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Antimicrobial Agents and Chemotherapy, January 1998, p. 65-71, Vol. 42, No. 1
Department of Microbiology and Immunology,
Queen's University, Kingston, Ontario K7L 3N6,
Canada,1 and
Department of
Microbiology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan2
Received 23 June 1997/Returned for modification 16 September
1997/Accepted 1 November 1997
The mexCD-oprJ and mexAB-oprM operons
encode components of two distinct multidrug efflux pumps in
Pseudomonas aeruginosa. To assess the contribution of
individual components to antibiotic resistance and substrate
specificity, these operons and their component genes were cloned and
expressed in Escherichia coli. Western immunoblotting
confirmed expression of the P. aeruginosa efflux pump
components in E. coli strains expressing and deficient in
the endogenous multidrug efflux system (AcrAB), although only the
Pseudomonas aeruginosa is
an opportunistic human pathogen characterized by an intrinsic
resistance to a variety of antimicrobial agents. Previously attributed
to a highly impermeable outer membrane (24), this property
is now recognized to result from the synergy between broadly specific
drug efflux pumps and low outer membrane permeability (22).
One such efflux system, encoded by the mexAB-oprM operon
(8, 32, 33), acts on a range of antibiotics, including tetracycline, chloramphenicol, quinolones, novobiocin, macrolides, trimethoprim, and, apparently, Expressed constitutively in wild-type cells, where it contributes to
intrinsic drug resistance (4, 17, 33), the
mexAB-oprM operon is hyperexpressed in nalB
mutants (34), producing elevated levels of resistance to
substrate antibiotics (8, 16, 17, 33). The MexAB-OprM efflux
system is highly conserved in serotype, clinical, and environmental
strains (2), indicating that it plays an important role in
the intrinsic resistance of all examples of this organism. Homologous
efflux systems encoded by the mexCD-oprJ (31) and
mexEF-oprN (15) operons have also been described. Apparently not expressed during growth under normal laboratory conditions, these systems are expressed in nfxB
(31) and nfxC (15) multidrug-resistant
mutants, respectively. nfxB strains are resistant to
chloramphenicol, tetracycline, quinolones, macrolides, novobiocin, and
"fourth generation" cephalosporins (such as cefepime and cefpirome)
but display hypersusceptibility to most The tripartite efflux pumps consist of an inner membrane component
(e.g., MexB, MexD, or MexF) which functions as an RND family H+ antiport exporter (23, 35), an outer
membrane, presumably channel-forming component (e.g., OprM, OprJ, or
OprN) (22, 25), and a so-called membrane fusion protein
predicted to link the membrane-associated efflux components (e.g.,
MexA, MexC, or MexE) (22, 25). Recent data suggest that the
operation of MexAB-OprM (and by analogy the remaining efflux systems)
is at least partially dependent upon the TonB energy-coupling protein
implicated in the opening of outer membrane gated channels responsible
for iron-siderophore uptake across the P. aeruginosa outer
membrane (45). Thus, the outer membrane components of these
efflux pumps may be gated channels.
Related efflux systems have been described in Escherichia
coli (acrAB [20, 22] and
acrEF [22], formerly called
envCD [14]) and Neisseria
gonorrhoeae (mtrCDE [9]). The AcrAB
efflux components appear to function in conjunction with the TolC outer membrane protein (5), a pore-forming protein (1)
previously implicated in hemolysin (42) and colicin V
(44) export across the outer membrane. The
acrAB-tolC-encoded efflux system is the primary known efflux
system contributing to intrinsic resistance in E. coli, and
acrAB deletion strains are markedly susceptible to a variety
of antimicrobial agents (21, 30).
In an effort to assess the role of individual efflux system components
in resistance and substrate specificity and to demonstrate that
antibiotic resistance attributed to the multidrug efflux pumps in
P. aeruginosa is a true indication of their efflux by these
pumps, and in particular that Bacterial strains and plasmids.
Bacterial strains and
plasmids used in this study are described in Table
1. For the selection of nfxB
mutants hyperexpressing MexCD-OprJ (e.g., K1111), overnight cultures of
P. aeruginosa ML5087 were plated on Luria broth-NaCl (LB
[see "Media and culture conditions" below]) plates containing 0.4 µg of ciprofloxacin/ml. MexAB-OprM-hyperexpressing nalB
mutants of this strain (e.g., K1112) were selected on 0.2 µg of
ciprofloxacin/ml and 8 µg of cefoperazone/ml. The strains were
confirmed as nfxB and nalB by antimicrobial
susceptibility testing and by Western immunoblotting of cell envelopes
with antibodies specific to OprJ and OprM, respectively (see Fig. 1).
The mexCD-oprJ operon was previously cloned into plasmid
pAK1900 on a ca. 10-kb BamHI fragment to yield pKMJ002. Restriction analysis of pKMJ002 revealed that mexCD-oprJ was
in an orientation opposite to that of the lac promoter
(plac) of this vector. To facilitate mexCD-oprJ
expression in E. coli, the 10-kb BamHI fragment
containing mexCD-oprJ was recloned into two other vectors,
pRK415 (yielding pRSP15) and pAK1900 (yielding pRSP45), such that the
operon was in the same orientation as plac. The
mexCD genes were also cloned into pRK415 in the same
orientation as plac (yielding pRSP25) on an 8.7-kb
BamHI fragment derived from pRSP23, a pKMJ002 derivative in
which oprJ had been eliminated by the deletion of two
internal EcoRV fragments (0.7 and 0.8 kb). This same
BamHI fragment was cloned into pAK1900, again with the mexCD genes in the same orientation as plac, to
yield pRSP46. Finally, oprJ was cloned into pRK415 in the
same orientation as plac on a 2.5-kb KpnI
fragment to yield pRSP06. An 8.5-kb HindIII fragment
containing the mexAB-oprM operon of pPV1 was previously cloned into pAK1900, and the resulting plasmid was designated pPV20.
Restriction analysis of pPV20 revealed that mexAB-oprM was
also transcribed in the opposite direction to the lac
promoter of this vector. To facilitate mexAB-oprM expression
in E. coli, the 8.5-kb HindIII fragment of
pPV20 was recloned into pAK1900 (to yield pRSP01) and pRK415 (to yield
pRSP17) in the same orientation as plac. pPV6, a pAK1900
derivative carrying mexAB on a ca. 5-kb HindIII fragment, was initially obtained by cloning a
mexAB-containing 9-kb XhoI fragment from pPV1
into the unique SalI site of pT7-6 (to yield pRSK01)
followed by its recovery on a ca. 5-kb HindIII fragment
(one HindIII site upstream of mexA and a
second in the multiple cloning site [MCS], downstream of the
SalI-XhoI hybrid site). The 5-kb
mexAB-containing HindIII fragment was
subsequently cloned from pPV6 into pRK415 in the same orientation as
plac to yield pRSP19. pRSP08 is a pRK415 derivative carrying
the oprM gene on a 4.2-kb PstI fragment of pRSP01
in the same orientation as plac.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Expression of Pseudomonas aeruginosa
Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a
Multidrug-Sensitive Escherichia coli Strain
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
acrAB strain, KZM120, demonstrated increased resistance
to antibiotics in the presence of the P. aeruginosa efflux
genes. E. coli KZM120 expressing MexAB-OprM showed
increased resistance to quinolones, chloramphenicol, erythromycin,
azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin,
and, significantly, several 
-lactams, which is reminiscent of the
operation of this pump in P. aeruginosa. This confirmed
previous suggestions that MexAB-OprM provides a direct contribution to
-lactam resistance via the efflux of this group of antibiotics. An
increase in antibiotic resistance, however, was not observed when MexAB
or OprM alone was expressed in KZM120. Thus, despite the fact that
-lactams act within the periplasm, OprM alone is insufficient to
provide resistance to these agents. E. coli KZM120
expressing MexCD-OprJ also showed increased resistance to quinolones,
chloramphenicol, macrolides, SDS, and crystal violet, though not to
most -lactams or novobiocin, again somewhat reminiscent of the
antibiotic resistance profile of MexCD-OprJ-expressing strains of
P. aeruginosa. Surprisingly, E. coli KZM120
expressing MexCD alone also showed an increase in resistance to these
agents, while an OprJ-expressing KZM120 failed to demonstrate any
increase in antibiotic resistance. MexCD-mediated resistance, however,
was absent in a tolC mutant of KZM120, indicating that
MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the
efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their
substrate specificity in E. coli.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
-lactams and -lactamase inhibitors (8, 16-18, 33). The -lactam--lactamase inhibitor
group of antibiotics are somewhat unique in that their cellular targets are within the periplasm. In contrast to the remaining antibiotic substrates, which act within the cytoplasm and are thus expected to
access the pump at the cytoplasmic face of the inner membrane, the
-lactam--lactamase inhibitor compounds, if truly eliminated by
MexAB-OprM, must interact differently with pump components. Possibilities include interaction with inner membrane constituents at
the periplasmic face of the inner membrane or efflux via the outer
membrane OprM alone. Still, it is not yet clear that MexAB-OprM and/or
the nalB mutation associated with MexAB-OprM overexpression (see below) does not influence -lactam resistance via an indirect effect on another resistance determinant within P. aeruginosa.
-lactam antibiotics
(10). nfxC strains elicit resistant to
chloramphenicol, trimethoprim, quinolones, and carbapenems, including
imipenem, although the last results from the loss of the porin
protein OprD in these mutants and not from overexpression of MexEF-OprN
(6, 15).
-lactams are truly substrates for
MexAB-OprM, the mexAB-oprM and mexCD-oprJ operons
were expressed in a multidrug-sensitive acrAB E. coli
strain. We report here that these systems were indeed expressed in
E. coli, where they contributed the expected resistance to
dyes, detergents, and antibiotics, including -lactams.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
TABLE 1.
Bacterial strains and plasmids
Media and culture conditions. Liquid media for routine culturing of E. coli and P. aeruginosa strains were prepared by dissolving 15.5 g of Miller's Luria broth base (Difco) and 2 g of NaCl per liter of H2O (LB). For the selection of chromosomal or plasmid antibiotic resistance markers in E. coli, ampicillin at 100 µg per ml, kanamycin at 50 µg per ml, and tetracycline at 10 µg per ml were used in the growth media. Solid media were obtained by the addition of Bacto Agar (1.5% [wt/vol]; Difco).
Molecular biological techniques.
Restriction endonuclease
digestions, ligations, and transformations were performed as described
by Sambrook et al. (36). E. coli DH5
was used
as the host for large-scale isolation of plasmid DNA with a plasmid
maxi kit (Qiagen). DNA fragments used in cloning were extracted from
agarose gels with Prep-A-Gene (Bio-Rad) according to the
manufacturer's instructions. E. coli cells were made
competent by the CaCl2 method (36) or, when
highly competent E. coli were required, by the method of
Inoue et al. (12).
Isolation of cell envelopes.
Twenty milliliters of bacterial
culture was grown in LB to an absorbance at 600 nm
(A600) of 1.00, harvested by centrifugation, and
stored at 
20°C. Cells were later thawed on ice, resuspended in 1 ml
of phosphate-buffered saline (1.67 mM
NaH2PO4 · H2O-8.09 mM
Na2HPO4-150 mM NaCl [pH 7.4]), and sonicated
until the cell suspension was clear. Following centrifugation
(8,000 × g; 10 min) to pellet unlysed cells and
debris, the resulting supernatant was centrifuged (300,000 × g; 15 min) and the cell envelope pellet was resuspended in
100 µl of H2O.
Purification of MexB and generation of rabbit polyclonal
antiserum.
To generate antibodies to MexB, the protein was
purified as polyhistidine-tagged MexB (MexB-His) following the cloning
and expression of the gene in plasmid pET21-d(+) (Pharmacia). To clone mexB into this vector so that the 3' end of the gene was in
frame with the polyhistidine-encoding sequences of pET21-d(+), the
following approach was taken. First, the bulk of mexB was
released from plasmid pPV6 by digestion with EcoRI and
KpnI, with EcoRI cutting upstream of the gene
within mexA and KpnI cutting 133 bp upstream of
the mexB terminus. The 133 bp at the 3' end of
mexB were then amplified with Vent polymerase (New England
Biolabs) and primers TK-1
(5'-GATCGGTACCGGCGTGATCGGCGGCATGGTCACTGCGACCGTCCTGGCGATCTTCTGGGTGCC-3') and TK-2 (5'-AATTCTCGAGTTGCCCCTTTTCGACGGACG-3'). TK-1
eliminates a KpnI site within the 3' region of
mexB as a result of an A-G change (highlighted in bold
italics) which does not alter the amino acid sequence of MexB but does
permit subsequent digestion of the PCR product with KpnI and
XhoI so that it can be cloned together with the
aforementioned mexB-containing
EcoRI-KpnI fragment to regenerate an intact
mexB gene. PCR mixtures (100 µl) contained 20 ng of pPV6,
1 µM each primer, 200 µM each deoxynucleoside triphosphate, 4 mM
MgSO4, 10% (vol/vol) dimethylsulfoxide, and 1 U of Vent
polymerase in 1× reaction buffer. Mixtures were heated at 94°C for 2 min followed by 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min a 72°C before finishing with 10 min at 72°C. The PCR product
was then purified with the Qiaquick PCR purification kit (Qiagen), digested with KpnI and XhoI, and cloned into
KpnI-XhoI-restricted pSL1180. The insert was
sequenced to ensure that the complete 133-bp 3' region had been cloned
and, except for the elimination of the KpnI site, that no
alterations in the mexB sequences had been introduced during
PCR. The mexB-containing 3.8-kb
EcoRI-KpnI fragment was then cloned into
EcoRI-KpnI-restricted pSL1180 carrying the cloned
PCR product, and the restored mexB gene was then recovered on a ca. 3.9-kb EcoRI-XhoI fragment. Following
the cloning of this fragment into
EcoRI-XhoI-restricted pET21-d(+), the resultant vector, pTK15, was introduced into E. coli BL21(DE3)
carrying the pLysS plasmid (strain K113) and expression of
mexB was induced with IPTG
(isopropyl--D-thiogalactopyranoside). Briefly, overnight cultures of pTK15-carrying K113 in LB containing appropriate
antibiotics were diluted 1:49 in the same medium (500 ml) and incubated
for 4 h, at which time IPTG was added (0.2 mM final
concentration). Two hours later, cells were harvested by centrifugation
(8,000 × g) and cell envelopes were prepared (as
above) and solubilized in 2 ml of 20 mM Tris-HCl (pH 8.0)-100 mM
NaCl-2% (vol/vol) Triton X-100. The MexB-containing Triton
X-100-soluble material (supernatant fraction) was then recovered,
following centrifugation (300,000 × g), and applied to
a 1.5-ml TALON (Clonetech Laboratories, Inc., Palo Alto, Calif.) column
equilibrated with 20 mM Tris-HCl (pH 8.0)-100 mM NaCl-0.5% (vol/vol)
Triton X-100 (column buffer). The column was washed with 15 ml of
column buffer, and bound protein was eluted with column buffer
containing 100 mM imidazole at a flow rate of 0.1 ml/min. Three
hundred-microliter fractions were collected, and MexB-containing
fractions, identified following analysis by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were pooled and
dialyzed against 2 liters of 20 mM Tris-HCl (pH 8.0)-0.5% (vol/vol)
Triton X-100. Antibodies to purified MexB-His were subsequently raised
in rabbits by L. Mutharia, Department of Microbiology, University of
Guelph.
SDS-PAGE and immunoblotting. SDS-PAGE was performed as described previously (19) with 10% (wt/vol) acrylamide in the running gel. Proteins resolved in gels were Coomassie blue stained or transferred electrophoretically to nitrocellulose membranes (BA85; Schleicher & Schuell). Electrophoretic transfer of proteins was as described previously (41) except that SDS (0.1% [wt/vol]) was included in the blotting buffer and transfer was carried out for 16 h at 4°C and 25 mA constant current. Blotted membranes were subsequently incubated in phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 (BDH) (PBST) and 10% (wt/vol) skim milk (Difco) for 60 min. Following two 5-min washes with PBST, the membranes were incubated for 60 min with primary antibody in PBST containing 1% (wt/vol) bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) and then washed four times for 10 min each time with PBST. Secondary antibody conjugated to horseradish peroxidase (HRP) in PBST containing 1% bovine serum albumin was then added to the membranes, which were subsequently washed four times for 10 min each time with PBST. Substrates for HRP were from the ECL Western blotting detection kit (Amersham) and were used according to the manufacturer's instructions. The enzymatic activity of HRP was detected as the emission of chemiluminescence after the exposure of blots to XAR 5 film (Kodak). All incubations and washings in the immunoblot procedure were carried out at room temperature with agitation. The primary antibodies anti-OprJ (11) and anti-OprM (7) were previously described mouse monoclonals, and anti-MexB was a rabbit polyclonal antiserum (see above). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson Laboratories and Amersham, respectively.
Antimicrobial susceptibility testing. The susceptibilities of E. coli strains to a number of antimicrobial agents were tested by inoculating 1 ml of LB containing serial twofold dilutions of each antimicrobial agent with 105 organisms as described previously (17). Bacterial inocula were derived from stocks prepared from overnight cultures grown in LB which had been harvested and resuspended in an equal volume of 10 mM MgSO4.
Antimicrobial agents. Ampicillin, penicillin G, ticarcillin, cefotaxime, cefoperazone, cefsulodin, cephaloridine, ceftriaxone, ciprofloxacin, norfloxacin, novobiocin, chloramphenicol, and tetracycline were purchased from Sigma. SDS and crystal violet were purchased from ICN Biochemicals, Inc., and Difco, respectively. Cefepime was provided by Bristol-Myers Squibb. Azithromycin was a gift from Pfizer. Sparfloxacin (Rhône-Poulenc Rorer) and pefloxacin (Laboratoire Roger Bellon) were gifts from Microcide Pharmaceuticals Inc., Mountain View, Calif. Cefpirome (Roussel Uclaf) and ceftazidime (Glaxo) were also gifts. Erythromycin (Abbott) and imipenem (Merck Sharpe & Dohme) were purchased from the pharmacy of the Kingston General Hospital.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Expression of MexAB-OprM in E. coli.
To assess the
operation of MexAB-OprM in E. coli, it was necessary to
express the P. aeruginosa multidrug efflux system in this
heterologous host. Using antibodies specific to MexB and OprM (Fig. 1A
and B, lanes 2), therefore, we examined
the production of pump components in E. coli. Initially,
mexAB-oprM was cloned into the low-copy-number,
broad-host-range vector pRK415 (to yield pRSP17) in the same
orientation as the lac promoter of this vector in case it
was necessary to induce expression of the operon in order to obtain
detectable expression of the pump components. Introduction of pRSP17
into E. coli DH5 (AcrAB+) (data not
shown) or KZM120 (AcrAB
) provided for substantial
expression of MexAB-OprM without IPTG induction, as evidenced by the
production of MexB (Fig. 1A, lane 8) and OprM (Fig. 1B, lane 8) in cell
envelopes of these strains. Similarly, E. coli KZM120
carrying the mexAB vector pRSP19 or the oprM
vector pRSP08 produced substantial levels of cell envelope-associated MexB (Fig. 1A, lane 9) and OprM (Fig. 1A, lane 7), respectively, without IPTG induction. Thus, E. coli carrying
mexAB-oprM (or mexCD-oprJ [see below]) or its
components was assessed for antibiotic susceptibility in the absence of
IPTG.
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Activity of MexAB-OprM in E. coli.
Despite the
expression of MexAB-OprM in E. coli DH5, the presence of
pRSP17 failed to enhance the antibiotic resistance of this strain to
any of the tested agents (data not shown) (Tables 2 and 3
list the agents tested). In contrast, the acrAB deletion strain KZM120 carrying pRSP17 exhibited elevated resistance to a
variety of agents, including the more hydrophobic quinolones, novobiocin, macrolides, chloramphenicol, detergents, dyes, and the
penicillin subgroup of the -lactam antibiotics (Tables 2 and 3).
Although the influence of pRSP17 on tetracycline resistance could not
be assessed (the vector carries a tet gene), KZM120 carrying
pRSP01, a bla vector with the mexAB-oprM operon,
did show a twofold increase in resistance to tetracycline (data not shown).
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-lactam
resistance in E. coli, for example, was generally limited to
the penicillins (e.g., ampicillin, penicillin G, and ticarcillin),
whose nuclei are more hydrophobic than those of the cephalosporins
(43). Moreover, although MexAB-OprM-mediated resistance to
the cephalosporins cefotaxime, ceftazidime, ceftriaxone, cefoperazone,
cefpirome, and cefepime was minimal or lacking in E. coli,
resistance to these cephalosporins is readily provided by this efflux
system in P. aeruginosa (17, 37, 38), indicating
that the lack of cephalosporin resistance in MexAB-OprM-expressing
E. coli is not due to a deficiency of the pump. The
observation that MexAB-OprM-expressing E. coli is less
susceptible (though still not resistant) to cefoperazone is likely
explained by the presence of an exceptionally bulky side chain
(43), which is expected to impede the drug's passage across
the outer membrane, thereby enhancing the contribution of efflux to
resistance to this agent.
Arguably, the most significant observation was that -lactam
(penicillin) resistance was afforded by MexAB-OprM in E. coli KZM120, since this confirmed that this efflux system can
indeed accommodate -lactams and that it is directly responsible for the -lactam resistance attributed to it in, e.g., nalB
strains. Interestingly, however, and despite the periplasmic location
of their targets, KZM120 expressing oprM alone did not
exhibit enhanced resistance to -lactams (or any other agent) (Tables
2 and 3), indicating that the inner membrane-associated components are
essential features of a -lactam-extruding pump and that OprM alone
is unable to facilitate -lactam efflux and, thus, resistance.
Similarly, MexAB alone (pRSP19) also failed to influence antibiotic
resistance in the acrAB strain (Tables 2 and 3),
demonstrating that a tripartite pump is the only efflux-competent
entity.
Although not previously reported for P. aeruginosa, the
observation that MexAB-OprM expression afforded resistance to crystal violet and SDS in E. coli indicated that, as with AcrAB, the
MexAB-OprM efflux pump can accommodate dyes and detergents.
Interestingly, recent comparisons of MexB-OprM+ and
MexAB-OprM strains of P. aeruginosa failed to
reveal any differences in susceptibility to either of these agents
(data not shown). The MexAB-OprM strain was, in fact,
very resistant to both SDS and crystal violet (data not shown),
presumably due to the presence of additional efflux systems in this
strain which are capable of exporting these compounds.
Expression of MexCD-OprJ in E. coli.
pRSP15 carrying
mexCD-oprJ was also introduced into E. coli
DH5 and KZM120, and expression of the efflux system was assessed by
immunoblotting with an available anti-OprJ antiserum. As above, E. coli DH5 (data not shown) and KZM120 carrying this
vector demonstrated OprJ production (Fig. 1C, lane 5) consistent with the expression of the mexCD-oprJ operon in this strain.
Interestingly, introduction of pKMJ002 (the original
mexCD-oprJ vector, which carries the efflux genes in an
orientation opposite to that of the lac promoter on this
plasmid) into KZM120 failed to elicit any antibiotic resistance,
indicating that, while IPTG induction may not have been necessary for
expression of efflux components, expression from the lac
promoter was probably critical. KZM120 carrying oprJ alone
on pRSP06 also demonstrated substantial OprJ production (Fig. 1C, lane
4); in fact, levels were markedly higher than those seen for KZM120
carrying pRSP15. This difference in OprJ expression may be due to the
presence of the nfxB gene, encoding a repressor of
mexCD-oprJ expression (28, 31), on pRSP15, which
might have limited mexCD-oprJ (and hence OprJ) expression from this vector. The ability of the nfxB gene to repress
expression of a mexC-lacZ fusion in E. coli has
been demonstrated (31). In contrast, the
mexAB-oprM-encoding plasmid pRSP17 lacks the mexR
repressor gene implicated in regulation of mexAB-oprM
(34), and accordingly, the levels of OprM detected in KZM120
carrying oprM alone (Fig. 1B, lane 7) or
mexAB-oprM (Fig. 1B, lane 8) were similar.
Activity of MexCD-OprJ in E. coli.
E. coli KZM120
carrying mexCD-oprJ on pRSP15 demonstrated elevated
resistance to a variety of agents, including macrolides, quinolones,
chloramphenicol, SDS, and crystal violet (Table 2). As was seen for
MexCD-OprJ in P. aeruginosa, this efflux system did not
facilitate resistance to novobiocin (Table 2) or most -lactams
(Table 3) in E. coli. The decreased susceptibility of
MexCD-OprJ-expressing E. coli to cefoperazone, however, was consistent with previous observations that nfxB strains of
P. aeruginosa show slightly reduced cefoperazone
susceptibility (38). Interestingly, although
MexCD-OprJ-expressing strains of P. aeruginosa display
resistance to the fourth generation cephalosporins cefepime and
cefpirome (17, 38), this efflux system failed to alter the
susceptibility of E. coli KZM120 to these agents (Table 3), again highlighting the point that the P. aeruginosa efflux
systems are not as effective in providing resistance to substrate
antibiotics in E. coli. The expression of OprJ
alone on pRSP06 (Fig. 1C, lane 4) did not increase the
resistance of KZM120 to any tested antimicrobial agent (Table 2),
although KZM120 expressing MexCD from pRSP25 exhibited the same profile
of resistances as the MexCD-OprJ-expressing KZM120(pRSP15) (Table 2).
The dispensability of OprJ suggests that some E. coli outer
membrane protein substitutes for OprJ in the operation of this efflux
system.
Role of TolC in MexCD-mediated antibiotic resistance in E. coli.
Previous studies have shown that deletion of oprJ
in a mexCD-oprJ-hyperexpressing nfxB strain
compromised the resistance afforded by the MexCD-OprJ system
(38), indicating that the MexCD components are not efflux
competent, at least in P. aeruginosa, in the absence of an
outer membrane pump constituent. The TolC outer membrane protein,
implicated as the outer membrane component of the AcrAB efflux system
(5), is one possible candidate for association with MexCD in
the reconstitution of an efflux system in E. coli. Precedents exist for the functioning of such so-called chimeric efflux
systems, since functional MexCD-OprM and MexAB-OprJ pumps have been
successfully constructed in P. aeruginosa (38).
To assess the possible involvement of TolC in OprJ-independent,
MexCD-mediated multidrug resistance, the mexCD genes were
introduced into E. coli LBB1201, a KZM120 derivative
carrying a tolC::Tn10 mutation. Because
of the Tn10-encoded tetracycline resistance of this strain, however, it was necessary to introduce these genes on the pAK1900-based bla (-lactam resistance) plasmid pRSP46 rather than on
the aforementioned tetracycline resistance vector, pRSP25. The pRSP46
vector failed to increase the resistance of LBB1201 to several agents,
including erythromycin, azithromycin, norfloxacin, crystal violet, and
SDS, although as expected, it did increase the resistance of the
TolC+ strain KZM120 to these agents (Table 2), indicating
that the observed MexCD-mediated multidrug resistance in
acrAB strains of E. coli requires TolC. Thus,
the latter protein likely functions as the outer membrane efflux system
component of a MexCD-TolC pump. Introduction of mexCD-oprJ
on the pAK1900-based vector pRSP45 also facilitated resistance to
erythromycin, azithromycin, norfloxacin, crystal violet, and SDS in
both KZM120 and LBB1201 (Table 2), consistent with the native
MexCD-OprJ efflux system being operational in E. coli,
independent of the AcrAB-TolC components, and with strain LBB1201 being
capable of reconstituting a functional MexCD-OprJ efflux system.
Interestingly, the MexCD-OprJ system appeared to function better in the
TolC strain LBB1201 than in the TolC+ strain
KZM120 (Table 2), suggesting that by virtue of its apparent ability to
associate with MexCD, TolC may interfere with the proper association
and activity of the MexCD-OprJ components.
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ACKNOWLEDGMENTS |
|---|
We are grateful to H. Nikaido and J. Fralick for providing strains.
This work was supported by a grant from the Canadian Cystic Fibrosis Foundation to K.P. R.S. was the recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Research Council (NSERC). K.P. is an NSERC University Research Fellow.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada. Phone: 613-545-6677. Fax: 613-545-6796. E-mail: poolek{at}post.queensu.ca.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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