INTRODUCTION

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Glycopeptide antibiotics vancomycin and teicoplanin inhibit cell wall synthesis by forming complexes with the peptidyl-D-alanyl-D-alanine (D-Ala-D-Ala) termini of peptidoglycan precursors at the cell surface (33). Acquired resistance to these antibiotics is mostly due to two types of gene clusters, designated vanA and vanB, that confer resistance by the same mechanism and encode related enzymes (Fig. 1) (10). In both cases, resistance is due to synthesis of peptidoglycan precursors ending in the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac) that bind to glycopeptides with reduced affinity (15, 17). The vanA and vanB clusters comprise three genes (vanHAX and vanH_BBX_B) that are necessary and sufficient for resistance (9) and appear to originate from glycopeptide-producing bacteria (32). These genes encode a dehydrogenase (VanH or VanH_B) that reduces pyruvate into D-Lac, a ligase (VanA or VanB) that synthesizes D-Ala-D-Lac, and a D,D-dipeptidase (VanX or VanX_B) that hydrolyzes D-Ala-D-Ala. The combined action of these three enzymes results in incorporation of D-Ala-D-Lac instead of D-Ala-D-Ala into peptidoglycan precursors (8). In addition, the vanA and vanB clusters encode a D,D-carboxypeptidase (VanY or VanY_B) that contributes to glycopeptide resistance by cleaving the C-terminal D-Ala of late membrane-bound peptidoglycan precursors (3). The latter precursors originate from incomplete hydrolysis of D-Ala-D-Ala by the VanX or VanX_B D,D-dipeptidases (34). Finally, the vanA and vanB clusters also contain two genes of unknown function (vanZ and vanW, respectively) that do not have significant sequence similarity to previously sequenced genes or to each other (17). The VanZ protein was found to confer low-level resistance to teicoplanin only (6), whereas the role of VanW in resistance has not been investigated.

Expression of the resistance genes of the vanA and vanB clusters is regulated by the VanRS and VanR_BS_B two-component regulatory systems, each composed of a membrane-associated sensor kinase (VanS or VanS_B) and a cytoplasmic response regulator (VanR or VanR_B) that acts as a transcriptional activator (Fig. 1) (9, 17). The regulatory and resistance genes are transcribed from distinct promoters that appear to be coordinately regulated. Namely, the VanRS regulatory system controls transcription of the vanRS and vanHAXYZ operons at the P_R and P_H promoters (5). Likewise, the VanR_BS_B system controls transcription of the vanR_BS_B and vanY_BWH_BBX_B operons at the P_RB and P_YB promoters (17, 35). The present review is focused on how the regulation of these promoters determines inducible expression of VanA- and VanB-type glycopeptide resistance. Background information on regulation of bacterial genes by two-component regulatory systems can be found elsewhere (25).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Events leading to transcriptional activation of the vanA and vanB gene clusters. The Van sensors contain two trans-membrane segments (TMS) that delineate an external loop (EL). The sequence of these regions of VanS and VanSB are not related, as is found for sensors that recognize different signals (25). A linker connects the membrane-associated domain to the catalytic cytoplasmic domain (hatched). The catalytic domain is conserved in all sensors and contains a conserved histidine residue (H) which is autophosphorylated. The phosphoryl group (P) is then transferred to an aspartate residue (D) of the effector domain of VanR or VanRB. The effector domain is also conserved in all response regulators (25). Phosphorylation of the effector domain activates the DNA binding domain leading to increased affinity for the regulatory regions of the target promoters. The DNA binding domains of VanR and VanRB are related to those of response regulators belonging to the OmpR subfamily (25).

	OVERVIEW OF EVENTS THAT LEAD TO INDUCTION OF RESISTANCE

The membrane-associated domains of VanS and VanS_B are thought to sense the presence of glycopeptides in the culture medium by an unknown mechanism (Fig. 1). The signal is transduced to the cytoplasmic catalytic domain of the sensors, leading to kinase stimulation, autophosphorylation of a specific histidine residue, and transfer of the phosphoryl group to a specific aspartate residue of the partner response regulators. The phosphorylated regulators bind to the regulatory region of the target promoters, resulting in transcriptional activation of the regulatory and resistance genes. Ultimately, induction brings about a switch from synthesis of precursors ending in D-Ala-D-Ala to precursors ending in D-Ala-D-Lac.

	INDIVIDUAL COMPONENTS OF SIGNAL TRANSDUCTION PATHWAY IN VANA-TYPE ENTEROCOCCI

Phosphotransfer reactions catalyzed by VanS and VanR in vitro. VanR and the cytoplasmic kinase domain of VanS fused to the maltose binding protein have been overerproduced in Escherichia coli, purified, and assayed for phosphotransfer reactions (40). VanS was found to catalyze ATP-dependent autophosphorylation of a histidine residue. Upon addition of VanR, the phosphoryl group was transferred from the phosphohistidine residue of VanS to an aspartate residue of the response regulator. In the absence of VanS, dephosphorylation of the phosphorylated form of VanR (phospho-VanR) was extremely slow compared to that of related response regulators, with a half-life of 10 h. VanS stimulated this reaction, indicating that the sensor has a so-called phosphatase activity.

In vitro binding of VanR to the regulatory region of P_R and P_H promoters. VanR and phospho-VanR bind to a similar 80-bp region of the regulatory region of the P_H promoter that contains two putative 12-bp VanR-binding sites (26) (Fig. 2A). The P_R promoter contains a single 12-bp binding site, and phosphorylation of VanR increases the size of the protected region from 20 to 40 bp. Phosphorylation of VanR increases the affinity of the response regulator for both promoters (Fig. 2B). The affinity is higher for P_H than for P_R, and the difference is more important for phospho-VanR than for VanR. These observations suggest that phospho-VanR may cooperatively bind to the P_H promoter.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Regulation of the PR and PH promoters. (A) Schematic representation of the vanA gene cluster showing details of the regulatory region of PR and PH. An amplification loop results from binding of phospho-VanR to PR, increased transcription of vanR, and accumulation of phospho-VanR following phosphorylation (4). (B) Determination of the effective concentrations of phospho-VanR and VanR required to saturate at 50% (EC50) DNA fragments carrying the PR and PH promoters based on gel shift experiments indicated that phosphorylation increases the affinity of the response regulator for DNA at both promoters and that the affinity is higher for PH than for PR (26). In spite of these differences, the PR and PH promoters were found to be coordinately regulated in vivo (C) and to have a similar strength (5).

In vivo activation of the P_R and P_H promoters in Enterococcus faecalis. Activation of the P_H and P_R promoters has been studied using various types of transcriptional fusions with reporter genes. Parallel determinations of D,D-dipeptidase activity and of the cytoplasmic pool of peptidoglycan precursors showed that expression of glycopeptide resistance is mainly, if not exclusively, regulated at the level of transcriptional initiation at these promoters (8). The P_H and P_R promoters were found to have a similar strength and to be similarly regulated (Fig. 2C) (5). The promoters are inactive in the absence of VanR and VanS, constitutively activated by VanR in the absence of VanS, and inducible by glycopeptides if the host produces both VanR and VanS. These results indicate that VanR is a transcriptional activator required for initiation at both promoters. VanS is not necessary for full activation of the promoters, because VanR can be activated (phosphorylated) independently from its partner sensor, presumably by a heterologous kinase encoded by the host chromosome. In contrast, VanS is required for negative control of the promoters in the absence of glycopeptides. The sensor is therefore thought to act as a phosphatase under noninducing conditions, preventing accumulation of phospho-VanR.

In vivo evidence for phosphatase activity of VanS. Sensor kinases of two-component regulatory systems display a highly conserved amino acid motif, called the H box, that contains the phosphorylated histidine residue (Fig. 1) (25). Replacement of this histidine by glutamine abolishes autophosphorylation of the sensors and, consequently, transfer of phosphoryl groups to the response regulators (27). However, the modified sensors retain phosphatase activity. Production of VanSH₁₆₄Q, i.e., VanS which carries a histidine (H)-to-glutamine (Q) substitution at the putative autophosphorylation site (position 164), prevented transcriptional activation in the absence of glycopeptides, indicating that the protein was acting as a phosphatase (Table 1) (4). VanSH₁₆₄Q also prevented transcription of the resistance genes in the presence of glycopeptides, suggesting that the phosphatase activity of this protein was not negatively modulated by the antibiotics. Negative control of VanR by VanS₁₆₄Q confirmed that the phosphorylated form of the response regulator is responsible for transcriptional activation.

Amplification of signal. As indicated above, phospho-VanR binds to the P_R promoter and activates transcription of the vanR and vanS genes. Thus, regulation of the vanA gene cluster involves not only a modulation of the relative concentration of VanR and phospho-VanR by the kinase and phosphatase activities of VanS but also a modulation of the absolute concentration of the response regulator (4, 5). An amplification loop results from binding of phospho-VanR to the P_R promoter, increased expression of vanR, and accumulation of phospho-VanR following phosphorylation (Fig. 2A). This may account for the high-level transcription of the resistance genes observed in vanS null mutants, since the amplification loop, in combination with the long half-life of phospho-VanR, may compensate for inefficient phosphorylation of the response regulator by the putative host kinase (5).

	ACQUISITION OF TEICOPLANIN RESISTANCE BY VANB-TYPE ENTEROCOCCI

Enterococci harboring clusters of the vanB class remain susceptible to teicoplanin, since this antibiotic does not trigger induction of the resistance genes (8, 17). However, teicoplanin selects mutants resistant to this antibiotic that belong to one of three phenotypic classes (11, 12) due to three distinct alterations of VanS_B functions (13, 14) (Table 1). Mutations leading to teicoplanin resistance also confer low-level resistance to the investigational glycopeptide LY333328 (7).

Inducible phenotype. Mutations leading to inducible expression of vancomycin and teicoplanin resistance introduced amino acid substitutions in the sensor domain of VanS_B (13). A minority of the substitutions were located between the two putative trans-membrane segments of VanS_B (Fig. 1). This portion of the sensor is located at the outer surface of the membrane and may therefore interact directly with ligands, such as glycopeptides, which do not penetrate into the cytoplasm. The majority of the substitutions were located in the linker that connects the membrane-associated domain to the cytoplasmic catalytic domain. Based on analogies with related sensor kinases, these substitutions may affect signal transduction (13). The analysis of the inducible mutants provides experimental evidence that the N-terminal domain of VanS_B is involved in signal recognition and also provides one of the rare examples of modification of specificity of a sensor kinase.

Constitutive phenotype. Mutations responsible for constitutive expression of the vanB cluster led to amino acid substitutions at two specific positions located on either side of the histidine at position 233, which corresponds to the putative autophosphorylation site of VanS_B (13) (Table 1). Constitutive expression of glycopeptide resistance is most probably due to impaired dephosphorylation of VanR_B by VanS_B, as similar substitutions affecting homologous residues of related sensor kinases impair the phosphatase but not the kinase activity of the proteins (13).

Heterogeneous phenotype. The heterogeneous mutants most probably harbor null alleles of vanS_B since the mutations introduced translation termination codons at various positions of the gene (Table 1). The antibiotic disk diffusion assay revealed the presence of inhibition zones containing scattered colonies that grew predominantly in 48 h (11). Since this phenotype suggested the presence of a mixture of susceptible and resistant bacteria, the P_YB promoter (Fig. 1) was fused to a green fluorescent protein reporter gene in order to analyze gene expression in single cells based on fluorescence microscopy and flow cytometry (14). Use of this reporter system revealed that cultures of heterogeneous mutants grown in the absence of glycopeptides contain a major nonfluorescent subpopulation and a smaller sub population with various fluorescence intensities. Thus, VanR_B was activating transcription of the resistance genes only in a minority of the bacteria. Attempts to grow fluorescent and nonfluorescent bacteria as pure cultures were unsuccessful, indicating that expression of the resistance genes was reversibly turned on and off under noninducing conditions, a phenomenon referred to as clonal variation (39). Vancomycin and teicoplanin induced expression of the reporter gene in bacteria of the major nonfluorescent subpopulation (14). These observations imply that clonal variation and induction both result from activation of VanR_B by a host kinase which is stimulated by glycopeptides.

In vivo emergence of teicoplanin resistance. Teicoplanin-resistant mutants have been observed in a patient (24) and in rabbits with experimental endocarditis treated with vancomycin or teicoplanin (11). In the animal model, the combination of gentamicin and teicoplanin was effective in reducing the number of surviving VanB-type E. faecalis organisms in cardiac vegetations and prevented the emergence of resistant mutants (11). Subsequent work revealed that two mutations were required to abolish the synergistic activity of teicoplanin and gentamicin against wild VanB-type strains: an initial mutation that allows expression of teicoplanin resistance by one of the three mechanisms discussed above and a second mutation conferring a moderate (two to fivefold) increase in the intrinsic level of gentamicin resistance (30). Simultaneous acquisition of the two types of mutations is probably a rare event that was not observed in the animal or in vitro (30). These laboratory investigations suggest that emergence of teicoplanin resistance may be responsible for therapeutic failure, although the use of drug combinations may prevent the selection of resistant mutants (11, 30).

Acquisition of teicoplanin resistance in two steps. VanB-type enterococci requiring vancomycin for growth (vancomycin-dependent phenotype) are readily selected by vancomycin in vitro (13), in a rabbit model of aortic endocarditis (11), and in patients (20, 37). These mutants harbor mutations in the host D-Ala-D-Ala ligase gene and depend entirely on the VanB ligase for peptidoglycan synthesis (13). Impaired D-Ala-D-Ala ligase activity accounts for vancomycin dependence, since the VanB protein is only produced under conditions of induction by this antibiotic. Impaired D-Ala-D-Ala ligase also results in increased resistance to vancomycin, since incorporation of D-Ala-D-Ala into peptidoglycan precursors is abolished (13). Mutations in vanS_B that lead to constitutive expression of the resistance genes are readily selected in vancomycin-dependent bacteria, not only on teicoplanin, but also on media devoid of antibiotics, since such mutations allow growth in the absence of the inducer (13, 37). Thus, vancomycin may indirectly select for constitutive teicoplanin resistance in two steps. This may account for the emergence of teicoplanin resistance in a patient treated with vancomycin (24) and may also account for constitutive expression of vanB-related gene clusters in clinical isolates of E. faecalis that harbor null mutations in the ddl D-Ala-D-Ala ligase gene (16).

	CROSS TALK AND REGULATION IN HETEROLOGOUS HOSTS

The chromosome of most bacteria encodes several related two-component regulatory systems, and analysis of the unfinished E. faecalis genome sequence available from The Institute for Genomic Research reveals the presence of at least 20 pairs of sensor kinases and response regulators. Sequence conservation in the kinase domain of the sensors and in the effector domain of response regulators can allow inefficient phospho-transfer reactions between nonpartner sensors and response regulators (25, 38). In vivo, such cross-reactivities (cross talk) usually lead to activation of response regulators by heterologous kinases in mutants that do not produce the partner sensor kinase. We shall compare activation of VanR and VanR_B by partner and nonpartner sensors in E. faecalis, E. coli, and Bacillus subtilis.

E. faecalis. Expression of the resistance genes in E. faecalis is inducible by glycopeptides in vanS_B and vanS null mutants if the latter harbor P_H on a multicopy plasmid (see above and Table 1) (4, 14). Induction occurs with the same set of antibiotics, indicating that the response regulators may be activated by the same kinase (Table 1) (4). These observations raise the possibility that E. faecalis harbors, as of yet, unidentified genes expressed under the control of this putative kinase in response to inhibition of peptidoglycan synthesis (4).

E. coli. The P_H and P_R promoters of the vanA gene cluster are specifically activated by VanR in E. coli (35). Likewise, the P_YB and P_RB promoters are specifically activated by VanR_B (35). Phosphorylation is required for promoter activation by both response regulators (22, 35). VanR and VanR_B belong to the OmpR subfamily of response regulators based on sequence similarity in the DNA binding domains of the proteins (9). Response regulators of this subfamily activate promoters that are recognized by the major form of RNA polymerase corresponding to E₇₀ in E. coli (25). Therefore, it is perhaps not surprising that there are the same requirements for promoter activation in distantly related bacteria such as E. coli and E. faecalis.

B. subtilis. Regulation of the P_H promoter was analyzed in B. subtilis hosts producing VanR, VanR and VanS, or neither (36). As expected, VanR was required for promoter activation. Glycopeptides induced P_H both in the presence and absence of VanS, although induction required higher drug concentrations in the absence of this sensor. Thus, activation of VanR by cross talk is regulated by glycopeptides in B. subtilis, indicating that this host produces a glycopeptide sensor as does E. faecalis. Of note, a glycopeptide-inducible transcriptional fusion was obtained by screening random insertions of a Tn917 derivative carrying a promoterless lac reporter gene in B. subtilis (28). Expression of the transcriptional fusion might be controlled by the kinase responsible for activation of VanR_B in the absence of VanS_B.

Cross talk between VanRS and VanR_BS_B regulatory systems. Production of the VanS sensor restored homogeneous high-level glycopeptide resistance of a heterogenous vanS_B null mutant and significantly increased synthesis of the VanX_B D, D-dipeptidase (4). These observations suggest that VanS can phosphorylate VanR_B. Activation of VanR_B by VanS could not be tested in E. coli since cross talk led to high-level activation of this response regulator (35).

	NATURE OF THE STIMULUS

The nature of the signal recognized by the VanS and VanS_B sensors is unknown. In fact, it has never been established that signal recognition involves a direct interaction between the sensors and a specific ligand such as glycopeptides or peptidoglycan precursors. Such specific interactions may not exist if signal recognition depends upon a physical constraint imposed on the membrane by the inhibition of peptidoglycan synthesis (36). The issue was addressed indirectly by determining which compounds, mostly antibiotics, trigger induction. There are discrepancies between the conclusions of different groups that used different reporter systems in different hosts, although there is a consensus that the vanA gene cluster is inducible by subinhibitory concentrations of all antibiotics that inhibit the transglycosylation reaction, i.e., the transfer of disaccharide-pentapeptide subunits from lipid intermediate II to the nascent peptidoglycan at the outer surface of the membrane. These antibiotics include glycopeptides and moenomycin, which are structurally unrelated and inhibit the transglycosylases by different mechanisms (12, 23).

Three groups found that VanA-type resistance is inducible by glycopeptides and moenomycin but not by drugs that inhibit the reactions preceding (e.g., ramoplanin) or following (e.g., bacitracin and penicillin) transglycosylation (12, 23, 31). This narrow specificity suggests that accumulation of lipid intermediate II, resulting from inhibition of transglycosylation, may be the signal recognized by the VanS sensor. This would account for induction by antibiotics that inhibit the same step of peptidoglycan synthesis but have different structures (12, 23). In contrast, the VanS_B sensor may interact directly with vancomycin since teicoplanin is not an inducer (12). Amino acid substitutions in the sensor domain of VanS_B allowed induction by teicoplanin but not by moenomycin (see above and Table 1) (12, 13). The primary amino acid sequence of the sensor domains of VanS and VanS_B are unrelated (17). These observations indicate that VanS and VanS_B may sense the presence of glycopeptides by different mechanisms (13).

Two groups reported induction of VanA-type resistance by inhibitors of late (e.g., glycopeptides, moenomycin, bacitracin, and ramoplanin) but not of early (e.g., fosfomycin and D-cycloserine) stages of peptidoglycan synthesis (1, 21). The broader specificity observed by these authors supports the concept that inhibition of peptidoglycan polymerization is critical for induction but does not imply that accumulation of a specific precursor is involved.

Lastly, one group analyzed regulation of the P_H promoter in B. subtilis instead of in enterococcal hosts and reported induction by bacitracin, penicillin, fosfomycin, and D-cycloserine as well as by treatment with lysosyme, mutanolysin, and lysostaphin (36). The authors concluded that it is unlikely that VanS is stimulated by the accumulation of peptidoglycan precursors.

Attempts to correlate antibacterial activity and induction using a large number of glycopeptide antibiotics indicated that binding to the peptidyl-D-Ala-D-Ala target and structural features of the antibiotic may both be important for induction (21). This observation may imply that regulation involves recognition of more than one signal (21) but is also compatible with the possibility that VanS interacts with a lipid intermediate II-glycopeptide complex at the outer surface of the membrane. Of note, two large-scale screenings identified 0.1% (29) and 1% (31) of inducers among 6,800 and 8,000 compounds, respectively, revealing induction of VanA-type resistance by a few additional compounds, including some membrane-active agents such as polymyxin B.

In vivo analysis is clearly not sufficient to associate an inducing signal with a specific kinase, since response regulators may be stimulated by cross talk. For example, it is possible that activation of VanR in response to moenomycin in wild VanA-type strains depends not upon a modification of the signaling status of VanS but upon stimulation of the putative heterologous kinase that was revealed by the analysis of vanS null mutants (Table 1). Cross talk is expected to vary in different hosts, and this may account for some of the discrepancies concerning the induction specificity of the VanS sensor.

	CONCLUSIONS

Top Introduction Conclusion References

Several aspects of regulation of the vanA and vanB gene cluster, including the phosphotransfer reactions (Fig. 1) and promoter activation (Fig. 2), are well characterized based on in vivo analysis of the impact of mutations in the regulatory genes (Table 1) and in vitro analysis of protein functions. In contrast, our understanding of signal recognition remains limited. Likewise, the importance of cross talk in regulation of glycopeptide resistance has been revealed by numerous analyses, although host factors involved in this process remain to be identified. Detection of inducible expression of glycopeptide resistance genes in the absence of the VanS or VanS_B sensors suggests that regulation of the vanA and vanB cluster may be more integrated in host regulation than was initially anticipated. This observation also suggests that specific host genes may be turned on in response to glycopeptides in susceptible bacteria.

Acquisition of teicoplanin resistance by VanB-type strains, in particular constitutive expression of the resistance genes associated with impaired host D-Ala-D-Ala ligase activity, appears to be a potential trend in the evolution of VanB-type resistance. This may ultimately lead to exclusive production of D-Lac-ending precursors in enterococci. However, production of such precursors is associated with hypersusceptibility to -lactam antibiotics in some enterococci, presumably because certain low-affinity penicillin binding proteins are unable to efficiently function with D-Lac-ending precursors (2, 13). This could contribute to counterselection of bacteria expressing glycopeptide resistance constitutively.