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. 2004 Jun;48(6):1953–1959. doi: 10.1128/AAC.48.6.1953-1959.2004

tcaA Inactivation Increases Glycopeptide Resistance in Staphylococcus aureus

Hideki Maki 1,†, Nadine McCallum 1, Markus Bischoff 1, Akihito Wada 2, Brigitte Berger-Bächi 1,*
PMCID: PMC415614  PMID: 15155184

Abstract

The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.

Vancomycin is commonly used as the antibiotic of last resort for the treatment of multiresistant methicillin-resistant Staphylococcus aureus infections. Following the first report of the failure of vancomycin therapy due to a vancomycin-intermediate-resistant S. aureus (which is synonymous with glycopeptide-intermediate-resistant S. aureus [GISA]) isolate in 1997 (21), an increasing number of strains with decreased glycopeptide susceptibilities, some of which caused glycopeptide therapy failures (1, 26, 30, 40), have been identified in various locations, including Japan, the United States, and Europe (15). The emergence of GISA isolates is preceded by the appearance of heterogeneous GISA (hGISA) isolates, which display lower levels of vancomycin resistance than GISA isolates and which contain a small subpopulation of subclones with higher levels of resistance. Prolonged glycopeptide therapy is thought to select for GISA from this hGISA subpopulation. In order to assess the clinical relevance of hGISA strains and evaluate the efficacy of vancomycin treatment, it is important to determine their prevalence (17). However, hGISA strains are difficult to distinguish from susceptible strains by standard resistance tests except by use of the time-consuming process of population analysis profiling (26, 40). Since intermediate glycopeptide resistance in staphylococci is not due to an acquired resistance element but is multifactorial and is generated endogenously by the accumulation of various mutations (4), molecular identification methods are not yet available.

One of the phenotypic characteristics of many hGISA and GISA strains is a thickened cell wall and an increased number of free, un-cross-linked d-Ala-d-Ala residues in the peptidoglycan which trap vancomycin and prevent the drug from reaching its lethal target, the membrane-associated lipid II-linked peptidoglycan precursor (5, 12). Both clinical GISA strains and GISA strains selected in vitro show multiple changes in cell wall metabolism and composition, such as (i) enhanced incorporation of N-acetylglucosamine, (ii) an increased size of the cytoplasmic pool of the murein monomer precursor UDP-N-acetylmuramyl-pentapeptide, (iii) increased levels of PBP 2 and PBP 2′ production (6), (iv) decreased levels of PBP 4 activity (13), (v) a decrease in the level of amidation of the muropeptide, (vi) reduced cross-linking of the cell wall peptidoglycan, and (vii) enhanced autolysis. A set of up- and down-regulated genes, including regulators, was reported (18, 19, 25, 29). The enhanced activity of the alternative transcription factor σB increases the level of teicoplanin resistance, although the relevant σB-controlled target genes involved in the resistance remain to be identified (3). A defective agr function has also been suggested to be associated with an increased probability of glycopeptide resistance formation upon pressure from vancomycin treatment (35).

Although a number of factors have been shown to be involved in the complex mechanism that evokes glycopeptide resistance, none of them can be attributed to hGISA or GISA strains specifically.

Deletion of a genomic region containing the tcaRAB locus was previously reported to increase the level of teicoplanin resistance in S. aureus (7). However, neither the mechanism of increased resistance nor the contributions of individual genes affected by the deletion are known. TcaR, which has been postulated to be a member of the MarR-like family of transcriptional regulators, has recently been identified as a regulator of virulence determinants in S. aureus (28). tcaA is predicted to encode a trans-membrane protein containing a C4-type metal-binding motif, and TcaB shares similarity with multidrug efflux pump proteins. In this study, we show that the tcaA gene is the major factor responsible for the increased levels of teicoplanin resistance. Additionally, we demonstrate that tcaA inactivation in experimental isolates as well as in clinical isolates contributes to glycopeptide resistance.

MATERIALS AND METHODS

Molecular biological and genetic methods.

Molecular biological manipulations were performed in accordance with standard protocols (36). Sequencing was performed with a Thermo Sequenase cycle sequencing kit (U.S. Biochemicals, Cleveland, Ohio) and an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, Calif.) by the protocol of Wada (39). Genetic manipulations of S. aureus were done as described earlier (23). The general transducing phage 80α was used for transductions.

Strains and growth conditions.

The strains and plasmids used in this study are listed in Table 1. Growth was in Luria-Bertani broth (Becton Dickinson, Sparks, Md.) at 37°C unless specified otherwise. Kanamycin at 50 μg/ml or chloramphenicol at 20 μg/ml was added to the medium when needed.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid Relevant genotype or phenotypea Reference or source
Strains
    E. coli DH5α F− Ω80 lacZΔM15 relA1 Invitrogen
    S. aureus
        BB255 Essentially the same as NCTC8325, rsbU 2
        RN4220 NCTC8325-4, r− m+, cured of prophages 22
        GP268 BB255, rsbU+ Tcr 16
        COL Oxr Tcr 7
        BB1372 COL Ω2027 ΔtcaRAB::Tn917 Emr Ter 7
        BB1380 BB255 Ω2026tcaA::Tn917 Emr Ter 7
        BB1440 GP268 Ω2026tcaA::Tn917 Emr Ter This study
        BB1456 BB1372, third-step mutant selected on increasing Te concentrations Ter This study
        BB938 BB255 rsbW Ter 4
        Mu3 hGISA Oxr NRSA2 (20)
        MI GISA tcaA Oxr NRSA3 (8)
        HIP06297 GISA Oxr NRSA17 (37)
        HIP09143 GISA Oxr NRSA24
        HIP09433 GISA Oxr NRSA27
        LIM-2 GISA Oxr NRSA36 (32)
        SA137/93A GISA Oxr 33
        SA137/93G SA137/93A tcaA::IS256 ΔSCCmec GISA Oxs 33
        BB1372-4 BB1372, selected on 4 μg of Vm/ml This study
        Mu3-4 Mu3, selected on 4 μg of Vm/ml This study
        COL-4 COL, selected on 4 μg of Vm/ml This study
Plasmids
    pAW17 E. coli-S. aureus shuttle vector; Kmr 34
    pAW17-tcaR KmrtcaR+ This study
    pAW17-tcaRAB KmrtcaR+tcaA+tcaB+ This study
    pAW17-tcaAB KmrtcaA+tcaB+ This study
    pAW17-tcaA KmrtcaA+ This study
    pAW17-tcaB KmrtcaB+ This study
    pMGS100 E. coli-S. aureus shuttle expression vector; Cpr 14
    pMGS100-tcaA CprtcaA+ This study
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a

Abbreviations: Ap, ampicillin; Cp, chloramphenicol; Em, erythromycin; Km, kanamycin; Ox, oxacillin; Tc, tetracycline; Te, teicoplanin; Vm, vancomycin; r, resistant; s, susceptible.

Resistance tests.

The MICs of antibiotics were determined by E-test (AB-Biodisk, Solna, Sweden) on brain heart infusion (BHI; BBL) agar plates with an inoculum of a 2 McFarland standard and incubation at 35°C for 48 h or by broth microdilution, as recommended by the NCCLS (31). For population analysis profiles, appropriate dilutions of an overnight culture were plated on BHI agar plates containing increasing concentrations of vancomycin. The numbers of CFU were determined after 48 h of incubation at 37°C. Stepwise selection for increased teicoplanin resistance was performed by swabbing an overnight culture on a teicoplanin-containing gradient plate and isolation and purification of colonies growing at the highest concentration.

Plasmid construction.

The series of plasmids used for complementation were constructed by cloning various fragments into pAW17 (34), as shown in Fig. 1. The open reading frame of tcaA was amplified by Pwo polymerase (Roche Diagnostics, Rotkreuz, Switzerland) PCR with primers HM7 (5′-TTTTTCGGCCGGCATGAAATCTTGCCCGAAGTGCG-3′) and HM8 (5′-ATTTTTCGCGATTTTTCTGATGTCTTGATTAAT-3′), with the underlined sequences showing the EagI and NruI cleavage sites, respectively. The amplification product was cloned into the EagI-NruI cleavage sites of an overexpression vector, pMGS100 (14), resulting in pMGS100-tcaA.

FIG. 1.

FIG. 1.

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Complementation of tcaRAB mutant BB1372. The nucleotide positions shown correspond to the sequence registered in GenBank and EMBL (accession number AY008833). Bold lines, fragments inserted into vector pAW17; dotted lines, deleted ClaI-ClaI fragment; asterisk, the BglII site, which was used to remove part of tcaA from pAW17-tcaAB to create pAW17-tcaB. The MICs of teicoplanin and vancomycin on BHI agar for strain BB1372 complemented with the indicated plasmids are shown on the right. The positions of the probes used for Northern blot analysis are indicated as bars above the open reading frames.

Northern blot analysis.

Total RNA was isolated as described by Cheung et al. (10). For induction, 10 μg of teicoplanin per ml was added to exponentially growing cultures at an optical density at 600 nm (OD600) of 0.5 for 30 min. Digoxigenin-labeled DNA probes, produced by using the PCR DIG Probe synthesis kit (Roche, Basel, Switzerland), were used for the detection of specific transcripts by Northern hybridization, according to the instructions of the manufacturer. The primers used for probe amplification were Dig-tcaAF (5′-GAATGCGATTGAGAATAATG-3′) and Dig-tcaAR (5′-TCTCAGTTGATTTCATAGCT-3′), Dig-tcaRF (5′-CGTTAACGCATTAACTGCAA-3′) and Dig-tcaRR (5′-GCGATGATTGATGACTTCTA-3′), and Dig-tcaBF (5′-GGAGCATTATCGATAGATAT-3′) and Dig-tcaBR (5′-CTGTTAATGATTCAGGTACT-3′).

Nucleotide sequence accession number.

The nucleotide sequence has been deposited in the EMBL and GenBank nucleotide sequence databases under accession number AY008833.

RESULTS

Complementation of the tcaRAB deletion mutant.

Deletion of the tcaRAB region is known to confer teicoplanin resistance to S. aureus (7). The gene responsible for the increase in teicoplanin resistance was determined by complementing tcaRAB deletion mutant BB1372 with pAW17-based plasmids containing a set of inserts covering various parts of the tcaRAB locus (Fig. 1). The teicoplanin and vancomycin MICs for BB1372 complemented with the empty pAW17 plasmid were 24 and 8 μg/ml, respectively. Only plasmids that included an intact tcaA gene were found to restore teicoplanin susceptibility to the original level for susceptible parent strain COL (Fig. 1). Neither tcaR nor tcaB alone influenced the resistance level, while tcaA by itself was sufficient to restore the original level of susceptibility.

Complementation of tcaA in a strain with high-level teicoplanin resistance.

A highly teicoplanin-resistant mutant, BB1456, was obtained by repeated stepwise selection of tcaRAB mutant BB1372 by growth on increasing concentrations of teicoplanin. After three selective steps, the teicoplanin MIC for the isolate on BHI agar increased from 24 to 96 μg/ml, while that of vancomycin rose from 8 to 32 μg/ml. Due to the multiple-step selection, strain BB1456 is likely to carry, besides the tcaRAB defect, several additional mutations contributing to the teicoplanin resistance level. Complementation of BB1456 with tcaA dramatically reduced the MIC of teicoplanin to 12 μg/ml and that of vancomycin to 6 μg/ml, demonstrating that the lack of tcaA had a major effect on the resistance level. However, when pAW17-tcaA was introduced into genetically distinct, teicoplanin-resistant strain BB938, whose teicoplanin resistance is based on increased σB activity plus at least one other uncharacterized mutation (4) unlinked to tcaA (A. Wada, unpublished data), only a moderate decrease in the teicoplanin MIC (from 24 to 12 μg/ml) was observed, while no effect at all was detectable for vancomycin.

Influence of overexpression of tcaA on resistance.

The open reading frame of tcaA was cloned into the expression vector pMGS100, an Enterococcus faecalis-Escherichia coli shuttle vector that is also functional in S. aureus (14). Northern blot analysis confirmed that strains containing the construct produced highly elevated amounts of the tcaA transcript compared to the amounts produced by wild-type strains (results not shown). In general, overexpression of tcaA increased the levels of teicoplanin susceptibility in all strains analyzed (Table 2), especially in derivatives harboring a tcaA mutation, suggesting that overexpression of tcaA confers teicoplanin hypersusceptibility. This trend toward hypersusceptibility was specific for teicoplanin but not for vancomycin, for which only the complementation effect was observable. Interestingly, tcaA overexpression also reduced the oxacillin MICs from 256 μg/ml to 64 to 32 μg/ml for highly and homogeneously methicillin-resistant strain COL and the other methicillin-resistant S. aureus strains tested, whereas it did not affect the oxacillin resistance levels of methicillin-susceptible S. aureus strains BB938 and SA137/93G.

TABLE 2.

Complementation by tcaA overexpression

Strain MIC (μg/ml)a
Teicoplanin
Vancomycin
pMGS100 pMGS100-tcaA pMGS100 pMGS100-tcaA
COL 4 2 3 3
BB1372 16 2 6 3
BB938 24 12 4 4
MI 32 6 12 6
SA137/93A 6 3 4 4
SA137/93G 32 4 8 4
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a

The media used for MIC determinations were supplemented with chloramphenicol. MICs were determined by E-test with a 2 McFarland inoculum on BHI agar.

Combination of inactivation of tcaA with σB activity.

We previously noted that the effect of the tcaRAB deletion on the glycopeptide resistance level is strain dependent, being significantly higher in strain COL than in strain BB255 (7). Derivatives of the widely used laboratory strain S. aureus NCTC8325, such as strain BB255, are known to carry a mutation in rsbU, which codes for a positive regulator of the alternative sigma factor σB (16). As a consequence, the σB activities of those strains are significantly lower than those of rsbU+ strains. cis complementation of NCTC8325 derivative BB255 with the rsbU+ allele from strain COL was shown to restore the activity of σB to levels found in strains carrying an intact sigB operon and to result in a slight decrease in teicoplanin susceptibility (3). To investigate whether a wild-type level of σB activity contributes positively to the glycopeptide resistance level of a tcaA mutant, we inactivated tcaA in BB255 and in its rsbU+ complemented derivative GP268 by transducing Ω2026tcaA::Tn917, yielding strains BB1380 and BB1440, respectively. While inactivation of tcaA in BB255 raised the teicoplanin MIC from 6 to only 8 μg/ml, corroborating previous findings (9), a clear increase, from 8 to 24 μg/ml, was observed for GP268 tcaA derivative BB1440. This increase was comparable to that for the tcaRAB deletion mutant of strain COL.

Expression of tcaA.

A previous report (7) proposed that tcaA forms part of an operon composed of the three genes tcaR, tcaA, and tcaB. Under normal growth conditions, weak transcripts hybridizing with the tcaA- and tcaR-specific probes suggested, according to their size, a background transcription of the three genes, which could be confirmed by reversed transcription-PCR (unpublished data). However, the transcriptional organization of these genes appears to be complex, with several bands that could not yet be assigned to the tcaA- and tcaR-specific probes found to hybridize, as indicated in Fig. 2A. When cells were stressed with teicoplanin, a strong tcaA-specific signal appeared (Fig. 2B), but this signal was not detected with either tcaR- or tcaB-specific probes (data not shown). The increase in tcaA transcript abundance, while not as large as that observed in strains containing the overexpression plasmid (data not shown), indicated that teicoplanin-mediated stress has the ability to induce the expression of a monocistronic tcaA transcript.

FIG. 2.

FIG. 2.

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Northern blot analysis of tcaRAB transcription. (A) Transcripts hybridizing to the probes specific for tcaR, tcaA, and tcaB; the positions of the probes within the corresponding open reading frames are shown in Fig. 1. Total RNA was harvested from COL at growth stages corresponding to the OD600 shown. (B) Total RNA extracted from COL, uninduced (−) and induced (+) with teicoplanin, probed with tcaA. Each lane contains 10 μg of RNA. The positions of 16S rRNA (1.5 kb) and 23S rRNA (2.9 kb) and the approximate sizes (in nucleotides) of potential transcripts are shown, followed by the putative compositions of the transcripts in parentheses. As an indication of RNA loading, the 16S rRNA band from the ethidium bromide-stained gel is shown.

Population analysis profiles.

Two to four first-step mutants of strains COL, BB1372, and Mu3 growing on plates containing 4 μg of vancomycin per ml were isolated and purified on drug-free plates, which prevented the selection of potential further mutations, to obtain single colonies. One representative of each strain, termed COL-4, BB1372-4, and Mu3-4, respectively, were monitored by population analysis profiling. The population analysis profile of tcaRAB mutant BB1372 showed, as was shown for strain Mu3, a typical hGISA resistance profile in the presence of vancomycin, which significantly differed from that of its parental strain, COL (Fig. 3). Interestingly, stepwise-selected mutants BB1372-4 and Mu3-4 showed higher levels of resistance to vancomycin than their respective parental strains; however, COL-4 retained the resistance profile of the parental strain.

FIG. 3.

FIG. 3.

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Population analysis profiles. Resistance profiles represent the number of CFU from an overnight culture plated on increasing concentrations of vancomycin after 48 h of incubation at 35°C.

tcaA sequences of clinical GISA strains.

We investigated the tcaA gene sequences of six clinical GISA isolates (isolates MI, HIP06297, HIP09143, HIP09433, LIM-2, and SA137/93A) and strain SA137/93G, a spontaneous derivative of strain SA137/93A which had become more glycopeptide resistant than its parent (33). In this collection of GISA strains, two strains had a mutated tcaA gene, as shown in Fig. 4: a premature stop codon resulting from the insertion of an adenine residue was identified in strain MI, and insertion of an insertion sequence (IS) element, IS256, was identified in strain SA137/93G. The mutation in strain MI generated a stop codon, truncating TcaA after the first N-terminal third of the protein. In SA137/93G, the IS256 insert was flanked by the characteristic 8-bp duplication accompanying the transposition of this IS element (27). The parent strain, SA137/93A, carried no insertion in the tcaA gene. The pronounced reductions in the levels of vancomycin and teicoplanin resistance after the introduction of pMGS100-tcaA confirmed the functional defect of TcaA in both MI and SA137/93G (Table 2).

FIG. 4.

FIG. 4.

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tcaA inactivation in GISA strains. The nucleotide sequence resulting from a nucleotide insertion in strain MI is shown in the box. The insertion of an additional adenine nucleotide, highlighted in bold, generated a stop codon (asterisk). The position of the IS256 insertion in strain SA137/93G is shown. This was accompanied by an 8-bp duplication at both ends of the element. The arrow indicates the direction of tnp within IS256. Numbers indicate the nucleotide positions from the 5′ terminus of tcaA.

DISCUSSION

In contrast to the mechanism of resistance of recently isolated vanA-based vancomycin-resistant S. aureus (9), intermediate glycopeptide resistance is endogenous and is acquired in a stepwise fashion by the accumulation of chromosomal mutations, each of which contributes to a two- to fourfold increase in MICs. The first steps reducing the susceptibility to glycopeptides usually occur unnoticed, since only a small subpopulation of such mutants is able to grow at higher glycopeptide concentrations. The number of cells analyzed in standard susceptibility tests is too small to ensure the inclusion of this subpopulation. hGISA strains and their characteristic heterogeneous glycopeptide resistance can be visualized reliably only by population analysis profiles. As the mutations accumulate, changes in levels of resistance are multiplied. They appear larger when preexisting glycopeptide resistance-promoting mutations are present. For instance, when BB1372, which harbors the tcaA deletion, was exposed to a given concentration of vancomycin, the glycopeptide MIC for the resulting subclones was significantly increased, whereas the COL-derived clones retained their parental resistance levels.

The effect of the tcaA deletion on resistance was previously found to be strain specific (7), with deletion of tcaA in COL resulting in a much more pronounced increase in the MIC than deletion of tcaA in BB255. A key difference in the genetic backgrounds of these two strains is the level of expression of the alternate sigma factor σB, which is significantly decreased in BB255 due to inactivation of rsbU, a positive regulator of σB activity. When the tcaA deletion was introduced into GP268, a derivative of BB255 containing an intact σB operon, the increase in teicoplanin resistance was almost identical to that seen upon tcaA deletion in COL. This suggests that levels of σB expression can be a factor contributing to the variability of resistance levels in strains with different genetic backgrounds, at least with respect to the resistance conferred by tcaA inactivation. However, microarray data obtained for a wild-type laboratory strain compared with that obtained for a σB mutant (M. Bischoff, submitted for publication) suggested that tcaA was not under the control of σB.

Exposure to vancomycin induces multiple changes in the staphylococcal transcriptome in both the glycopeptide-susceptible S. aureus (24) and the GISA (29) backgrounds. However, expression of few of these differentially regulated genes has been demonstrated experimentally to correlate with increased glycopeptide resistance, such as overexpression of PBP 2 or the VraRS two-component signal transducer (25). We add to this list TcaA, a transmembrane protein with a predicted intracellular N-terminal metal-binding motif and a large extracellular domain. Its biochemical functions still need to be determined, but it is thought to be involved in some way in cell wall metabolism and may act as a sensor and/or signal transducer. TcaA has been reported to be upregulated in the presence of vancomycin (24) and oxacillin (38) and was shown here to be strongly upregulated by teicoplanin. However, in contrast to other described resistance markers, resistance increases when the TcaA protein is inactivated. Interestingly, while tcaA trans-complementation from its own promoter was efficient in tcaA mutants, it had only a minor effect on the teicoplanin resistance levels of strains with a functional tcaA allele. However, overexpression of tcaA was more effective in reducing the teicoplanin resistance level than expressing tcaA from it own promoter, suggesting that the level of TcaA produced might influence the degree of susceptibility. The rather complex regulation of tcaA, expected from the various transcripts seen in Northern blots, may indicate that it is under a complex control.

The induction of tcaA transcription in the presence of cell wall-active antibiotics would therefore theoretically increase the susceptibilities of these strains to teicoplanin. This may create a selective pressure for the inactivation of TcaA in the presence of glycopeptides.

In a preliminary screen of a small number of clinical GISA isolates, strain MI was found to carry a point mutation that truncated TcaA. Strain SA137/93G was also found to have gained teicoplanin resistance as a result of an IS256 insertion into the tcaA gene. This is one of a number of examples in which IS256 has been shown to drive staphylococcal virulence and resistance formation. IS256 is known to be intimately involved in modulation of the ica genes causing phase variation of virulence (41). It was also shown to activate the llm gene through its outward-pointing promoter (27) and to regulate the expression of the mecA gene in a Staphylococcus sciuri strain (11).

Overexpression of tcaA in both SA137/93G and MI was found to decrease significantly the teicoplanin and vancomycin MICs, thereby proving that glycopeptide resistance resulting from tcaA inactivation, a phenotype originally identified by in vitro experimentation, is also of clinical relevance.

Two major tasks remain to provide answers to lingering questions. One is to determine the prevalence of tcaA mutations among all clinical isolates. The other is the unraveling of the physiological function of TcaA and its relationship with the other factors reported to play a role in glycopeptide resistance.

Acknowledgments

We are grateful to G. Bierbaum, Universität Bonn, for the generous gift of S. aureus SA137/93A and SA137/93G and to S. Fujimoto, Gunma University School of Medicine, for kindly providing pMGS100. We thank E. Huf for technical assistance with the susceptibility tests.

Strains NARSA-2, -3, -17, -24, -27, and -36 were provided by the network on antimicrobial resistance in Staphylococcus aureus (http://www.narsa.net/content/default.jsp). This study was supported by the Swiss National Science Foundation grant (NRP 4049-063201).

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