(Received for publication, May 20, 1997, and in revised form, June 26, 1997)
From the Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-11, Japan
The MexA-MexB-OprM efflux pump of
Pseudomonas aeruginosa consists of two inner membrane
proteins, MexA and MexB, and one outer membrane protein, OprM. We
investigated the role of the components of this drug extrusion system
by evaluating the repercussions of deleting these subunit components on
the accumulation of several fluorescent probes. Fluorescence
intensities of positively charged 2-(4-dimethylaminostyryl)-1ethylpyridinium and uncharged
N-phenyl-1-naphtylamine were 7 and 4 times higher,
respectively, in the mutant lacking OprM and 4 and 1.7 times higher,
respectively, in the mutants lacking MexA or MexB than in the wild type
strain. This order of fluorescence intensity was fully consistent with
a previously reported minimum inhibitory concentration of antibiotics
such as tetracycline, chloramphenicol, and fluoroquinolones. Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times
faster than the rate in the wild type cells. This result is in accord
with the minimum inhibitory concentration of -lactam antibiotics.
These results suggest that the fluorescence probes could be
successfully used in real time monitoring of the function of the drug
extrusion machinery in Gram-negative bacteria. The downhill extrusion
kinetics of
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which
orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded
by a slow increase in fluorescence intensity, whereas the wild type
cell immediately released the dye. This observation was explained by a
slow trans-cytoplasmic membrane crossing of intracellular dye in the
mutants. These results reflected higher accumulation of the probe in
the cytoplasmic membrane in the mutants and strengthened the hypothesis
that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM
mainly takes place from the interior of the cytoplasmic membrane.
Multiple antibiotic resistance in bacteria has been associated with the overexpression of endogenous efflux genes (for reviews see Refs. 1 and 2). In Pseudomonas aeruginosa, active extrusion (3, 4) combined with a tight diffusion barrier at the outer membrane (5, 6) are the main reasons for low specific resistance against diverse antibiotics and other toxic elements (7).
Multidrug resistance in P. aeruginosa is now attributed mainly to the overexpression of three sets of operons. Overexpression of the operon mexA-mexB-oprM is characteristic for nalB type multidrug-resistant mutants, (8, 9), whereas overexpression of the operon mexC-mexD-oprJ (10) is responsible for multidrug resistance in nfxB type mutants (11). A third type designated as nfxC has also been associated with a diminished intracellular drug accumulation (12). The genes coding for the proteins involved in the drug extrusion have been characterized recently (13) and named mexE-mexF-oprN.
A common characteristic of multiantibiotic-resistant mutants in P. aeruginosa is their broad resistance to quinolones, and hence one method generally used to assess multiantibiotic-resistant-type resistance mutations is based on determination of the time course of quinolones accumulation inside the cell. A critical review of the methods used for measuring the accumulation of quinolones discusses the weaknesses of these methods (14). A striking point observed by us and others is the fact that an increase in the MIC1 of quinolone of several orders of magnitude is often reflected by only about a 2-fold decrease in drug accumulation (3, 11, 12). A high background due to unspecific surface binding combined with the release of a substantial fraction of accumulated quinolone during the long washing period are responsible for these results.
Fluorescent probes, which change their spectroscopic properties upon entering the cell are particularly suitable for uptake experiments because they fluoresce weakly in aqueous environments, but become strongly fluorescent in nonpolar or hydrophobic environments. The possibility of energized extrusion of fluorescent membrane probes was suggested previously (15). Sedgwick and Bragg (16) used DMP to study the role of the cell envelope and of efflux systems in the uptake of lipophilic cations in Escherichia coli acrA mutants that showed hypersusceptibility toward acridine, cationic dyes, detergents, and antibiotics (17, 18) and lacked the ability to extrude these compounds in an energy-dependent manner (19). More recently the combination of several fluorescent probes provided more insight into the extrusion process of the LmrA protein of Lactococcus lactis, which is a member of the ATP-binding cassette superfamily (20).
We previously reported the construction of mutants lacking one, two, or all three components of the efflux system formed by the mexA-mexB-oprM gene products (21). In contrast to MexA-, MexB-, and OprM-deficient mutants constructed by others that were reported to be equally hypersusceptible to many antibiotics (22), we were able to divide our mutants into two groups with different antibiotic susceptibility profiles. All the mutants deficient in OprM exhibited 8-16 times higher susceptibility against fluoroquinolone antibiotics, chloramphenicol, and azthreonam than the parent strain, whereas mutants deficient in MexA, MexB, or MexA,B were only 2-4 times more susceptible to these antibiotics than the parent strain. The findings of the ciprofloxacin accumulation experiments were in agreement with the MIC results, but this method provided little information about uptake kinetics. The use of fluorescent probes combined with genetically defined mutants lacking the subunit protein(s) is therefore a powerful approach to obtaining more insight into the extrusion mechanism associated with mexA-mexB-oprM operon products. In this study, we investigated the response of DMP, TMA-DPH, NPN, and ethidium bromide to the lack of mexA, mexB, or oprM gene product in P. aeruginosa.
The following reagents were obtained from the indicated sources and used without further purification: DMP, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and carbonylcyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma, and ethidium bromide (EtBr) and NPN were purchased from Wako Chemicals.
Bacterial Strains and GrowthStrains used in this study are
listed in Table I. All the strains were
grown at 37 °C in LB medium (1% tryptone, 0.5% yeast extracts,
0.5% NaCl, 5 mM MgCl2, pH 7.2). Experimental
cultures inoculated at 10% with a fully grown overnight culture were
rotated at 200 rpm at 37 °C for 4 h. The mutants lacking MexA,
MexB, OprM, MexA-MexB, and MexA-MexB-OprM were abbreviated to MexA,
MexB,
OprM,
MexA,B, and
MexA,B-OprM, respectively.
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Cells were harvested at 7000 × g for 10 min, washed once with 100 mM NaCl-50 mM sodium phosphate buffer (pH 7.0), and suspended again in the same buffer at A600 = 0.1 in the presence of 0.05% of glycerol. Cell suspension at a higher cell density leads to rapid oxygen depletion in the uptake medium, resulting in respiration-related artifacts, especially during NPN and DMP accumulation experiments (23, 24). Experimental measurements were generally performed within 2 h after cell preparation.
Fluorescence MeasurementsFluorescence measurements were performed at 30 °C. DMP, TMA-DPH, and NPN were dissolved in absolute methanol and EtBr in water to the final concentrations indicated in the figure legends. CCCP was also dissolved in absolute methanol. Control experiments indicated that the presence of methanol at the maximal experimental concentration of 2.5% did not have a significant effect. Fluorescence emission intensity was measured with a Hitachi 650-10S fluorometer equipped with a Lauda RM6 circulating water bath, and the data were registered on a Hitachi 056 recorder. Excitation and emission wavelengths for DMP were, respectively, 467 and 557 nm, for TMA-DPH 350 and 425 nm, for EtBr 520 and 590 nm, and for NPN 340 and 420 nm. Slit widths were all set at 5 nm for excitation and at 10 nm for emission.
The neutral probe NPN has been
used to monitor changes in bacterial membranes from the energized to
nonenergized state (23) or to monitor outer membrane permeabilization
(25). The addition of NPN to the cell suspension leads to a rapid
increase in the fluorescence intensity at 420 nm in comparison with
fluorescence without cells (Fig.
1A). Initial rapid increments
within a period of less than 1 min were higher in the deletion mutants
when compared with the parent strain, but this level was nearly the
same for all mutants. A second slower incremental phase at 0.5-4 min
could be observed in all mutants. Eventual fluorescence intensity in the OprM and MexA or MexB mutants were, respectively, 3.7 and 1.7 times
higher than that in the parent strain. This second phase was absent in
the parent strain due to active efflux of the dye. In parallel with the
increase in emission intensity, a blue shift in the emission spectra
was observed (Fig. 1B)
reflecting the movement of the probe to a more hydrophobic environment.
These results suggested that the lowest fluorescence of NPN in the wild type cell may be due to lower accumulation of the dye resulting from
extrusion of NPN by the MexA,B-OprM efflux system. Conversely, the
highest and intermediate fluorescence intensities in the mutant lacking
OprM (also MexA,B-OprM) and
MexA or
MexB, respectively, may
have been attributable to a total or partial dysfunction of the Mex
pump.
DMP Fluorescence Response in the Wild Type Strain and MexA-, MexB-, or OprM-deficient Mutants
Increase of DMP fluorescence intensity
in the presence of cells is attributed to movement of the dye molecules
from the head group region of the cytoplasmic membrane bilayer into the
region of the fatty acyl chains (24). An instantaneous rise in
fluorescence emission at 567 nm upon addition of the probe to the cell
suspension was followed by a slower phase in the OprM,
MexA, or
MexB (Fig. 2A). The rate of increment for
OprM was
significantly higher than for
MexA or
MexB, whereas no
significant variation in the fluorescence intensity was detected in the
parent strain. The final fluorescence level attained in the OprM mutant
was about twice as high as that in the
MexA or
MexB mutants and 7 times higher than that in the parent strain. These results suggest that DMP may have also been efficiently extruded in the wild type strain. In
the mutants, the dye accumulates at higher levels and fluoresces strongly, which is most likely attributable to pump dysfunction. These
observations strongly suggest the involvement of
mexA-mexB-oprM gene products in DMP and NPN extrusion. In
contrast to NPN, the fluorescence emission spectrum and the increase in
DMP fluorescence in the deletion mutants was not accompanied by a shift
of the emission maximum (Fig. 2B). The differences in NPN
and DMP fluorescence intensity increment among various kinds of mutants
was correlated with the MICs of the antibiotics in these cells,
reported elsewhere (21).
Before the discovery of active drug efflux
systems, the outer membrane barrier was considered the main cause of
low specific antimicrobial resistance in Gram-negative bacteria
including P. aeruginosa (26). To evaluate the importance of
the outer membrane barrier and the role of OprM in the overall
accumulation rate, the impact of outer membrane permeabilization was
investigated. DMP accumulations in PAO4290 and its Mex operon knock-out
mutants in the presence and the absence of EDTA are presented in Fig. 3. If we assume that DMP fluorescence
emission observed in the parent strain to be the minimum value and the
emission in the OprM strain to be the maximum value, the addition of
EDTA to PAO4290 resulted in a fluorescence increment of only 15% of
the maximum value. The
MexA,B mutant reached a fluorescence
intensity of about 45% of that in the
OprM mutant, which in the
presence of EDTA was increased to 90%. The effect of EDTA on DMP
fluorescence in the
OprM mutant is reflected by a rise of only 10%.
Small fluorescence increments in the presence of EDTA for PAO4290 and
OprM were attributable to destruction of the outer membrane barrier. Furthermore, a small increment of DMP fluorescence in the parent strain
after the addition of EDTA suggests that the ability to extrude the
substrate compound is still maintained under these conditions. These
results suggest that increased accumulation of the dye in
OprM
mutant is not due to a higher periplasmic dye concentration, because
outer membrane permeabilization allows instantaneous equilibration
between the outer medium and the periplasmic space. Consequently, we
concluded that MexA,B and OprM form a functional unit during the
extrusion process, in which OprM plays an indispensable role. We
previously explained the lower MICs of antibiotics in
OprM mutants
compared with
MexA and
MexB by assuming the presence of
unidentified inner membrane components that act as substitutes for
MexA,B. Addition of EDTA showed the highest effect in the MexA,B
mutant, leading us to believe that the interaction between OprM and the
putative inner membrane pump is susceptible to this treatment.
TMA-DPH May Be Taken up from the Lipid Domain of the Cytoplasmic Membrane
The amphiphilic character arising from attachment of the
positively charged phenyltrimethylammonium group to diphenylhexatriene allows orientation of this probe in the lipid bilayer perpendicular to
the plane of the cytoplasmic membrane. Therefore, partitioning of
TMA-DPH into the lipid bilayer was shown to be a biphasic process that
resulted from rapid insertion of the dye into the outer leaflet of the
cytoplasmic membrane, followed by slower transbilayer movement to the
inner leaflet of the membrane (20). Addition of TMA-DPH to the cell
suspensions resulted in rapid increase of the fluorescence intensity,
followed by a slower incremental phase leading to the maximum level
within about 2 min in the MexA or
MexB mutant and the wild type
strain (Fig. 4A). The mutant
lacking OprM took a much longer time than the others to equilibrate.
The first component of the biphasic process is similar in all strains
and the difference in the fluorescence increase mainly occurs during
the second phase. The slower second phase was absent in the parent
strain, high in the
OprM mutant and at an intermediate level in the
MexA and
MexB strains (Fig. 4). The results clearly showed that
TMA-DPH is efficiently extruded via the Mex extrusion machinery,
probably from the inner leaflet of the cytoplasmic membrane. Raising
the TMA-DPH concentration by the factor of two or more in the parent strain brought the fluorescence intensity to the same level as that
caused by half that concentration in the
M mutant (Fig. 4B). This means that accumulation in the parent strain takes
place only in the outer leaflet of the cytoplasmic membrane, whereas the dye accumulates in the
OprM mutant in both the inner and outer
leaflet of the inner membrane.
To address the question of whether or not TMA-DPH accumulates in the
inner leaflet of the cytoplasmic membrane and is then extruded from
this site by the extrusion system, we performed downhill efflux
experiments. Cells were preloaded with concentrated TMA-DPH, and then
the suspension was rapidly diluted. We observed a very rapid
single-phase decrease in fluorescence of the wild type strain (Fig.
5). On one hand, the fluorescence
decrease in ABM cells was preceded by a transient rise in
fluorescence; then there was a slow decrease until the main
equilibration was reached (Fig. 5). We interpreted these results to
mean that transient increase in fluorescence represents slow movement
of the dye from the cytoplasm to the inner leaflet then to the outer
leaflet, because the mutant lacking the extrusion proteins cannot
extrude the dye. In the wild type strain, the Mex pump takes up the dye from the inner leaflet very rapidly, and therefore, a sharp decrease in
fluorescence represents only the diffusion from the outer leaflet that
also occurs in the
MexAB-OprM mutant. A downhill extrusion profile
of TMA-DPH in the potassium cyanide-poisoned wild type cell is similar
but slightly higher than that in the
MexAB-OprM mutant, confirming
that the extrusion was driven by the cellular energy.
Ethidium Bromide Accumulation Experiment Confirms That Extrusion Is a Proton Motive Force-driven Process
In contrast to DMP, NPN, and
TMP-DPH uptake, which monitors mainly accumulation of the dye to the
cytoplasmic membrane, EtBr uptake reflects the accumulation of this dye
into the cytoplasmic compartment. Because the MIC of EtBr in the wild
type strain of P. aeruginosa is over 200 µg/ml, it is
conceivable that a fraction of this high resistance is due to efflux.
All the mutants showed markedly higher fluorescence than in the wild
type strain (Fig. 6). In contrast to the
hydrophobic membrane probes, EtBr did not show intermediate levels of
uptake rates in the MexA,
MexB, or
MexA,B mutant. These
results suggest the following possibilities: (i) the Mex extrusion
machinery takes up the substrate also from the cytoplasm; and (ii)
intact extrusion assembly is necessary for mediating extrusion from the
cytoplasm. Addition of CCCP to the mixture induces a sharp
increase in EtBr accumulation, confirming the proton motive force
dependence of the extrusion process (Fig. 6, curve d).
Concentration-dependent DMP Accumulation
To
determine whether MexA,B-OprM-mediated efflux of the fluorescent dyes
was saturable, we examined the effect of external dye concentration on
the fluorescence increase (Fig. 7).
Fluorescence intensity of DMP in the buffer solution remained constant
in the range of 20-80 µM. A maximum saturation in
fluorescence increase was reached in the case of the OprM or
MexA,B-OprM mutant at a concentration of 50 µM.
Increasing external dye concentration further results in the quenching
of fluorescence. Fluorescence intensity in the parent strain reaches a
steady saturation level at an external concentration of about 50-60
µM, which is at about half the fluorescence intensity of
the
ABM mutant. We calculated the apparent Km
value of DMP extrusion from the difference in the fluorescence
intensity between the
ABM mutant and the wild type strain and found
the value to be about 10 µM. An apparent Km of 0.2 mM for a putative
carrier-mediated active efflux system was determined by Cohen et
al. (27) by using proton motive force-dependent uptake
of norfloxacin into everted inner membrane vesicle from E. coli.
Fluorescence probes with different intracellular binding modes were used to assess the role of the subunit protein of the Mex extrusion machinery in substrate extrusion by monitoring real time accumulation. The increase in fluorescence with DMP, NPN, and TMA-DPH were shown to be directly related to their accumulation in the cytoplasmic membrane (23, 28). In the present study, the highest level of NPN, DMP, and TMA-DPH accumulation was observed in the mutant lacking OprM, the intermediate level was observed in the mutant lacking MexA or MexB, and the lowest level was observed in the wild type strain (Figs. 1, 2, 3, 4). This order of fluorescence probe accumulation was a perfect match with the MICs of fluoroquinolones, chloramphenicol, and tetracycline in these mutants and the parent strain (21), suggesting the possibility that a low level of antibiotic accumulation may be a major factor causing the resistance. A correlation between the deletion of mexA-mexB-oprM operon products and MIC results was firmly established previously (21).
A conspicuous fact is that permeabilization of the outer membrane by
EDTA treatment has a minor effect on the induction of a higher level of
DMP accumulation in the wild type or OprM when compared with the
effect on
MexA,B (Fig. 3). This result indicates that the
synergistic effect between the outer membrane barrier and extrusion
activity in protecting the cell against lipophilic compounds is still
valid, but the role of this barrier in the case of hydrophobic low
molecular weight molecules, like DMP (molecular weight, 380) and NPN
(molecular weight, 219), is less extensive than expected and defers the
outer membrane permeability to a secondary role.
The outer membrane component of efflux systems in P. aeruginosa is thought to act as a sort of exit channel, because
the whole extrusion machinery dysfunctions in the mutants carrying at
least OprM. Furthermore, the intermediate level in the MIC of
antibiotics in
MexA,B suggested the possibility of a two-step
extrusion by which OprM can extrude drugs directly from the periplasm.
However, DMP accumulation experiments in outer membrane permeabilized
cells demonstrated that MexA,B-OprM forms a tight functional unit
during extrusion, because the EDTA treatment of the wild type strains only increased fluorescence a little (see "Results" and Fig.
3).
The mechanism of extrusion includes at least two steps. In the first
step, the dyes and antibiotics located in the inner membrane are
recognized by the functional unit composed of MexA and MexB. The second
step leads to the release of the drug into the outer medium through
OprM. Interaction between the inner membrane components (MexA and MexB)
and OprM is the crucial step in triggering the transport of these
compounds. We previously suggested the existence of other proteins
located in the cytoplasmic membrane, capable of substituting the
function of MexA,B (21). The putative functional homologue of MexA,B
differs probably in specificity toward different substrates. In the
case of ethidium bromide, the accumulation proceeded at the same rate
and extent in all mutants (Fig. 6), suggesting that this putative
exporter does not recognize this cytoplasmic probe. The MIC test of
several hydrophilic -lactam antibiotics like azthreonam, cefsulodin,
and cefoperazone in the MexA,B mutants did not show intermediate
susceptibility, suggesting that the complementary system might not be
able to initiate the extrusion of these compounds (21).
The interaction between outer and inner membrane proteins during extrusion is not specific to P. aeruginosa. Although suspected for some time, there is now a growing amount of evidence that outer membrane protein TolC is the functional counterpart of OprM for AcrA,B-mediated extrusion in E. coli (29). Deletion of TolC results in hypersensitivity to hydrophobic agents, even in AcrA,B overexpressed mutants.
The fact that membrane probes are particularly "sensitive" to mex-operon products is an indication that a crucial step in active extrusion occurs during the membrane crossing step. Bolhuis et al. (20) showed previously that the fluorescent membrane probe TMA-DPH is distributed into the phospholipid bilayer via a biphasic process. A first rapid entry of TMA-DPH into the outer leaflet is followed by a slower transbilayer movement to the inner leaflet. The second component of this process is absent when extrusion occurs in energized L. lactis cells. A similar behavior of TMA-DPH accumulation was observed in P. aeruginosa in which the slower transfer to the inner leaflet was notably higher in the knock-out mutants (Figs. 4 and 5). The role of MexA,B-OprM in removing membrane bound TMA-DPH was particularly obvious in the downhill extrusion experiments (Fig. 5). The slow diffusion step of the amphiphilic dye from the inner leaflet to the outer leaflet before rapid release from the membrane surface was absent in the parent strain. These results suggest the existence of a common mode of action between MexA,B-OprM and LmrA, the ATP-dependent multidrug resistance pump of L. lactis, which is the extrusion of dye that takes place from the inner leaflet of the cytoplasmic membrane.
Because most of the extruded compounds are hydrophobic, it is assumed that the recognition site of the extrusion system possesses lipophilic properties. A recognition site for hydrophobic compounds located in a lipophilic environment has many advantages when compared with a site located in an aqueous environment. Lipophilic interaction sites exposed to the aqueous phase would require complex solvations to stabilize the recognition site (30). Initiation of the extrusion process would necessitate complex interactions between the extruded substrate, the water molecules, and the recognition site. This would in turn impose strict structural conditions on the extruded compound. Hence, a hydrophobic site located in a hydrophobic environment can exist without solvation and stay accessible to a broad range of substrates.
Our results agree with previously reported observations on the human multidrug transporter P-glycoprotein (31, 32) for the MDR-related protein MRP (33) and finally the ATP-dependent drug extrusion in L. lactis. The MDR system driven by the MexA,B-OprM machinery is probably more complex than that described above, because the proteins connecting functions of two membranes are involved. However, well characterized genetic and biochemical systems in P. aeruginosa serve as excellent models for studying the extrusion of noxious compounds. We believe that these studies eventually contribute to understanding of MDR in all living organisms. We assume that a common mechanism drives the first step of the extrusion in all of these systems.