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Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15,1 Université Paris VI, INSERM U655-Laboratoire de Recherche Moléculaire sur les Antibiotiques, Paris,2 Service de Microbiologie and EA 2128 Relations Hote et Microorganismes des Epitheliums, CHU Côte de Nâcre, Université de Caen-Basse Normandie, Caen,France3
SUMMARY INTRODUCTION REGULATION OF RESISTANCE EXPRESSION BY TWO-COMPONENT SYSTEMS IN GRAM-POSITIVE BACTERIA Two-Component Regulatory Systems Resistance to Glycopeptides in Enterococci Two-component regulatory systems in Van-type enterococci. Phosphotransfer reactions catalyzed by VanRS and VanRBSB two-component systems. In vitro binding of VanR and VanRB to promoter regulatory regions. VanRB-P recruits the RNA polymerase to the regulatory and resistance gene promoters. In vivo activation of the PR and PH promoters in VanA-type strains. Acquisition of teicoplanin resistance by VanB-type enterococci. (i) Inducible phenotype. (ii) Constitutive phenotype. (iii) Heterogeneous phenotype. Resistance to Glycopeptides in Staphylococcus aureus Resistance to ß-Lactams in Enterococcus faecalis Resistance by Efflux Resistance to quinolones in Staphylococcus aureus. Resistance to multiple drugs in gram-negative bacteria. (i) Acinetobacter baumannii. (ii) Stenotrophomonas maltophilia. (iii) Pseudomonas aeruginosa. (iv) Escherichia coli. ROLE OF IS ELEMENTS AND INTEGRONS IN THE MODULATION OF RESISTANCE GENE EXPRESSION Effects of IS Elements on the Expression of Resistance General characteristics of IS elements. IS-mediated effects on resistance-conferring and resistance-modulating genes. (i) Activation of resistance genes by promoter alterations. (ii) Disruption of resistance-modulating genes. Modulation of Resistance Gene Expression in Class 1 Integrons General characteristics of integrons. Transcriptional control of resistance gene expression in class 1 integrons. (i) Impact of the integron-borne promoter region. (ii) Impact of the cassette-borne 59-be. (iii) Transcription independent of integron-specific sequences. Translational control of gene expression in class 1 integrons. POSTTRANSCRIPTIONAL (TRANSLATIONAL) ATTENUATION Inducible Expression of Macrolide Resistance The erm(C) paradigm. Control of expression of other erm genes. Phenotypes of inducible MLSB resistance. Constitutive Expression of erm Genes Clinical Implications of Inducible MLSB Resistance What is the clinical evidence for failure of clindamycin treatment? Implications for the clinical microbiology laboratory. CONCLUSION ACKNOWLEDGMENTS REFERENCES
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| REGULATION OF RESISTANCE EXPRESSION BY TWO-COMPONENT SYSTEMS IN GRAM-POSITIVE BACTERIA |
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Glycopeptide resistance in enterococci results from the production of modified peptidoglycan precursors ending in D-Ala-D-Lac (VanA, VanB, and VanD) or D-Ala-D-Ser (VanC, VanE, and VanG), to which glycopeptides exhibit low binding affinities, and from the elimination of the high-affinity precursors ending in D-Ala-D-Ala and synthesized by the host Ddl ligase (17, 218). In enterococci with the VanA, VanB, or VanD phenotype, the synthesis of D-Ala-D-Lac requires the presence of a ligase (VanA, VanB, or VanD) of altered specificity compared to the host Ddl ligase and of a dehydrogenase (VanH, VanHB, or VanHD) that converts pyruvate to D-Lac (Fig. 2)(19). In VanC-, VanE-, and VanG-type strains, the ligase genes (vanC, vanE, or vanG) encode a protein catalyzing the synthesis of D-Ala-D-Ser (218), and the production of D-Ser is due to a membrane-bound serine racemase (VanT, VanTE, or VanTG) (Fig. 2) (1, 11, 63).
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Classification of glycopeptide resistance is based on the primary sequence of the structural genes for the resistance-mediating ligases. VanA-type strains display high-level inducible resistance to both vancomycin and teicoplanin, whereas VanB-type strains have variable levels of inducible resistance to vancomycin only, since teicoplanin is not an inducer (15, 211). VanD-type strains are characterized by constitutive resistance to moderate levels of both glycopeptides (66, 67). VanC, VanE, and VanG are resistant to low levels of vancomycin but remain susceptible to teicoplanin (63, 80, 135). VanC- and VanE-type strains are inducibly or constitutively resistant (2, 187). In several constitutive strains of these types, various mutations in VanS could, as in VanB-type strains, account for constitutivity (26, 65).
Although all six types of resistance involve genes encoding related enzymatic functions, they can be distinguished by the location of the genes and by the various modes of regulation of gene expression (Fig. 2). The vanA and vanB operons are located on plasmids or in the chromosome (20), whereas the vanD (42, 66, 67), vanG (63), vanE (1), and vanC (10) operons have so far been found exclusively in the chromosome.
Two-component regulatory systems in Van-type enterococci. Among the ubiquitous two-component systems that constitute one of the largest families of transcriptional regulators in bacteria, the VanS/VanR-type systems are the only ones that control the expression of genes that mediate antibiotic resistance. Expression of VanA-, VanB-, VanD-, VanC-, VanE-, and VanG-type resistance is regulated by a VanS/VanR-type two-component signal transduction system composed of a membrane-bound histidine kinase (VanS, VanSB, VanSD, VanSC, VanSE, or VanSG) and a cytoplasmic response regulator (VanR, VanRB, VanRD, VanRC, VanRE, or VanRG) that acts as a transcriptional activator (Fig. 2) (1, 10, 18, 42, 63, 66, 67, 77). In the vanA, vanB, vanD, and vanG operons, the genes for the two-component regulatory system (vanRS, vanRBSB, vanRDSD, and vanRGSG) are present upstream from the structural genes for the resistance proteins (20, 42, 63, 67), whereas in the vanC and vanE clusters, vanRCSC and vanRESE are located downstream (Fig. 2) (1, 10). The regulatory and resistance genes in the vanA, vanB, and vanD operons are transcribed from distinct promoters, PR, PRB, and PRD and PH, PYB, and PYD, respectively, that are coordinately regulated (13, 14, 43, 65, 77). The vanC and vanE clusters are cotranscribed from a single upstream promoter (Fig. 2) (1, 2, 187).
The vanRG and vanSG genes have the highest homology with vanRD and vanSD, respectively (Fig. 2). Additionally, vanUG encodes a predicted transcriptional activator (63), and a protein of this type has not previously been associated with glycopeptide resistance. Thus, as opposed to the other van gene clusters, the vanG operon contains three genes, vanUG, vanRG, and vanSG, for a putative regulatory system that are cotranscribed constitutively from the PUG promoter, whereas inducible transcription of the vanYG, vanWG, vanG, vanXYG, and vanTG resistance genes is initiated from the PYG promoter (Fig. 2) (63).
Phosphotransfer reactions catalyzed by VanRS and VanRBSB two-component systems. Despite the fact that the VanS/VanR and VanSB/VanRB two-component systems are only distantly related, they catalyze similar reactions. The two response regulators are 34% identical, whereas the histidine kinases possess only 23% sequence identity, with unrelated amino-terminal sensing domains (Fig. 2). VanS-type sensors comprise an N-terminal sensor domain with two membrane-spanning segments and a C-terminal cytoplasmic kinase domain (Fig. 1) (18, 269). Following a signal related to the presence of a glycopeptide in the culture medium, the cytoplasmic domain of VanS or VanSB catalyzes ATP-dependent autophosphorylation of a specific histidine residue at positions 164 and 233, respectively, and transfers the phosphate group to an aspartate residue at position 53 of VanR or VanRB present in the effector domain (Fig. 3) (13, 18, 269).
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In vitro binding of VanR and VanRB to promoter regulatory regions. There is sequence similarity between VanR and VanRB and response regulators of the OmpR/PhoB subclass in both the effector and DNA binding domains, with VanR being closer to OmpR (37% similarity) than to PhoB (35%), whereas VanRB is closer to PhoB (32% similarity) than to OmpR (26%). Phosphorylation of VanR and VanRB increases their DNA affinity, but VanR-P (112) appears to be more stable than VanRB-P (64). The promoters in the vanA and vanB operons have common features, with a single binding site in the PR and PRB promoters and two sites in the PH and PYB promoters (64, 112). However, the positionings of these sites in the promoter regions differ: in the case of VanR, the binding site is upstream from the –35 region (112), whereas it overlaps the –35 region for VanRB (Fig. 4) (64). The binding site is centered at –54.5 for VanR in PR and at –32.5 for VanRB in PRB. In the PH and PYB promoter regions, the sites are centered at –53.5 and –86.5 for VanR (112) and at –33.5 and –55.5 for VanRB (64), respectively. The two copies of the binding sites at PH and PYB are 33 bp (112) and 22 bp (64) apart, respectively, suggesting that since these figures differ almost exactly by three or two helical turns of B-DNA (10.5 bp/turn), they both lie on the same face of the DNA helix. VanR and VanRB bind with higher affinity to the corresponding PH and PYB promoters controlling the resistance genes than to the PR and PRB promoters for the regulatory genes (Fig. 4) (64). Phosphorylation increases the affinity for PH by 40-fold but increases the affinity for PYB by only 10-fold, indicating that the cooperativity is higher at PH than at PYB (Fig. 4) (64, 112). A direct relationship between the binding cooperativity of VanRB-P to its sites and the expression of the resistance genes may exist, since the levels of induction of the resistance genes are lower with VanRB than with VanR.
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A 21-bp consensus was identified within the binding regions of PRB and PYB, which consists of two and four direct repeats of the CTACAG(G/A)heptanucleotide, respectively (64). A similar organization has been observed in other response regulators such as CtsR (68) and PhoP (74, 270) from Bacillus subtilis and DcuR from Escherichia coli (3). The heptanucleotides, which correspond to the VanRB recognition sequence, are separated by four nucleotides, and at each site, the protected guanines are 10 bp apart and are thus positioned on the same face of the B-DNA helix (64). This tandem symmetry is consistent with the notion that VanRB binds to DNA as a head-to-tail dimer, as reported previously for PhoB (35). The consensus sequence of PRB and PYB is not present in the promoter regions of the other van operons. In contrast, sequence comparison of the PYG promoter, controlling the resistance genes in the vanG operon; the PYD promoter, controlling those of the vanD operon; and the PH promoter revealed a 12-bp consensus sequence, (T/C)CGTAXGAAA(T/A)T, similar to T(T/C)GTA(G/A)GAAA(T/A)T, corresponding to the regions protected by VanR and VanR-P in the vanA operon (112) that is present three times in the PYG region (63) and twice in the PYD region.
VanRB-P recruits the RNA polymerase to the regulatory and resistance gene promoters.
As mentioned above,
VanRB and VanRB-P bind specifically to
the same regions of the PRB and PYB promoters, and although not
essential for binding, phosphorylation of the regulator significantly
increases the affinity for the DNA targets
(64). Treatment with
acetylphosphate converts VanRB from a monomer with low
affinity for its binding site into a homodimer with higher DNA affinity
(64). Activation of gene
expression in vivo most likely requires the phosphorylation and
consequently the dimerization of VanRB to raise the binding
affinity to physiologically relevant levels. In order to switch on the
positive autoregulatory loop that leads to the expression of the
vancomycin resistance genes, a VanB-type strain needs to synthesize a
minimum number of VanRB and VanSB molecules even
in the absence of antibiotic. VanRB-P has a higher affinity
for its targets than VanRB and appears to be more
efficient than VanRB in promoting an open complex formation
with PRB and PYB
(64). The RNA polymerase
is able to interact with the PRB promoter region in
the absence or presence of VanRB but is able to interact
with PYB only in the presence of VanRB
and in both cases with an increased affinity when VanRB is
phosphorylated. In vitro transcription assays showed that
VanRB-P activates PYB more strongly than
PRB
(64). The higher affinity
of VanRB for PYB relative to
PRB may result from PYB having
two heptanucleotide direct repeats, possibly resulting in the
cooperative binding of the regulator to the two adjacent sites, which
may serve as recognition sites for VanRB and
VanRB-P binding. Although the regions protected by
VanRB and VanRB-P encompass the –35
regions of the promoters, VanRB-P is able to recruit the RNA
polymerase at the promoters and allows efficient open complex
formation. Unlike the situation with PhoB, the C-terminal domain of the
RNA polymerase 
subunit is required for transcription
activation from the PRB and PYB
promoters, possibly by making direct contact with the activator or by
being mandatory for promoter binding
(64).
In vivo activation of the PR and PH promoters in VanA-type strains. In VanA-type strains, the activation of the PR and PH promoters has been studied using various transcriptional fusions with reporter genes (13, 14). Determinations of D,D-dipeptidase activity and of the cytoplasmic pool of peptidoglycan precursors show that the expression of glycopeptide resistance is regulated at the level of transcriptional initiation at these promoters. The PR and PH promoters have similar strengths and are regulated similarly. They are not activated in the absence of VanR and VanS, are induced by glycopeptides when VanR and VanS are present, and are constitutively activated by VanR in the absence of VanS due, presumably, to phosphorylation of VanR by host kinases (13, 14). Consequently, VanR is a transcriptional activator required for initiation at both promoters, whereas VanS is not necessary for the full activation of the promoters since VanR can be phosphorylated independently of its partner sensor. However, VanS is required for negative control of the promoters in the absence of glycopeptides, acting as a phosphatase under noninducing conditions, thus preventing the accumulation of VanR-P. VanR-P binds to the PR promoter and activates the transcription of the vanR and vanS genes. Regulation of the vanA gene cluster therefore involves not only a modulation of the relative amounts of VanR and VanR-P by the kinase and phosphatase activities of VanS but also a modulation of the concentration of the response regulator. An amplification loop results from the binding of VanR-P to the PR promoter with a resultant increased expression of vanR and accumulation of VanR-P following phosphorylation. This may explain 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 VanR-P, may compensate for the inefficient phosphorylation of the response regulator by the putative host kinase.
Acquisition of teicoplanin resistance by VanB-type enterococci. As mentioned above, enterococci harboring clusters of the vanB class remain susceptible to teicoplanin since this antibiotic is not an inducer (15). However, mutations in the vanSB sensor gene have been obtained in vitro (26) and in vivo in animal models (21) following selection by teicoplanin, which have resulted in three phenotypic classes (constitutive, teicoplanin-inducible, or heterogeneous expression of the resistance genes) due to three types of alterations of VanSB function. Mutations leading to teicoplanin resistance also confer low-level resistance to the glycopeptide oritavancine (LY333328) (16). Derivatives of VanB-type strains that are resistant to teicoplanin have been isolated from two patients following treatment with vancomycin (103) or teicoplanin (125), but the isolates were not studied further.
(i) Inducible phenotype. Substitutions in the sensor domain of VanSB lead to inducible expression of resistance by vancomycin and teicoplanin (Fig. 5) (26). A minority of the mutations are located between the two putative transmembrane segments of VanSB. 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 are located in the linker that connects the membrane-associated domain to the cytoplasmic catalytic domain. The N-terminal domain of VanSB is thus involved in signal recognition and is associated with alterations of specificity that allow induction by teicoplanin but not by the nonglycopeptide moenomycin, which also inhibits the transglycosylation reaction (13, 25).
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(ii) Constitutive phenotype. In the VanS-type sensors, five blocks (H, N, G1, F, and G2) of the kinase domain are highly conserved (Fig. 5). The H block is responsible for both autophosphorylation and kinase/phosphatase activities, and G1 and G2 correspond to ATP binding blocks. Mutations responsible for constitutive expression of the vanB cluster result from amino acid substitutions at two specific positions located on either side of the histidine at position 233, which is the putative autophosphorylation site in VanSB (Fig. 5) (26). Constitutive expression of glycopeptide resistance is most probably due to impaired dephosphorylation of VanRB by VanSB, as similar substitutions affecting homologous residues of related sensor kinases impair the phosphatase but not the kinase activity of the proteins (26, 65). These observations confirm that dephosphorylation of VanRB is required to prevent the transcription of the resistance genes (13).
A VanB-type Enterococcus faecium strain that was resistant to vancomycin and susceptible to teicoplanin was isolated from a patient, and 2 weeks later, a derivative that was constitutively resistant to high levels of both glycopeptides was isolated from the same patient (65). Increased resistance in the derivative was shown to be due to the combination of a frameshift mutation leading to the loss of the Ddl ligase activity and the constitutive synthesis of pentadepsipeptide precursors by the loss of VanSB phosphatase activity following a six-amino-acid deletion, which partially overlaps the conserved G2 ATP-binding domain (Fig. 5) (65).
(iii) Heterogeneous phenotype. The heterogeneously resistant derivatives most probably harbor null alleles of vanSB since the mutations introduce translation termination codons at various positions in the gene (Fig. 5) (27). The antibiotic disk diffusion assay revealed the presence of inhibition zones containing scattered colonies of resistant bacteria that grew predominantly in 48 h (21, 27).
The overproduction of PBP2 significantly increases resistance to teicoplanin, whereas the reduction in teicoplanin resistance is observed in vraSR null mutants, which agrees well with a loss of PBP2 induction (97). PBP2 possesses transglycosylase activity that catalyzes the elongation of the nascent peptidoglycan chains (195). However, elongation of the chains is not completely abolished after the inactivation of the transglycosylase domain of PBP2, indicating that other transglycosylases also catalyze the elongation reaction. The VraSR system positively regulates the sgtA and sgtB glycosyltransferase genes. The deduced proteins show significant similarity with transglycosylase domains and, consequently, may be involved in glycopeptide resistance in S. aureus (109). It is considered that increased transglycosylase activity contributes to resistance either by competing with glycopeptides for the capture of the membrane-bound murein monomers or by increasing the production of nascent peptidoglycan chains to provide more D-Ala-D-Ala that serves as a false target for vancomycin. High copy numbers of the vraSR genes do not increase the transcription of pbp2 and sgtB and require the presence of cell wall synthesis inhibitors to induce the expression of the genes (131). This indicates that the signal that activates the VraS sensor kinase could be generated by the inhibition of cell wall synthesis.
L-Ala-L-Ala-Lys3
peptidoglycan cross-bridge. Thus, the CroRS two-component regulatory
system is essential for ß-lactam resistance mediated by PBP5 in
enterococci. However, CroRS is not required for the production of
low-affinity PBP5, suggesting that it controls other,
as-yet-unidentified, factors essential for the activity of this
low-affinity penicillin binding protein. Recently, to gain a more comprehensive view of the role of two-component signal transduction pathways in the biology of E. faecalis, each of the 18 response regulators previously identified in E. faecalis V583 was targeted by insertion mutagenesis (99). An insertion in croR led to susceptibility to the cephalosporins, bacitracin, and vancomycin despite the presence of a functional vanB operon in strain V583. CroR is thus involved in resistance to a wide range of cell wall-active agents, indicating that this system may have a role in the regulation of cell wall synthesis.
The envelope of gram-negative bacteria comprises two membranes, the inner or cytoplasmic membrane and the outer membrane, which are separated by the periplasmic space, whereas gram-positive bacteria possess a single membrane. The membrane-located transporters can be grouped into the following five families based on sequence homology, mechanisms, and molecular characteristics: the ATP binding cassette (ABC) family, the major facilitator superfamily (MFS), the multidrug and toxin extrusion family, the resistance-nodulation-division (RND) family, and the small multidrug resistance (SMR) family (Fig. 6). In gram-negative bacteria, the efflux machinery is complex, comprising a cytoplasmic membrane-located transporter, a periplasmic membrane adaptor protein, and an outer membrane channel protein. Genomes of gram-negative bacteria usually encode multiple members of each family of multidrug transporters (192). To date, only the ABC, MFS, and SMR families have been described in gram-positive organisms.
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Resistance to quinolones in Staphylococcus aureus. NorA was the first chromosomally encoded S. aureus pump to be identified. Based on its sequence, the cloned norA gene of a fluoroquinolone-resistant clinical strain was predicted to encode a typical MFS-type protein with 12 membrane-spanning alpha helices. NorA has the highest degree of identity with the Bmr MFS pump of Bacillus subtilis (44%) and only 20 to 25% identity with several tetracycline-specific efflux proteins of gram-negative bacteria (121). Cloning of norA in a plasmid in either S. aureus or E. coli results in fluoroquinolone resistance, particularly to hydrophilic molecules. NorA has a broad substrate specificity, including hydrophilic fluoroquinolones, biocides, and dyes. In addition, the substrates of NorA are typical of those of MDR pumps, namely, amphipathic cations. NorA activity is inhibited by reserpine, a compound known to act as an inhibitor of the function of many MDR efflux proteins. Resistance associated with NorA occurs only when the structural gene for this protein is either amplified or overexpressed as a result of regulatory mutations (121).
Regulation of NorA expression depends on at least two systems, ArlRS and MgrA (formerly NorR) (83, 84, 255). MgrA is composed of 147 residues, has modest similarity with other regulatory proteins such as MarR in E. coli and SarR in S. aureus, and, when overexpressed, causes increased expression of norA. It binds upstream from the norA promoter, and experimental data suggest that repeats of the TTAATT consensus sequence may be involved in the binding of this protein (255). Four such hexamers are located upstream from the –35 motif of the norA promoter. MgrA is not a specific regulator of norA expression but, rather, is a global regulator, since it also regulates autolytic activity and the expression of several virulence factors, including alpha toxin, nuclease, and protein A (153). MgrA is transcribed from two promoters, positively regulates its own expression, and acts at the transcriptional level to enhance the expression of numerous genes. Recently, two novel efflux transporters, NorB and Tet38, that confer resistance to multiple drugs including quinolones and tetracycline, respectively, have been shown to be negatively regulated by MgrA (254).
The ArlR-ArlS two-component regulatory system is involved in adhesion, autolysis, and extracellular proteolytic activity of S. aureus (85). The binding of MgrA to the norA promoter is modified in a strain with a disrupted arlS such that increased norA expression is observed (83, 84, 255). Overexpression of mgrA in a strain producing the ArlS sensor results in increased transcription of norA and reduced susceptibility to various NorA substrates. These data suggest that a mutation in arlS increases the effect of MgrA on the norA promoter and that wild-type levels of MgrA have little effect on norA expression. Highly fluoroquinolone-resistant strains of S. aureus in which norA expression is enhanced in the absence of any modification in arlR-arlS or change in mgrA expression have been reported, indicating that other loci must be involved in the regulation of norA expression.
Resistance to multiple drugs in gram-negative bacteria. The synthesis of the tripartite efflux systems of gram-negative bacteria (Fig. 6) depends on regulatory genes, implying individual control and thus distinct functions in the cell (180). Two-component systems are not commonly involved in the regulation of drug efflux transporters, although such systems have recently been associated with RND-type pumps, such as AdeABC in Acinetobacter baumannii (154), SmeABC in Stenotrophomonas maltophilia (145), and MdtABC in E. coli (28).
Intrinsic resistance of gram-negative bacteria is due to multidrug efflux by RND pumps that are widely distributed and act in synergy with the outer membrane barrier. The wide substrate range of these transporters often includes ß-lactams and aminoglycosides, which are rarely subjected to efflux by other pump classes. RND transporters form a multiprotein complex with members of the outer membrane factor family and of the periplasmic linker membrane fusion protein family. These complexes allow the excretion of drugs directly into the medium. Chromosomally encoded multidrug RND efflux systems appear to be most important for resistance to antimicrobials in P. aeruginosa and other gram-negative pathogens.
(i) Acinetobacter baumannii. A. baumannii is one of the predominant bacteria associated with outbreaks of nosocomial infections that are often very difficult to treat because of the frequent resistance of this species to multiple antibiotics. Aminoglycosides can be used successfully in combination with a ß-lactam, and combinations of a ß-lactam with either a fluoroquinolone or rifampin have also been proposed. Partial resistance of A. baumannii to ß-lactams is due to the synthesis of a species-specific cephalosporinase (258).
The chromosomally encoded three-component AdeABC pump in A. baumannii is composed of the membrane fusion homolog AdeA, the RND superfamily member AdeB with 12 transmembrane segments, and AdeC an outer membrane protein similar to OprM of P. aeruginosa (154). Insertional inactivation of adeB indicates that the corresponding protein is responsible for resistance not only to aminoglycosides but also to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, and trimethoprim. Thus, this efflux pump recognizes a wide spectrum of substrates including hydrophobic, amphiphilic, and hydrophilic molecules, which can be either positively charged or neutral. When the adeC gene is inactivated, resistance to the various substrates of the AdeABC pump is unaltered (161), suggesting that AdeAB can utilize another outer membrane constituent, as already observed for MexXY from P. aeruginosa (see below).
The
expression of multidrug transporters is commonly controlled by specific
regulatory proteins. Their structural genes are most often adjacent to
those encoding the efflux system. The adeABC genes are
cotranscribed and adjacent to the adeS and adeR genes
that are transcribed in the opposite direction and encode a sensor and
a regulator, respectively (Fig.
7) (161). Inactivation of
adeS leads to aminoglycoside susceptibility, indicating that
this gene is required for the expression of the adeABC operon.
Spontaneous aminoglycoside-resistant derivatives that have mutations in
the AdeS sensor or in the AdeR regulator can be obtained in vitro. The
T153M substitution in AdeS, downstream from histidine 149,
the putative site of autophosphorylation, is presumably responsible for
the loss of phosphatase activity of the sensor, as observed for EnvZ
(T247R), PhoR (T220N), and VanSB
(T237K). In AdeR, the P116L mutation
at the first residue of the 5 helix of the receiver domain is
involved in interactions that control the output domain of response
regulators. These mutations result in the constitutive expression of
the AdeABC pump, which is otherwise cryptic in wild-type A.
baumannii due to stringent control by
AdeRS.
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The SmeABC multidrug efflux system, a homolog of the mexAB-oprM efflux operon of P. aeruginosa (see below), is regulated by the SmeSR two-component system (Fig. 7) (145). A strain in which the smeABC genes are overexpressed displays resistance to aminoglycosides, ß-lactams, and the fluoroquinolones. Deletions in smeC but not in smeB decrease resistance, suggesting that SmeC only, which possesses its own promoter, contributes to multidrug resistance. Thus, SmeABC does not function as a multidrug efflux system, but it rather appears that SmeC plays a role in antimicrobial resistance independently of SmeAB, possibly as the outer membrane factor component of another unidentified multidrug efflux system (145).
As has been observed for the AdeABC system of A. baumannii, two genes, smeR and smeS, upstream from the smeABC operon and transcribed in the opposite direction, encode a regulatory system composed of a sensor (SmeS) and a regulator (SmeR) (Fig. 7) (145). SmeR positively regulates both smeABC and its own smeSR operon.
(iii) Pseudomonas aeruginosa. P. aeruginosa is a ubiquitous aerobic gram-negative opportunistic pathogen and one of the most common causes of nosocomial infections. Treatment of P. aeruginosa infections is complicated by the intrinsic resistance of this organism to many antimicrobial agents, which results from the synergistic activity of the outer membrane barrier with that of various broad-substrate-range multidrug efflux systems. In addition to intrinsic resistance, multidrug efflux (Mex) systems promote acquired resistance by overexpression of the structural genes for the pumps following mutational events.
Six RND efflux systems in P. aeruginosa have been characterized (Table 1) (4, 5, 50, 111, 129, 206, 207). The efflux operons each encode an inner membrane RND transporter (MexB, MexD, MexF, MexX, MexK, or MexI), a periplasmic membrane fusion protein (MexA, MexC, MexE, MexY, MexJ, or MexH), and, in certain cases, an outer membrane channel protein (OprM, OprJ, OprN, or OpmD). All these RND operons are similar in their genetic organizations but not with respect to regulation, and the corresponding pumps differ in their substrate specificities (Fig. 7 and Table 1). The antibiotic substrate spectrums of these systems are very wide (Table 1). MexAB-OprM, which exhibits an extraordinarily broad substrate range, is constitutively produced in wild-type bacteria and plays a major role in the intrinsic resistance of P. aeruginosa (Table 1) (128). The MexCD-OprJ, MexEF-OprN, and MexJK-OprM systems are not expressed in wild-type P. aeruginosa (50, 129, 206). Expression of many RND multidrug pumps is controlled by local regulators (Table 1), mostly repressors (Fig. 7). With the exception of MexAB-OprM, the expression of most of these efflux systems is tightly regulated.
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(iv) Escherichia coli. Certain multidrug efflux pumps in E. coli are regulated by two-component systems. BaeSR is involved in the expression of the RND transporter MdtABCD that pumps out novobiocin and deoxycholate (28, 178). The baeS and baeR genes are immediately downstream from the mdtABCD genes and together probably form an operon.
BaeR and BaeS exhibit in vitro phosphotransfer in the presence of ATP (28), but the nature of the stimulus recognized by the BaeS sensor is not known. The BaeR response regulator binds to the mdtA promoter, and its overexpression strongly stimulates the transcription of the mdtABCD gene cluster, leading to an increase in resistance to novobiocin and deoxycholate. The presence of the BaeS sensor kinase is not required for the full activity of overexpressed BaeR in intact cells. BaeR could be phosphorylated by other sensor kinases present in E. coli, since such cross talk occurs particularly when one of the noncognate partners is present in excess. Cross-regulation has been observed between the various two-component regulatory systems, BaeSR, PhoBR, which is implicated in phosphate metabolism, and CreBC, which is implicated in carbon and energy metabolism (181).
Many of the two-component signal transduction systems in E. coli control the expression of multiple target genes. BaeR modulates the expression of mdtABCD but also that of acrD, which encodes a multidrug exporter system conferring resistance to ß-lactams and novobiocin (108).
| ROLE OF IS ELEMENTS AND INTEGRONS IN THE MODULATION OF RESISTANCE GENE EXPRESSION |
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IS elements may be present in one or several copies and localized on the chromosome, on plasmids, or on both and must reside on conjugative elements for intercellular transfer. Many transpose readily, and others, such as IS200, transpose rarely (32). There is great variability in the distribution of the IS elements of the different families among bacterial species, with some of them restricted to few hosts, such as IS6110, which has been found only in mycobacteria of the tuberculosis complex (156, 250).
IS elements are typically bounded by short repeat sequences of up to ca. 40 bp in an indirect orientation. These inverted repeats are specific for each element, and their presence and integrity are required for transposition, which may or may not be site specific. Upon insertion into the target DNA, a repeat sequence, 2 to 14 bp in length and characteristic for each element, is generated in a direct orientation (Fig. 8). Many elements carry a single, transposase-encoding open reading frame (ORF) covering most of the element, while others carry several ORFs, on a single strand or on both strands, the products of which may also play a role in the regulation of the transposition process. Of particular interest in the present context, IS elements may contain partial or complete promoters, often located at their extremities and in an outward orientation and capable of activating the expression of neighboring genes (Fig. 8) (46, 155).
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(i) Activation of resistance genes by promoter alterations. The molecular mechanisms responsible for altered, IS-mediated expression are not specific for resistance genes. Transcriptional activation may result from IS insertion into a region carrying a weak, an incomplete, or no promoter. Therefore, a hybrid promoter with an alternative or new IS-borne –35 region may be generated, or a complete IS-borne promoter containing both the –35 and the –10 regions may be acquired (Fig. 8). With few exceptions (see below), these two regions conform to the canonical consensus sequences TTGACA and TATAAT, respectively, with a spacing distance of 17 bp for optimal promoter activity as determined for E. coli (149).
(a) Resistance gene activation by IS-mediated formation of hybrid promoters. An IS-mediated rearrangement of the promoter region of the ampC gene of E. coli was shown in an experimental setup (118) only shortly before the observation of similar events affecting resistance genes in clinical isolates. It was found that the insertion of IS2, of which E. coli carries five chromosomal copies, into the –10 region of the artificially plasmid-borne ampC gene resulted in concomitant, ca. 20-fold increases in ampC transcription, ß-lactamase production, and ampicillin resistance levels. While the –10 region remained unaltered and the –35 region was changed to a sequence with less homology with the consensus sequence than that of the natural ampC promoter, the critical event was concluded to be the change of the spacer region from 16 to 17 bp. Despite the efficiency of this rearrangement in increasing the resistance level and although IS2 belongs to the family that is most widely distributed among bacterial species (156), this element does not seem to have been involved similarly in clinical isolates. Another IS2 insertion, with the creation of a putative hybrid promoter upstream from the efflux pump-encoding acrEF gene and its increased expression in an E. coli laboratory mutant, facilitated the determination of the substrate profile of the pump (119). Probably the first observation of an IS-mediated formation of a hybrid promoter for an antibiotic resistance gene in a clinical isolate was made by Bräu et al. (39) in Salmonella. They found the plasmid-borne aac(3)-IV and aph(4) genes, coding for gentamicin and hygromycin B resistance, respectively, in an operon-like arrangement downstream from IS140 (IS26), which provided the –35 region.
IS-mediated rearrangements of promoters driving the transcription of genes encoding extended-spectrum ß-lactamases belonging to several families of the class A or class D enzymes (117) have been observed (Table 2). The IS26 element has been reported to contribute to the formation of a hybrid promoter for a chromosome-borne SHV-2A gene in P. aeruginosa and for a similar, plasmid-borne gene in a resistance operon (downstream from an aminoglycoside 3'-O-phosphotransferase gene) in Klebsiella pneumoniae, with the new –35 region in each case at the optimal distance of 17 bp from the respective resistance gene-specific –10 region (137, 177). The gene of TEM-6, as identified in a ceftazidime-resistant strain of E. coli, acquired a –35 region after the insertion of an IS1-like element into the spacer region of its "natural" promoter, P3, the strength of which was increased by a factor of 10 (89). It was speculated that this element, which was found to be widespread among ß-lactamase-producing and non-ß-lactamase-producing Enterobacteriaceae, had been derived from IS1 through a substantial deletion of its central region as well as by point mutations in the remainder, which did not affect the –35 region. In a laboratory mutant, the replacement of the –35 region of the same P3 promoter of the TEM-1 gene carried on plasmid pBR322 by a similar IS1-borne region had previously been shown to result in decreased promoter strength, which was considered to be related to a lesser degree of homology between this region and the –35 consensus sequence (209).
In Acinetobacter species 13, aminoglycosideresistance is conferred by the species-specific 6'-N-acetyltransferase-encoding gene, aac(6')-Ij, which may be expressed at various levels (133). The activation of silent copies of the aac(6')-Ij gene in this species by the creation of a putative hybrid promoter with an IS18-borne –35 region appears to occur at a low frequency, at least as judged from the in vitro selection of tobramycin-resistant mutants of a susceptible clinical isolate (229).
The IS256 and IS257 elements have a proven role in the activation of resistance gene transcription in staphylococci. IS256 belongs to a large family with members in gram-negative and gram-positive bacteria (http://www-IS.biotoul.fr). It flanks the composite aminoglycoside resistance transposon Tn4001 and related elements and is involved in their dissemination in staphylococci, enterococci, and streptococci (158). IS256 is infrequently observed in the animal commensal species Staphylococcus sciuri, in which two-thirds of the isolates are susceptible to ß-lactam antibiotics including methicillin, although they carry a close homolog of the mecA gene, the primary drug resistance determinant in methicillin-resistant S. aureus (58). Analysis of a heterogeneously methicillin-resistant human clinical isolate of S. sciuri (with MICs of methicillin of between 25 and 800 µg/ml as opposed to between 3 and 6 µg/ml for a mecA-positive, IS256-negative control strain) revealed the insertion of an IS256 copy into the upstream region of mecA with the creation of a powerful hybrid promoter. This led to the speculation that the S. sciuri isolate had acquired IS256 in a clinical environment where the activation of mecA had then been selected under drug pressure (59). In that same study, mecA activation in S. sciuri was obtained in vitro, and the –35 region of the hybrid promoter was the same as that previously identified as being responsible for the transcriptional activation of llm, a gene of S. aureus encoding a putatively membrane-associated protein that contributes to methicillin heteroresistance in this species in an as-yet-unknown manner (159).
A variation
on the theme of hybrid promoter formation has been found in S.
aureus in connection with the IS257-dependent effects on
the levels of trimethoprim resistance resulting from the association of
a constant, IS-borne –35 region with variable –10
regions upstream from the resistance gene. Trimethoprim resistance in
S. aureus occurs at low or high levels (with MICs of 50 to 300
µg/ml or 
1,000 µg/ml, respectively) and is
mediated by the dihydrofolate reductase gene dfrA, which
resides in the center of a three-gene operon carried by Tn4003
(or Tn4003-like elements), a composite transposon flanked by
three copies of IS257
(138,
225). The promoter of
this operon overlaps the right end of the left copy of the element
IS257L, with its –10 sequence located in the central
region of the transposon and the –35 sequence in the right
terminus of IS257L. Low-level resistance was found to be
associated with various deletions that extend ca. 10 to 300 bp away
from the right end of IS257L and unmask alternative
–10 regions differing in sequence or distance to the
–35 box, or both, from the corresponding region in the
high-level resistance-conferring form of the transposon. Such deletion
variants exist in S. aureus as well as in coagulase-negative
staphylococci. It is believed that IS257 itself is involved in
the generation of the flanking deletions and that the transposon
variants that carry them may have established themselves by imposing
less strain on the fitness of their hosts while conferring levels of
resistance that are still advantageous
(138). IS257
has also been found to affect the level of tetA(K)-dependent
efflux-mediated tetracycline resistance in S. aureus
(237). Analysis
