and S. epidermidis BM3302, carrying vga(A)-related genes (Table 2). The vga(A) gene conferred high-level resistance to SGA (MIC = 32 μg/ml) in S. aureus, whatever the vector used. The vga(A)v gene, which is more prevalent than vga(A) among clinical isolates of this species (17), was not more efficient at conferring resistance (MIC = 32 μg/ml). These findings were described earlier (4, 15). The MICs of SGA due to the presence of vga(A) or vga(A)v were the same for S. epidermidis. Interestingly, we observed that vga(A) or vga(A)v was able to confer low-level resistance to LC (MICs = 4 and 8 μg/ml for S. aureus and S. epidermidis, respectively). This resistance-conferring trait of vga(A) was not described in previous analyses because of the presence of a constitutively expressed erm(C) gene in the vector pOX300 (4). Each gene, vga(A) or vga(A)v, conferred a slight increase in the basal level of resistance to CLI (MIC from 0.125 to 1 μg/ml for S. epidermidis and from 0.125 to 0.25 μg/ml for S. aureus). This phenotype is similar to that conferred by the lsa gene in Enterococcus faecalis, although the levels of resistance to lincosamides were much lower for staphylococci with the vga(A) and the vga(A)v genes (38). We suggest, therefore, that vga(A) or its variant, vga(A)v, is involved in the LSA phenotype occasionally found among staphylococcal isolates (14, 17, 24).

Neither vga(A) nor vga(A)v was able to confer resistance to PT, which is a mixture of 70% SGA and 30% SGB. Conversely, we found that vga(B) determines a large increase in the level of resistance to PT (MIC from 0.125 to 2 μg/ml for S. epidermidis), while it confers low-level resistance to SGA (MIC = 4 μg/ml). To the best of our knowledge, this has never been described. The detection of this gene in clinical strains might therefore be of importance for prediction of the in vivo activity of quinupristin-dalfopristin (2, 11, 17, 24).

A search for protein sequences similar to the sequences of the Vga proteins led us to identify a L. monocytogenes protein-coding ORF, Lmo0919, which displays 41% amino acid identity with Vga(A) (Fig. 2). This ORF is chromosomally borne, and no gene encoding transmembrane proteins was found in its vicinity. When lmo0919 was cloned on pRB474, it was able to confer resistance to SGA (MIC = 8 μg/ml for both hosts) and LC (MIC = 4 μg/ml for S. epidermidis). This phenotype is similar to that conferred by vga(A) and vga(A)v in the same hosts, although the levels of resistance are lower. The slight difference might be due to the less efficient expression of lmo0919 in the staphylococcal background. A less efficient interaction with a putative partner encoded by the host chromosome could be another explanation. Nevertheless, we postulate that Lmo0919 contributes to the intrinsic resistance of Listeria strains to SGA compounds and lincosamide antibiotics (5, 40). We are attempting to inactivate the lmo0919 gene in L. monocytogenes CLIP72401 to further document the antibiotic resistance-conferring properties of this chromosomal gene.

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FIG. 2.

Alignment of the proteins studied. Identical residues are boxed. Highly conserved functional motifs of ABC proteins (WALKER A, LID, SIGNATURE, WALKER B, and SWITCH) are indicated. Vga(A), Vga(A)v, and Lmo0919 are characterized by shorter helical domains encompassing residues between the LID and the SIGNATURE motifs of the N-terminal ABC domain.

The amino acid sequences of the five proteins analyzed in the present study exhibited from 45 to 85% similarity in pairwise comparisons. Conserved regions found universally in ABC proteins were present, such as the Walker A and B motifs and the signature motif involved in ATP binding and hydrolysis; the switch motif, which could also be involved in ATP hydrolysis; and the LID motif, which has been shown to be essential for the interaction of the MalK ABC ATPase with its membrane partners, MalF and MalG, in the E. coli maltose transporter (29). Except for Msr(A), which is 488 amino acids (aa) in length, all the other Vga(A)-related proteins had a lysine- and aspartate-rich C-terminal region. One of the common traits of Vga(A), Vga(A)v, and Lmo0919 (522, 524, and 523 aa, respectively) is the 22-aa deletion found in a region between the LID motif and the signature motifs of the N-terminal ABC domain. Vga(B) and Msr(A) do not present such deletions (Fig. 2). Despite this, it is rather difficult to correlate the sequence-structure traits of these proteins with their specificities for MLS antibiotics. We suspect, however, that the two halves of the Vga(A)-related proteins are involved in separate functions, given their structural dissimilarities.

Immune detection and subcellular localization of Vga(A) protein.Since many ABC systems are known to be involved in the import or export of a wide variety of substances, the Vga(A) protein and similar systems have been thought to be part of classical transporters that expel the MLS antibiotics from bacteria (11, 34). However, this assumption has not been confirmed by establishment of the membrane localization of these proteins or by characterization of any partner that might anchor the ABC proteins in the bacterial membranes. We thus analyzed the subcellular localization of Vga(A) by using the antiserum raised against MalE-Vga(A).

Lysates obtained from S. aureus RN4220 and BM3327 and S. epidermidis BM3302, BM10385, and IPF69 were probed with the antibodies. Because of smearing problems due to protein A, only the S. epidermidis strains were used for Western blot analyses. There were few cross-reactive bands in S. epidermidis BM3302 (Fig. 3, lane 2). Lysates of strain BM3302 transformed by vga(A) gave, as expected, a band of 58 kDa that reacted with the antibodies (Fig. 3, lane 3). This band was probably the Vga(A) protein, since its size corresponded to that predicted. It was observed that Vga(A)v strongly cross-reacted with the polyclonal antiserum (Fig. 3, lane 5), whereas Vga(B) and Lmo0919 did not (Fig. 3, lanes 4 and 6).

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FIG. 3.

Analysis of lysates extracted from S. epidermidis BM3302 transformants and probed with antibodies raised against MalE-Vga(A). Lanes: 1, 10 ng of purified MalE protein; 2, strain BM3302 transformed by pRB474; 3, strain BM3302 transformed by pIP1845 expressing Vga(A); 4, strain BM3302 transformed by pIP1861 expressing Vga(B); 5, strain BM3302 transformed by pIP1863 expressing Vga(A)v; 6, strain BM3302 transformed by pIP1867 expressing Lmo0919.

Fractionation of cells from clinical isolates BM10385 and IPF69, which express the vga(A) and the msr(A) genes, respectively, from their native promoters, showed that Vga(A) colocalized with the β subunit of the F₁-F₀ ATPase in the membrane fraction (Fig. 4). No trace of Vga(A) was detectable in the cytosolic or the ribosomal fractions analyzed. To the best of our knowledge, there are no previous reports of the membrane localization of Vga(A)-related systems in bacteria of medical interest. Since Msr(A) does not cross-react with the antibodies raised against Vga(A), we cannot draw a conclusion about the subcellular localization of Msr(A) in staphylococci.

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FIG. 4.

Analysis of membranes (M) and ribosomes (R) extracted from two S. epidermidis clinical isolates and probed with various immune sera. The first part (lanes 1) of each panel corresponds to strain BM10385 carrying vga(A), and the second part (lanes 2) corresponds to strain IPF69 carrying msr(A). (A) Immunoblot probed with antibodies raised against MalE-Vga(A); (B) immunoblot probed with antibodies raised against the β subunit of the F₁-F₀ ATPase; (C) immunoblot probed with antibodies raised against the L24 protein.

A similar experiment was performed with laboratory strain BM3302 transformed with pIP1845, which expresses vga(A) under the control of the constitutive vegII promoter. Most of the antibody-reacting material of this strain was detected in the membrane fraction (Fig. 5). However, it is worth mentioning that approximately 10 to 20% of the Vga(A) protein was also detected in the cytosolic fraction (Fig. 5, lanes 3 and 7). This result was probably due to overexpression of the vga(A) gene from vegII and might suggest that the putative membrane proteins, which were expected to form a canonical ABC transporter with Vga(A), are present in limiting amounts. Olano et al. (30) showed that Ole(B) partitions equally between the membrane and the cytosolic fractions in Streptomyces. Their observation and our findings give credence to the hypothesis that Vga(A) and, probably, Msr(A) are able to interact with the membranes of staphylococci.

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FIG. 5.

Analysis of the cytoplasmic (C) and membrane (M) fractions from S. epidermidis BM3302 transformants probed with antibodies raised against MalE-Vga(A). Lanes: 1 and 5, strain BM3302 transformed by pIP1835 expressing the first half of Vga(A); 2 and 6, strain BM3302 transformed by pIP1837 expressing the second half of Vga(A); 3 and 7, strain BM3302 transformed by pIP1845 expressing Vga(A); 4 and 8, strain BM3302 transformed by pRB474, used as a control.

Membrane targeting and expression of resistance.It has been reported that either one of the two ABC domains of Ole(B) is sufficient to confer resistance to oleandomycin (30). Moreover, it was demonstrated that the N-terminal ABC domain of Ole(B) is able to recognize oleandomycin (8). To test the possibility that each ABC domain of Vga(A) would behave similarly in conferring resistance to SGA, we constructed truncated versions of vga(A) by subcloning each half of the gene on separate plasmids. When the two plasmids were transformed into S. aureus and S. epidermidis, neither was able to confer resistance to any of the antibiotics tested (Table 2). In this respect, Vga(A) behaves like Msr(A), whose N-and C-terminal domains were found to be unable to promote erythromycin resistance (35). Moreover, each half of Vga(A) was expressed but was found exclusively in the cytosolic fraction of the cells (Fig. 5, lanes 1, 2, 5, and 6). These truncated proteins were unable to reach the membrane, probably because signals present in both moieties are needed to target the membrane. We also constructed plasmid pIP1846, in which the two halves were coexpressed in the same cells. This plasmid is also unable to confer resistance to the antibiotics tested, suggesting that the two halves of Vga(A) cannot interact together in order to reconstitute a functional system.

The Vga(A) protein consists of two ABC domains fused into a single polypeptide. In order to establish whether ATP hydrolysis is needed for resistance to SGA, we mutated an aspartate residue in either one of the two Walker B motifs of Vga(A). This quasi-invariant residue is in close contact with the γ-phosphate of ATP in crystallized ABC proteins, such as HisP (20). Vga(A) proteins carrying substitutions of D104 or D409 for lysine were unable to confer resistance to SGA (Table 2). This result demonstrates that the two ATP-binding sites of Vga(A) are needed for the protein to function. To further document this finding, we analyzed the subcellular localization of the plasmid-encoded proteins in S. epidermidis BM3302. Approximately 80% of the Vga(A) proteins with mutations in either one of the two Walker B motifs were found in the cell membrane fractions, as was also found for the wild-type protein (Fig. 6). Susceptibility to SGA and lincosamide antibiotics was therefore due to inactive rather than misexpressed or mislocated proteins.

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FIG. 6.

Analysis of the membrane fractions from S. epidermidis BM3302 transformants probed with antibodies raised against MalE-Vga(A). Lanes: 1, 10 ng of MalE protein; 2, strain BM3302 transformed by pRB474; 3, strain BM3302 transformed by pIP1838 expressing Vga(A) mutated in the second Walker B motif; 4, strain BM3302 transformed by pIP1845 expressing a wild-type Vga(A) protein; 5, strain BM3302 transformed by pIP1877 expressing the chimera Msr(A)-Vga(A); 6, strain BM3302 transformed by pIP1879 expressing the chimera Vga(A)-Msr(A); 7, strain BM3302 transformed by pIP1887 expressing Vga(A) mutated in the first Walker B motif.

Vga(A) a