Journal of Antimicrobial Chemotherapy (2000) 46, 885-893
© 2000 The British Society for Antimicrobial Chemotherapy
Influence of the MexA-MexB-OprM multidrug efflux system on expression of the MexC-MexD-OprJ and MexE-MexF-OprN multidrug efflux systems in Pseudomonas aeruginosa
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Of the Pseudomonas aeruginosa multidrug efflux systems, MexAB-OprM is expressed in wild-type cells, while MexCD-OprJ is not, and MexEF-OprN shows variable, strain-specific expression. In defined mutant strains, MexCD-OprJ expression increased with decreases in MexAB-OprM and was generally inversely related to MexAB-OprM expression. In so-called wild-type strains expressing MexEF-OprN, MexAB-OprM hyperexpression correlated with a decline in MexEF-OprN expression, while loss of MexAB-OprM was associated with increased expression of MexEF-OprN, also indicative of an inverse correlation between MexAB-OprM and MexEF-OprN expression. Still, the increases in MexCD-OprJ and MexEF-OprN failed to compensate for the loss of MexAB-OprM with respect to antibiotic resistance. Nonetheless, these data suggest that the overall complement of these MDR efflux systems is monitored and that alterations in the level of one efflux system may effect compensatory changes in the levels of the others.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Multidrug resistance (MDR) in bacteria has been associated with overexpression of endogenous efflux genes. Pseudomonas aeruginosa is an opportunistic human pathogen characterized by intrinsic resistance to a variety of antimicrobial agents. This property results from the interplay between broadly specific drug efflux systems and the low outer membrane permeability of P. aeruginosa.1–3 Four such efflux systems have been described in P. aeruginosa, including MexAB-OprM,4,5 MexCD-OprJ,6 MexEF-OprN7 and MexXY-OprM.8–10 These tripartite efflux systems belong to the resistance–nodulation–cell division (RND) family of transporters,11 and consist of an inner membrane RND-type chemiosmotic efflux pump (MexB, MexD, MexF or MexY), a presumed outer membrane channel-forming protein (OprM, OprJ or OprN) and a membrane fusion protein predicted to link the inner/outer membrane-associated efflux components (MexA, MexC, MexE or MexX).2 The MexAB-OprM efflux system contributes to the intrinsic resistance of this organism to quinolones, tetracycline, chloramphenicol, novobiocin, macrolides and most ß-lactams,5 and its hyperexpression is responsible for the elevated MDR of nalB mutants.5,12 Apparently not expressed during growth under laboratory conditions, the MexCD-OprJ and MexEF-OprN systems are expressed in nfxB and nfxC multi- drug-resistant mutants, respectively.6,7 Mutant nfxB strains are resistant to quinolones, chloramphenicol, tetracycline and newer cephems, but display increased susceptibility to most conventional ß-lactam antibiotics.6,13,14 Mutant nfxC strains exhibit resistance to quinolones, chloramphenicol, trimethoprim and carbapenems.7,13 Expression of MexXY is associated with the natural resistance of P. aeruginosa to aminoglycosides,9,10 although the cloned mexXY genes also afford resistance to erythromycin and fluoroquinolones in Escherichia coli and P. aeruginosa.8,9 Although some of the genes regulating expression of the P. aeruginosa MDR efflux systems are known,2 conditions responsible for induction of MexCD-OprJ and MexEF-OprN are unknown and expression of these apparently occurs only in mutants. In this report we demonstrate that expression of MexCD-OprJ and MexEF-OprN can occur in response to changes in the levels of MexAB-OprM, independent of nfxB and nfxC mutations, respectively.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bacterial strains, plasmids and growth conditions
Strains and plasmids used in this study are listed in Table I
.5,15–19 Luria–Bertani (LB) broth [1% (w/v) Difco tryptone, 0.5% (w/v) Difco yeast extract and 0.5% (w/v) sodium chloride] was used throughout the study and bacteria were cultivated at 37°C. Introduction of plasmids pVLT31 and pXZL34 from E. coli DH5
to the OprM-deficient P. aeruginosa strains was performed by conjugation (i.e. triparental mating) as described previously17 and the P. aeruginosa conjugants were obtained on LB agar containing tetracycline (10 mg/L) and chloramphenicol (25 mg/L), or tetracycline (10 mg/L) and imipenem (0.5 mg/L). Both chloramphenicol and imipenem were used to counterselect E. coli cells. Successful conjugation was confirmed by preparation of plasmids from the tetracycline-resistant P. aeruginosa strains.
|
Construction of mexAB-oprM deletion mutants
The mexAB-oprM deletion mutants were constructed using gene replacement vector pRSP21 (for PAO4098- and K1034-derived strains) or pELCT04 (for PAO4098E-derived strains) as described previously17,19 with some modifications. Briefly, pRSP21 or pELCT04 was mobilized from E. coli S17-1 into P. aeruginosa strains by conjugation (i.e. biparental mating).17 pRSP21- or pELCT04-containing P. aeruginosa were selected on LB agar supplemented with kanamycin (1500 mg/L) or mercuric chloride (15 mg/L), respectively, and tetracycline (10 mg/L, to counterselect E. coli S17-1). Kanamycin- or mercuric chloride-resistant transconjugants were recovered and re-streaked on LB agar containing 10% (w/v) sucrose. Sucrose-resistant colonies were screened for loss of kanamycin and/or mercuric chloride resistance, and those carrying a mexAB-oprM deletion were identified by their drug susceptibility phenotype and loss of MexA, MexB and OprM as assayed by Western immunoblotting (see below) with antibodies specific for MexA (X.-Z. Li and K. Poole, unpublished data), MexB20 and OprM.15
SDS–polyacrylamide gel electrophoresis and Western immunoblotting of membrane proteins
Cell envelopes of P. aeruginosa were prepared from the cells grown to the exponential phase by sonic disruption followed by differential centrifugation.21 Outer membranes were prepared by extraction of cell envelopes with 1.5% (w/v) sodium N-lauroyl sarkosinate (sarkosyl; Sigma, Oakville, Ontario, Canada) as described previously.21 Protein contents of cell envelopes and outer membranes were determined by the method of Lowry et al. using bovine serum albumin as the standard.22 The membrane proteins were subsequently analysed using slab sodium dodecyl sulphate–polyacrylamide (11% w/v) gel electrophoresis (SDS–PAGE).23 Each sample containing 50 µg of proteins (cell envelopes) or 30 µg of proteins (outer membranes) was heated at 100°C for 5 min before being subjected to SDS–PAGE. Following electrophoresis, proteins were transferred onto an Immobilon-P membrane (Millipore Corp., Bradford, MA, USA) at 20 mA for 16 h at 4°C. Membranes were processed as described previously.15 Antibodies (anti-OprM,15 anti-OprJ6 and anti-OprN7) and a horseradish peroxidase-coupled donkey anti-rabbit or anti-mouse immunoglobulin G were used as primary and secondary antibodies, respectively. Blots were developed with the enhanced chemiluminescence system (Amersham, Pharmacia Biotech, Bai d'Urfé, Québec, Canada) according to the manufacturer's instructions.
Antibiotic susceptibility assays
Drug susceptibility testing was carried out in LB broth using the two-fold serial broth dilution method with an inoculum of 5 x 105 cells/mL. Data were reported as MICs, which reflected the lowest concentration of antibiotic inhibiting visible growth after overnight incubation at 37°C. Antibiotics were obtained from the following sources: carbenicillin, cefoperazone, ciprofloxacin, tetracycline, chloramphenicol and novobiocin from Sigma–Aldrich Canada Ltd (Oakville, Ontario, Canada); cefpirome from Roussel UCLAF (Paris, France); and imipenem from Merck Sharp Dohme Canada (Montreal, Canada).
RT–PCR
Total bacterial RNA was isolated from late-log-phase cultures (1–2 mL) of P. aeruginosa strains using the Qiagen RNeasy Mini Kit (Qiagen Inc., Mississauga, Ontario, Canada), treated with RNase-free DNase (Promega, Madison, WI, USA) (1 U of enzyme/µg RNA for 60 min at 37°C) and re-purified using the same kit. A 0.2 µg sample of DNase-treated RNA was used as template for reverse transcription–polymerase chain reaction (RT–PCR) with the Qiagen OneStep RT–PCR kit (Qiagen Inc.) according to a protocol supplied by the manufacturer. Primer pairs specific for and internal to mexA [jt-18, 5'-ACCTACGAGGCCGACTACCAGA-3' (forward); jt-12, 5'-GTTGGTCACCAGGGCGCCTTC-3' (reverse)], mexC [mexc1xz, 5'-AGCCAGCAGGACTTCGATACC-3' (forward); mexc2xz, 5'-ACGTCG-GCGAACTGCAAC-3' (reverse)] and mexE [mexe1xz, 5'-GTCATCGAACAACCGC-TG-3' (forward); mexe2xz, 5'-GTCGAAGTAGGCGTAGACC-3' (reverse)] were used to amplify and quantitate the corresponding mRNA, as a measure of mexAB-oprM, mexCD-oprJ and mexEF-oprN expression. The mRNA of the constitutively expressed rspL gene was amplified and quantitated by RT–PCR using primers rspl1xz [5'-GCAACTATCAACCAGGCTG-3' (forward)] and rspl2xz [5'-GCTGTGCTCTTGCAGGTTGTG-3' (reverse)].10 Thirty picomoles of each primer was used per reaction (final volume of 50 µL), which involved a 30 min incubation at 50°C, followed by 15 min at 95°C, and 40 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, before finishing with 10 min at 72°C. A 15 µL sample of each reaction product was analysed by agarose (1.4% w/v) gel electrophoresis for the expected RT–PCR products (rpsL, 220 bp; mexA, 252 bp; mexC, 314 bp; mexE, 516 bp).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Influence of the MexAB-OprM status of P. aeruginosa on MexCD-OprJ expression
Strains reportedly wild type with respect to the MDR efflux systems of P. aeruginosa failed to express detectable levels of MexCD-OprJ in Western immunoblots (Figure 1b, lanes 1, 5 and 9, and data not shown). Consistent with this, deletion of this operon failed to alter the intrinsic drug susceptibility of wild-type strains.6,17 To assess whether MexCD-OprJ might respond to the loss of the MexAB-OprM efflux system, expression of MexCD-OprJ was examined in mexAB-oprM or oprM deletion derivatives of PAO1 strain K767 (K1119), PAO6609 (K1032), PAO4098 (K1232) and ML5087 (K1110 and K1121). In all instances, loss of MexAB-OprM or OprM (confirmed by immunoblotting with MexA-, MexB- and/or OprM-specific antisera; Figure 1a, lanes 2, 6 and 10, and data not shown) correlated with an increase in MexCD-OprJ, as assessed using an OprJ-specific antiserum (Figure 1b, lanes 2 and 6, and Figure 2b, lanes 2 and 8). These deletion strains were hypersusceptible to multiple antibiotics, including those known to be substrates for MexCD-OprJ (Table II). As expected, hyperexpression of MexAB-OprM in nalB strains K1034, PAO4098E and K1112 (Figure 1a, lanes 3 and 7, and Figure 2a, lane 3) correlated with a lack of MexCD-OprJ expression (Figure 1b, lanes 3 and 7, and Figure 2b, lane 3), while mexAB-oprM (or oprM) deletion derivatives of these nalB strains (i.e. K1230, K1234 and K1113) (Figure 1a, lanes 4 and 8, Figure 2a, lane 4) did express MexCD-OprJ (Figure 1b, lanes 4 and 8, and Figure 2b, lane 4). Again, these deletion derivatives were drug hypersusceptible (Table II).
|
|
|
To determine whether the increase in OprJ (MexCD-OprJ) was a response to the loss of the MexAB-OprM system and not simply a compensatory increase in OprJ as a result of the decline in OprM or the result of a mutation, the cloned wild-type oprM gene (on plasmid pXZL34) was introduced into OprM-deficient derivatives of ML5087 (K1110) and the nalB strain K1112 (K1113) and a MexAB-OprM-deficient derivative of ML5087 (K1121). As expected, introduction of pXZL34 into the OprM-deficient strains K1110 and K1113 completely reversed the increase in OprJ (Figure 2b
, lanes 5 and 6). In contrast, the MexAB-OprM-deficient strain K1121 produced decreased, though still detectable levels of OprJ upon the introduction of the oprM plasmid pXZL34 (Figure 2b, lane 10). Influence of the MexAB-OprM status of P. aeruginosa on MexEF-OprN expression
Western immunoblotting revealed that different strains apparently wild-type with regard to the MDR pumps expressed different levels of OprN (and, thus, probably MexEF-OprN). P. aeruginosa PAO1 strain K767 (data not shown) and PAO4098 (Figure 1c, lane 5) produced undetectable levels of OprN, and strains PAO6609 and ML5087 (Figure 1c, lanes 1 and 9) produced modest levels of this protein. OprM levels were generally consistent in the aforementioned strains (Figure 1a and data not shown). Elimination of MexAB-OprM or OprM in those strains producing modest levels of MexEF-OprN (PAO6609 and ML5087) yielded strains K1032, K1110 and K1121, respectively, which exhibited increased expression of MexEF-OprN (Figure 1c, lanes 2 and 10, and Figure 2c, lane 8). MexEF-OprN remained undetectable in those mexAB-oprM deletion strains (K1119, data not shown; K1232, Figure 1c, lane 6) which were derived from strains originally producing undetectable OprN (K767 and PAO4098).
In all instances, where OprN was detectable in the original wild-type strains (PAO6609 and ML5087), nalB derivatives of these exhibited undetectable levels of OprN (Figure 1c, lanes 3 and 11). Again, elimination of mexAB-oprM or oprM in the above nalB strains (efflux system is absent but the nalB mutation remains) restored MexEF-OprN expression in K1230, a mexAB-oprM deletion of the nalB strain K1034 (Figure 1c, lane 4), and in K1113, the oprM deletion of nalB strain K1112 (Figure 1c, lane 12). It indicates, therefore, that in these strains there is an inverse correlation between MexAB-OprM and MexEF-OprN, much like that seen for MexAB-OprM and MexCD-OprJ. In spite of this, the OprN level of K1113 was less than that of K1110, suggesting the nalB mutation itself seems to be impacting on MexEF-OprN, independent of any effect on MexAB-OprM expression. The mutation responsible for the nalB phenotype in K1112 (parent of K1113) occurs within the mexR gene encoding a repressor of mexAB-oprM expression.24 Still, introduction of a plasmid-borne wild-type mexR gene (on plasmid pRSP5524) into K1113 failed to restore OprN (i.e. MexEF-OprN) production (data not shown), suggesting that additional mutations are also contributing the nalB phenotype in this strain. In any case, the reduced amount of MexEF-OprN in K1113 was consistent with antibiotic resistance data showing this strain to be markedly more susceptible to ciprofloxacin, chloramphenicol and novobiocin than K1110 (Table II). Both strains are ML5087 derivatives lacking OprM, although only K1110 hyperexpresses MexEF-OprN (Figure 1c, lane 10) and this efflux system is known to accommodate both ciprofloxacin and chloramphenicol.7,13
As for MexCD-OprJ (above), it was important to establish that changes in MexEF-OprN (measured as OprN changes) were a response to loss of a functional MexAB-OprM MDR efflux system. Again, introduction of the cloned wild-type oprM gene (pXZL34) into K1110 (ML5087 
oprM), completely reversed the increase in OprN (Figure 2c, lanes 5). In contrast, the oprM plasmid had no effect on OprN levels in the MexAB-OprMdeficient strain K1121, which already produced substantial amounts of OprN (Figure 2c, lanes 9 and 10).
Efflux status of the multidrug hypersusceptible P. aeruginosa mutant Z61
The drug hypersusceptibility (attributed to increased outer membrane permeability)18,25 and efflux-deficiency of P. aeruginosa Z61 are now well known,12 although the nature of the defect in this strain has yet to be elucidated. Western immunoblotting with an anti-OprM and anti-MexB antisera revealed that, in contrast to its parent strain (Figure 1a, lane 13), this mutant lacks OprM (Figure 1a, lane 14) but still produces MexB (data not shown). Thus, Z61 was similar to the OprM-deficient strain K1110 in terms of the MexAB-OprM expression. Intriguingly, like K1110, this strain exhibited elevated production of both OprJ (Figure 1b, lane 10) and OprN (Figure 1c, lane 14) compared with the parent strain K799 (Figure 1b, lane 9, and Figure 1c, lane 13), indicating an enhanced production of the MexCD-OprJ and MexEF-OprN efflux systems in response to loss of OprM (i.e. loss of a functional MexAB-OprM pump) in this strain.
Influence of MexAB-OprM status on mexCD-oprJ and mexEF-oprN expression
To assess if the presence or absence of MexAB-OprM was influencing expression of the mexCD-oprJ and mexEF-oprN genes, and not impacting solely on production of the OprJ and OprN outer membrane proteins, RT–PCR was employed using internal primers specific for the first gene of each of these operons. As seen in Figure 3b, RT–PCR was an accurate measure of mexA (as a measure of mexAB-oprM) expression, with a mexA-specific RT–PCR product absent only in strains lacking the mexAB-oprM genes (Figure 3b, lanes 3 and 7). Using mexC-specific primers, it was clear that strains wild-type with respect to efflux expressed barely detectable levels of mexC (as a measure of mexCD-oprJ) mRNA (Figure 3c, lanes 2, 6 and 8) while the nfxB strain K1111, as expected, hyperexpressed this gene (Figure 3c, lane 5). In contrast to the wild-type strains, substantial expression of mexC was evident in their mexAB-oprM (Figure 3b, lanes 3 and 7) and oprM (Figure 3b, lane 4) derivatives. Similarly, the Z61 strain, shown above to lack OprM and thus, a functional MexAB-OprM efflux system, showed increased expression of mexC relative to its OprM+ parent strain (Figure 3b, lane 9, cf. lane 8). Similar results were observed with the mexE-specific RT–PCR, with weak mexE (as a measure of mexEF-oprN) expression evident for wild-type strains (Figure 3d, lanes 2, 6 and 8) and substantially increased expression seen in their MexAB-OprM-/OprM-deficient derivatives (Figure 3b, lanes 3–5, 7 and 9). RT–PCR using primers for the constitutively expressed rpsL gene served as a control and confirmed that the differences cited above were not due to variability in RNA recovery from the strains being examined (Figure 3a).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Using a variety of defined pump mutant strains, we investigated the influence of expression of MexAB-OprM, the predominant MDR efflux pump in wild-type P. aeruginosa, on the expression of the MexCD-OprJ and MexEF-OprN multidrug efflux systems. In all strains tested, either wild-type or MexAB-OprM-overproducing nalB strains, the loss of the MexAB-OprM efflux system correlated with an increase in MexCD-OprJ, suggesting that the absence of MexAB-OprM turned on the expression of MexCD-OprJ. Nonetheless, all MexAB-OprM or OprM deletion strains were hypersusceptible to multiple antibiotics, including the substrates for MexCD-OprJ (Table II
), indicating that the increase in OprJ (MexCD-OprJ) in these strains did not functionally compensate for the loss of MexAB-OprM in antibiotic resistance. This is, perhaps, not surprising, in as much as the levels of OprJ seen in the mexAB-oprM deletion strains were markedly lower than for an nfxB mutant (Figure 1b, lane 11), where MexCD-OprJ levels are sufficient to impact on antibiotic resistance.17 Introduction of the cloned oprM gene (pXZL34) into the OprM-deficient strain K1110 completely reversed the increase in OprJ, while the MexAB-OprM-deficient strain K1121 harbouring the cloned oprM gene produced decreased but still detectable levels of OprJ. That OprJ is still produced under these circumstances implies that OprJ (MexCD-OprJ) expression is responding to a lack of a functional MexAB-OprM efflux system in oprM and mexAB-oprM deletion strains. That a MexAB– -OprM+ strain (K1121/pXZL34) produces less OprJ (MexCD-OprJ) than a MexAB–-OprM– strain (K1121) (but still more than a MexAB+-OprM+ strain), when both lack a functional MexAB-OprM system, can probably be explained by the presence of the MexXY MDR efflux system, which will be inoperable in K1121 (since MexXY requires OprM for activity8,9), but will be operable in K1121/pXZL34. In essence, then, the expression of MexCD-OprJ in the oprM and mexAB-oprM strains is a response to the loss of two MDR efflux systems (i.e. MexAB-OprM and MexXY-OprM), while MexCD-OprJ expression in K1121 carrying pXZL34 is a response to the lack of the MexAB-OprM system only. It is not surprising that the former would elicit a greater compensatory increase in MexCD-OprJ than the latter. All these data indicated that increased OprJ (MexCD-OprJ) expression was a response to the loss of the MexAB-OprM system and not simply a compensatory increase in OprJ as a result of the decline in OprM or the result of a mutation. The fact, too, that mexC expression, as measured using RT–PCR, also increases in the mexAB-oprM/oprM strains clearly demonstrates that mexCD-oprJ gene expression increases in response to the absence of a functional MexAB-OprM efflux system. Thus, the increase in MexCD-OprJ occurs at the level of gene expression.
Similarly, decreases in MexAB-OprM were paralleled by a compensatory increase in MexEF-OprN (in K1032, K1110 and K1121, where the parent strains produced modest levels of MexEF-OprN), while hyperexpression of MexAB-OprM in strains K1034 and K1112 correlated with a seemingly compensatory decrease in MexEF-OprN. MexEF-OprN remained undetectable in those mexAB-oprM deletion strains (K1119 and K1232) that were derived from strains originally producing undetectable OprN (K767 and PAO4098). In the case of K1119, this may relate to the presence, within the mexT gene of its parent K767, of two mutations (K. Poole, unpublished data) that probably abrogate the function of this mexEF-oprN activator gene.26 Possibly, any increase in MexEF-OprN seen upon deletion of mexAB-oprM requires MexT activation of mexEF-oprN expression. That restoration of MexAB-OprM in the MexAB+-OprM– strain (K1110) by adding back OprM (pXZL34) reversed the increase in OprN while lack of a functional MexAB-OprM pump in K1121/ pXZL34 (MexAB–-OprM+) did not reverse the increase in OprN indicates that MexEF-OprN was responding solely to the presence or absence of a functional MexAB-OprM efflux system. Moreover, the observed increase in mexE expression in strains lacking MexAB-OprM indicates that this OprN increase is a result of enhanced mexEF-oprN gene expression and, thus, reflective of an increase in MexEF-OprN pump production. The higher expression of MexEF-OprN in K1110 (ML5087 oprM) than that in K1113 (ML5087 nalB oprM) indeed correlates with the increased resistance to ciprofloxacin, chloramphenicol and novobiocin (four- to 16-fold in MICs; Table II), indicating a clinical significance for the differential MDR pump expression.
The examination of the well-studied hypersusceptible P. aeruginosa Z61 and its parent strain demonstrated that Z61 lacks a functional MexAB-OprM system since it lacks OprM expression. This result confirms our previous conclusion that the mutant Z61 is deficient in antibiotic efflux12 and, thus, hypersusceptible to multiple antibiotics.12,18,25 Interestingly, the elevated production of both OprJ and OprN in the mutant Z61 is in agreement with the aforementioned compensatory changes seen in MexCD-OprJ and MexEF-OprN expression in response to the loss of a functional MexAB-OprM system. Again, too, this appears to occur at the level of gene expression.
The influence of the MexAB-OprM status on the expression of MexCD-OprJ and MexEF-OprN suggests that the cell can assess the status of its MDR efflux systems and provide for compensatory changes in the levels of one system in response to increases or decreases in another, perhaps to maintain a basal (if not optimal) level of efflux gene expression. Consistent with this, it was noted previously that nfxB strains hyperexpressing MexCD-OprJ produced decreased levels of MexAB-OprM.27 In spite of this, a mexCD-oprJ mexAB-oprM double mutant of ML5087 was no more susceptible to antibiotics than was a mexAB-oprM strain of ML5087 (data not shown), indicating that the increased expression of MexCD-OprJ in the absence of MexAB-OprM is insufficient to provide meaningful resistance to antibiotics. It is possible, however, that the elevated expression of MexCD-OprJ and MexEF-OprN complements, to some extent, the lack of MexAB-OprM with respect to its role in the export of some cell-associated compound(s). Given the very similar antibiotic substrate profiles of MexAB-OprM and MexCD-OprJ, it is likely that the latter could effectively replace the former with respect to export of whatever cell-associated compound(s) are the probable natural substrates for these MDR efflux systems.
The compensatory changes in MexCD-OprJ and MexEF-OprN upon the loss of functional MexAB-OprM may serve to maintain net expression of this family of efflux systems at some possibly pre-determined overall level. It is unclear whether this reflects some form of global regulation of MDR efflux transporters in P. aeruginosa. Alternatively, the loss of MexAB-OprM could promote increased accumulation of certain compounds within the cell and that these could then trigger expression of the other MDR systems via their own regulatory circuits. Given the broad and often overlapping substrate specificity of the MDR efflux systems of this organism, it would not be surprising to find these pumps to be somewhat interchangeable within the cell. In any case, it is obvious that the cell possesses mechanisms by which it maintains a certain level of expression of these efflux pumps, suggesting that these pumps play an important role in the cell, one that is probably independent of any contributions to antibiotic resistance.
|
Acknowledgments |
|---|
The authors wish to thank N. Gotoh for providing anti-OprJ and anti-OprN antibodies, and strain PAO2375 and its nfxC mutant. This research was supported by an operating grant from the Canadian Cystic Fibrosis Foundation (CCFF). X.-Z. L. acknowledges a studentship from the CCFF. K. P. is the CCFF Martha Morton Scholar.
|
Notes |
|---|
* Corresponding author. Tel: +1-613-533-6677; Fax: +1-613-533-6796; E-mail: poolek{at}post.queensu.ca
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 . Nikaido, H. (1989). Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrobial Agents and Chemotherapy 33, 1831–6.
2
.
Nikaido, H. (1996). Multidrug efflux pumps of gram-negative bacteria. Journal of Bacteriology 178, 5853–9.
3
.
Li, X.-Z., Zhang, L. & Poole, K. (2000). Interplay between the MexA-MexB-OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 45, 433–6.
4
.
Poole, K., Krebes, K., McNally, C. & Neshat, S. (1993). Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. Journal of Bacteriology 175, 7363–72.
5 . Li, X.-Z., Nikaido, H. & Poole, K. (1995). Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1948–53.[Abstract]
6 . Poole, K., Gotoh, N., Tsujimoto, H., Zhao, Q., Wada, A., Yamasaki, T. et al. (1996). Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Molecular Microbiology 21, 713–24.[Web of Science][Medline]
7 . Köhler, T., Michea-Hamzehpour, M., Henze, U., Gotoh, N., Curty, L. K. & Pechere, J. C. (1997). Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Molecular Microbiology 23, 345–54.[Web of Science][Medline]
8
.
Mine, T, Morita, Y., Kataoka, A., Mizushima, T. & Tsuchiya, T. (1999). Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 415–7.
9
.
Aires, J. R., Köhler, T., Nikaido, H. & Plésiat, P. (1999). Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrobial Agents and Chemotherapy 43, 2624–8.
10
.
Westbrock-Wadman, S., Sherman, D. R., Hickey, M. J., Coulter, S. N., Zhu, Y. Q., Warrener, P. et al. (1999). Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrobial Agents and Chemotherapy 43, 2975–83.
11 . Saier, M. H., Tam, R., Reizer, A. & Reizer, J. (1994). Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport. Molecular Microbiology 11, 841–7.[Web of Science][Medline]
12
.
Li, X.-Z., Livermore, D. M. & Nikaido, H. (1994). Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrobial Agents and Chemotherapy 38, 1732–41.
13 . Masuda, N., Sakagawa, E. & Ohya, S. (1995). Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 645–9.[Abstract]
14 . Masuda, N., Gotoh, N., Ohya, S. & Nishino, T. (1996). Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 40, 909–13.[Abstract]
15
.
Zhao, Q., Li, X.-Z., Srikumar, R. & Poole, K. (1998). Contribution of outer membrane efflux protein OprM to antibiotic resistance in Pseudomonas aeruginosa independent of MexAB. Antimicrobial Agents and Chemotherapy 42, 1682–8.
16
.
Li, X.-Z., Zhang, L., Srikumar, R. & Poole, K. (1998). Betalactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 399–403.
17
.
Srikumar, R., Li, X.-Z. & Poole, K. (1997). Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa. Journal of Bacteriology 179, 7875–81.
18
.
Zimmermann, W. (1980). Penetration of ß-lactam antibiotics into their target enzymes in Pseudomonas aeruginosa: comparison of a highly sensitive mutant with its parent strain. Antimicrobial Agents and Chemotherapy 18, 94–100.
19
.
Evans, K, Passador, L., Srikumar, R., Tsang, E., Nezezon, J. & Poole, K. (1998). Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa. Journal of Bacteriology 180, 5443–7.
20
.
Srikumar, R., Kon, T., Gotoh, N. & Poole, K. (1998). Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain. Antimicrobial Agents and Chemotherapy 42, 65–71.
21
.
Zhang, L., Li, X.-Z. & Poole, K. (2000). Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system. Antimicrobial Agents and Chemotherapy 44, 287–93.
22
.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 265–75.
23 . Lugtenberg, B., Meijers, J., Peters, R., van der Hoek, P. & van Alphen, L. (1975). Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K-12 into four bands. FEBS Letters 58, 254–8.[Web of Science][Medline]
24
.
Srikumar, R., Paul, C. J. & Poole, K. (2000). Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa. Journal of Bacteriology 182, 1410–4.
25
.
Angus, B. L., Carey, A. M., Caron, D. A., Kropinski, A. M. B. & Hancock, R. E. (1982). Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant. Antimicrobial Agents and Chemotherapy 21, 299–309.
26
.
Köhler, T., Epp, S. F., Curty, L. K. & Pechere, J. C. (1999). Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa. Journal of Bacteriology 181, 6300–5.
27
.
Gotoh, N., Tsujimoto, H., Tsuda, M., Okamoto, K., Nomura, A., Wada, T. et al. (1998). Characterization of the MexC-MexD-OprJ multidrug efflux system in mexA-mexB-oprM mutants of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 1938–43.
Received 2 August 2000; accepted 4 September 2000
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  P. D. Lister, D. J. Wolter, and N. D. Hanson Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms Clin. Microbiol. Rev., October 1, 2009; 22(4): 582 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Strateva and D. Yordanov Pseudomonas aeruginosa - a phenomenon of bacterial resistance J. Med. Microbiol., September 1, 2009; 58(9): 1133 - 1148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Wolter, J. A. Black, P. D. Lister, and N. D. Hanson Multiple genotypic changes in hypersusceptible strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients do not always correlate with the phenotype J. Antimicrob. Chemother., August 1, 2009; 64(2): 294 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Baughman The Use of Carbapenems in the Treatment of Serious Infections J Intensive Care Med, July 1, 2009; 24(4): 230 - 241. [Abstract] [PDF]
|
|
|
|
 |
|
 L. E. P. Dietrich, T. K. Teal, A. Price-Whelan, and D. K. Newman Redox-Active Antibiotics Control Gene Expression and Community Behavior in Divergent Bacteria Science, August 29, 2008; 321(5893): 1203 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Damier-Piolle, S. Magnet, S. Bremont, T. Lambert, and P. Courvalin AdeIJK, a Resistance-Nodulation-Cell Division Pump Effluxing Multiple Antibiotics in Acinetobacter baumannii Antimicrob. Agents Chemother., February 1, 2008; 52(2): 557 - 562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mesaros, Y. Glupczynski, L. Avrain, N. E. Caceres, P. M. Tulkens, and F. Van Bambeke A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa J. Antimicrob. Chemother., March 1, 2007; 59(3): 378 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Pumbwe, O. Ueda, F. Yoshimura, A. Chang, R. L. Smith, and H. M. Wexler Bacteroides fragilis BmeABC efflux systems additively confer intrinsic antimicrobial resistance J. Antimicrob. Chemother., July 1, 2006; 58(1): 37 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Sobel, S. Neshat, and K. Poole Mutations in PA2491 (mexS) Promote MexT-Dependent mexEF-oprN Expression and Multidrug Resistance in a Clinical Strain of Pseudomonas aeruginosa J. Bacteriol., February 15, 2005; 187(4): 1246 - 1253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Maseda, I. Sawada, K. Saito, H. Uchiyama, T. Nakae, and N. Nomura Enhancement of the mexAB-oprM Efflux Pump Expression by a Quorum-Sensing Autoinducer and Its Cancellation by a Regulator, MexT, of the mexEF-oprN Efflux Pump Operon in Pseudomonas aeruginosa Antimicrob. Agents Chemother., April 1, 2004; 48(4): 1320 - 1328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Van Bambeke, Y. Glupczynski, P. Plesiat, J. C. Pechere, and P. M. Tulkens Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy J. Antimicrob. Chemother., May 1, 2003; 51(5): 1055 - 1065. [Full Text] [PDF]
|
|
|
|
|
|
D. Hocquet, C. Vogne, F. El Garch, A. Vejux, N. Gotoh, A. Lee, O. Lomovskaya, and P. Plesiat MexXY-OprM Efflux Pump Is Necessary for Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides Antimicrob. Agents Chemother., April 1, 2003; 47(4): 1371 - 1375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Dean, M. A. Visalli, S. J. Projan, P.-E. Sum, and P. A. Bradford Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1 Antimicrob. Agents Chemother., March 1, 2003; 47(3): 972 - 978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. H. Jo, F. S. L. Brinkman, and R. E. W. Hancock Aminoglycoside Efflux in Pseudomonas aeruginosa: Involvement of Novel Outer Membrane Proteins Antimicrob. Agents Chemother., March 1, 2003; 47(3): 1101 - 1111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Z. Li, K. Poole, and H. Nikaido Contributions of MexAB-OprM and an EmrE Homolog to Intrinsic Resistance of Pseudomonas aeruginosa to Aminoglycosides and Dyes Antimicrob. Agents Chemother., January 1, 2003; 47(1): 27 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Grkovic, M. H. Brown, and R. A. Skurray Regulation of Bacterial Drug Export Systems Microbiol. Mol. Biol. Rev., December 1, 2002; 66(4): 671 - 701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Aendekerk, B. Ghysels, P. Cornelis, and C. Baysse Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium Microbiology, August 1, 2002; 148(8): 2371 - 2381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhang, X.-Z. Li, and K. Poole Fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia J. Antimicrob. Chemother., October 1, 2001; 48(4): 549 - 552. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||