From the School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The multidrug efflux pump QacA from
Staphylococcus aureus confers resistance to an extensive
range of structurally dissimilar compounds. Fluorimetric analyses
demonstrated that QacA confers resistance to the divalent cation
4',6-diamidino-2-phenylindole, utilizing a proton motive
force-dependent efflux mechanism previously demonstrated
for QacA-mediated resistance to the monovalent cation ethidium. Both
the ionophores nigericin and valinomycin inhibited QacA-mediated export
of ethidium, indicating an electrogenic
drug/nH+ (n The phenomenon of multidrug resistance is widespread in
prokaryotic and eukaryotic organisms and is characterized by the
ability of a single transmembrane protein to mediate the extrusion of a
broad spectrum of structurally disparate toxic substances from the
cell. Multidrug resistance pumps operate via an active efflux mechanism
and are typically ATP- or
PMF1-dependent.
Currently, all known ATP-dependent multidrug efflux systems
belong to the ABC superfamily of transporters, and the best
characterized of these is human P-glycoprotein, which confers resistance to an extensive array of cytotoxic, chemotherapeutic agents
used in human cancer cells (1, 2). Another example is LmrA, a
P-glycoprotein homolog that has been identified in Lactococcus
lactis and represents the first ATP-dependent
multidrug efflux system found in bacteria (3).
PMF-dependent multidrug efflux proteins have been
classified into three distinct families of membrane transport proteins
based on comparative sequence analysis: the major facilitator
superfamily (4-6), e.g. Bmr from Bacillus subtilis (7) and QacA from Staphylococcus aureus (8,
9); the small multidrug resistance family (10), e.g. EmrE
from Escherichia coli (11) and Smr from S. aureus
(12, 13); and the resistance/nodulation/cell division family (14),
e.g. MexB from Pseudomonas aeruginosa (15) and
AcrB from E. coli (16).
The multidrug resistance gene qacA is encoded on
multiresistance plasmids from clinical isolates of S. aureus
and confers resistance to a wide range of antimicrobial organic
cations, including various dyes, Qacs, diamidines, biguanidines, and
guanylhydrazones (8, 9, 17). qacA encodes a membrane
protein, QacA, with 14 TMS (18) providing resistance to ethidium via a
PMF-dependent export mechanism (8). Expression of the
qacA gene is negatively regulated by a divergently
transcribed regulator, QacR (19). QacA and a closely related protein,
QacB, also found in clinical isolates of S. aureus, mediate
high levels of resistance to monovalent cations, including a number of
intercalating dyes and Qacs. However, QacB characteristically differs
from QacA in that it confers significantly reduced levels of resistance
to divalent cationic drugs, such as diamidines and biguanidines (8,
18). Sequence analysis and mutagenesis studies have revealed that the
difference in substrate specificity between QacA and QacB is due to a
single amino acid substitution at position 323 in TMS 10, where the
presence of an acidic residue in QacA is essential for high levels of
resistance to diamidines and biguanidines (18). The wider substrate
specificity of QacA compared with QacB may have evolved in response to
the clinical use of divalent cations as chemotherapeutic agents
(20).
In this study, fluorescence transport assays have been utilized to
measure various aspects of the bioenergetics of
qacA-mediated multidrug transport. Kinetic analyses of
transport of monovalent and divalent fluorescent substrates show for
the first time that QacA interacts with each of its substrates with
high affinity. Interactions between various substrates and QacA using
competition analysis suggest that there are distinct binding sites for
monovalent and divalent cations. Transport assays utilizing a
membrane-specific fluorescence probe provide evidence that QacA
extrudes its substrates from the inner leaflet of the cytoplasmic membrane.
Bacterial Strains and Plasmids--
The S. aureus
strain SK982 was used as the host strain for the
qacA-encoding plasmid pSK1 and the qacB-encoding
plasmid pSK23, as described previously (8). The E. coli K-12
strain BHB2600 (F'803 supE supF hsdR met) (21) was used as
the host strain for the plasmid constructs pSK4219 and pSK4270, which
contain the qacA and qacB genes cloned into
pBluescript SK+, respectively (18). All strains were
cultured at 37 °C in LB containing, where appropriate, 25 µg/ml
gentamicin or 100 µg/ml ampicillin.
Chemicals--
Diamidinodiphenylamine and propamidine were
provided by Rhône-Poulenc Rorer (Dagenham, United Kingdom).
Benzalkonium, CCCP, chlorhexidine, DAPI, DiOC3, ethidium,
nigericin, pyronin Y, reserpine, tetraphenylarsonium, TMA-DPH,
valinomycin, and verapamil were all purchased from Sigma Aldrich.
Nigericin and valinomycin were dissolved in 100% chloroform. Reserpine
was prepared as a 1 mg/ml stock solution in 9:1 chloroform:methanol.
Verapamil and TMA-DPH were solubilized in 50% and 100% methanol,
respectively. CCCP was prepared as a solution in 1 mg/ml ethanol and
then diluted to 0.1 mg/ml in 10 mM NaOH as described
previously (22). All other chemicals were prepared as stock solutions
of 1 or 10 mg/ml in water, depending on solubility, immediately prior
to use.
Determination of Sensitivity to Potential Substrates and
Inhibitors--
MICs of DAPI, DiOC3, reserpine, TMA-DPH,
and verapamil were performed in 96-well microtiter plates. 100 µl of
LB was added to each well together with increasing concentrations of
the antimicrobial agent within the range of susceptibility and 1 × 10 Fluorimetric Analysis of QacA- and QacB-mediated Efflux of DAPI,
DiOC3, Ethidium, and Pyronin Y--
Fluorimetric assays of
QacA- and QacB-mediated efflux of DAPI, DiOC3, ethidium,
and pyronin Y were carried out essentially as described previously for
ethidium (8). These substrates bind to specific cellular components and
in so doing undergo a change in fluorescence that can be used to
indicate how much of the substrate is retained by the cell,
e.g. ethidium and DAPI bind to double-stranded DNA resulting
in a substantial increase in fluorescence (22, 23), DiOC3,
when bound to the cytoplasmic side of hyperpolarized membranes, is
highly fluorescent (24), and pyronin Y binds to RNA resulting in
fluorescence quenching (25). The S. aureus strain SK982,
carrying the qacA-encoding plasmid pSK1 or the
qacB-encoding plasmid pSK23, was grown for 16 h in 10 ml of LB (with selection) from which 1 ml was subcultured into 10 ml of
fresh LB and grown to OD650 = 0.85 (~2.5 h). The E. coli strain BHB2600, carrying pSK4219 (qacA), pSK4270
(qacB) or the vector (pBluescript SK+), was
grown for 16 h in 10 ml of LB (with selection) from which 1 ml was
subcultured into 10 ml of fresh LB and grown to OD650 = 0.6 (~3.5 h). Cells were harvested by centrifugation, washed twice and
resuspended in 10 ml of 20 mM HEPES (pH 7.0). Aliquots (1 ml) of cells were then loaded with each individual substrate (10 µM DAPI, 2.5 µM DiOC3, 15 µM ethidium, or 15 µM pyronin Y) by
incubating for 1.5 h at 37 °C in the presence of 10 µM CCCP. Loaded cells were collected by centrifugation,
washed three times and resuspended in 20 mM HEPES (pH 7.0).
Energy-dependent efflux was assayed following the addition
of sodium formate to a final concentration of 125 mM. 10 µM CCCP was used as an inhibitor in some experiments.
Fluorimetric measurements were performed at 37 °C using a Hitachi
4500 fluorimeter (slit widths of 5 nm). The excitation and emission
wavelengths used for each of the fluorescent compounds are as follows:
DAPI, 364 nm and 454 nm, respectively; DiOC3, 485 nm and
520 nm, respectively; ethidium, 530 nm and 610 nm, respectively; and
pyronin Y, 500 nm and 570 nm, respectively.
Determination of the Kinetic Parameters Km and
Vmax--
Increasing substrate concentrations were used to
establish the kinetics of QacA- and QacB-mediated drug efflux in
E. coli and in S. aureus. Aliquots (1 ml) of
cells were prepared as described above and loaded with the appropriate
range of concentrations for each substrate: DAPI, 1-25
µM; DiOC3, 0.1-10 µM;
ethidium, 0.1-25 µM; and pyronin Y, 1-50
µM. From these concentrations, a series of efflux curves
were generated and the initial velocity was calculated by averaging the
linear part of each curve. The range of substrate concentrations was
selected to be equally spread above and below the
Km, and a minimum of 10 substrate concentrations
were utilized for each experiment. The non-linear least squares method
(v = Vmax·[S]/([S] + Km)); where [S] represents substrate
concentration, was the method of choice to obtain estimates of
Km and Vmax. Software for the non-linear least squares method was kindly provided by Dr Ray Ritchie
(School of Biological Sciences, University of Sydney) and uses a
general least squares fitting technique to determine accurate estimates
of Km and Vmax, followed by
matrix algebra to refine and calculate the errors of these estimations (26). Km and Vmax
calculations were confirmed by double-reciprocal plots where 1/velocity
(v) is plotted as a function of 1/[S].
Inhibition of QacA-mediated Drug Export--
Aliquots (1 ml) of
S. aureus cells were prepared as described above, loaded
with 15 µM ethidium, washed three times, and resuspended in the appropriate buffer. To measure inhibition by the ionophores nigericin and valinomycin, cells were resuspended in a potassium phosphate buffer (50 mM KH2PO4 and
1.0 mM MgSO4, pH 7.0). Nigericin or
valinomycin, at a final concentration of 4 µM, was added
to loaded cells and incubated for 1 min followed by the addition of
sodium formate, to a final concentration of 125 mM, to
energize transport. To measure inhibition by reserpine and verapamil,
cells were resuspended in 20 mM HEPES (pH 7.0) and 50 µM reserpine or 150 µM verapamil. Loaded
cells were washed three times and resuspended in 20 mM
HEPES (pH 7.0), and fluorimetric measurements were obtained after the
addition of sodium formate to energize transport.
Competition Analysis--
Competition analyses were performed by
determining the kinetics of QacA- and QacB-mediated export of ethidium
(0.5-25 µM) in the presence of various fixed
concentrations of non-fluorescent substrates, including the monovalent
cations benzalkonium (2-10 µM) and tetraphenylarsonium
(5-100 µM), and the divalent cations chlorhexidine
(2-10 µM), propamidine (5-100 µM), and
diamidinodiphenylamine (5-150 µM). Aliquots of S. aureus cells were loaded with both substrates in the presence of
10 µM CCCP for 1.5 h and harvested by
centrifugation. Export of ethidium was measured fluorimetrically as
described above. The results were illustrated graphically by double-reciprocal plots using the computer program Cricket Graph (Computer Associates International) to obtain a line of best fit.
Uptake of TMA-DPH into Whole Cells--
TMA-DPH specifically
fluoresces in lipid environments (27) with an excitation and emission
spectra of 350 nm and 425 nm, respectively (24). S. aureus
strains, harboring plasmids pSK1, pSK23, or no plasmid, were prepared
as described above for fluorimetric analysis. 2.5 µM
TMA-DPH was added to cell suspensions, and accumulation of this
substrate was measured fluorimetrically. Cells were also preloaded with
2.5 µM TMA-DPH in the presence of 10 µM
CCCP as for other fluorescent substrates and efflux generated by the
addition of sodium formate to a final concentration of 125 mM. 10 µM CCCP was used as an inhibitor in
some experiments.
QacA Mediates Export of Monovalent and Divalent Cations--
The
multidrug export protein QacA confers resistance to more than 30 compounds that belong to 12 distinct chemical families (Table
I; Refs. 8, 9, and 17). To establish if
QacA and the closely related staphylococcal protein QacB mediate the
efflux of structurally dissimilar compounds via a common mechanism,
fluorimetric transport analyses of the monovalent substrates ethidium
and pyronin Y, and two newly identified substrates, the monovalent
cation DiOC3 (S. aureus MICs: SK982, <0.25
µg/ml; pSK1 or pSK23 in SK982, 8 µg/ml) and the divalent cation
DAPI (S. aureus MICs: SK982 or pSK23 in SK982, 20 µg/ml;
pSK1 in SK982, >80 µg/ml), all from different chemical families
(Table I), were performed (Fig. 1). Formate-driven efflux of DAPI, DiOC3, and ethidium are
shown as a rapid decrease in fluorescence, whereas efflux of pyronin Y is observed as a rapid increase in fluorescence (see "Experimental Procedures"). QacA-mediated efflux of all four substrates tested was
abolished by the addition of CCCP (data not shown), an uncoupler of the
PMF, which suggests that QacA mediates the extrusion of structurally dissimilar monovalent and divalent substrates via a common
PMF-dependent efflux mechanism (Fig. 1). Similarly,
QacB-mediated efflux of the monovalent substrates DiOC3,
ethidium and pyronin Y was inhibited by CCCP. QacB demonstrated no
efflux of the divalent diamidine DAPI (Fig. 1), in accord with the MIC
data and previous observations that QacB confers little or no
resistance to the diamidines (8, 17).
QacA- and QacB-mediated Drug Export Conforms to Michaelis-Menten
Kinetics--
There are various hypotheses that attempt to explain the
broad substrate range of multidrug transporters, e.g. the
transporter possesses one or more high affinity binding sites, which
enables it to interact directly and specifically with its substrates, (28-30); the transporter has an indiscriminate binding mechanism, which would enable it to recognize many different structures; or the
transporter is involved in altering some biophysical parameter of the
cell, such as the magnitude of the
Examination of the relationship between increasing substrate
concentration and the transport velocity indicated that QacA- and
QacB-mediated export of monovalent and divalent cations conformed to
classical Michaelis-Menten kinetics (Fig.
2), with the exception that export of
DAPI by QacB was not observed at any concentration (Fig. 2B
and Table II). The kinetic parameters,
Km and Vmax, for transport of
the fluorescent substrates DAPI, DiOC3, ethidium, and
pyronin Y were determined (Table II). Similar Km values were obtained from fluorimetric analyses performed in the natural host S. aureus, and in the heterologous host
E. coli, suggesting that these proteins function similarly
in both organisms. Both QacA and QacB demonstrated a high affinity for
their substrates with Km values in the low
micromolar range (<20 µM; Table II). QacA-mediated
export of monovalent cations, e.g. DiOC3,
ethidium, and pyronin Y, displayed an increased
Vmax, but not Km, compared
with QacB-mediated efflux of these compounds (Table II), suggesting
that although QacA and QacB bind monovalent substrates with equally
high affinity, QacA may transport them at a faster rate.
Competitive and Non-competitive Inhibition of QacA- and
QacB-mediated Export of Ethidium--
Fractional inhibitory
concentration analysis has indicated that QacA confers resistance to
two compounds at a rate proportional to the overall amount of substrate
present, thereby implying that the presence of one compound inhibits
the transport of another (17). Fluorimetric competition studies were
performed in S. aureus to examine QacA-mediated transport of
ethidium in the presence of various non-fluorescent substrates (Fig.
3). The monovalent cations benzalkonium
and tetraphenylarsonium competitively inhibited QacA-mediated export of
ethidium (Fig. 3, A and B), whereas the divalent
cations chlorhexidine and propamidine non-competitively inhibited
QacA-mediated export of ethidium (Fig. 3, C and
D). The ability of the monovalent cations benzalkonium
and tetraphenylarsonium to competitively inhibit ethidium transport
suggests that these substrates either share a common binding site or
have unique but overlapping binding sites. In contrast, the divalent
cations propamidine and chlorhexidine appear to bind at a distinct
site(s) in QacA compared with ethidium.
QacB-mediated export of ethidium was competitively inhibited by the
monovalent cations benzalkonium and tetraphenylarsonium within a
similar concentration range as shown for QacA (data not shown). These
data, together with the fact that QacA and QacB differ by only 7 amino
acids (18) and display similar binding constants for monovalent cations
(Table II), imply that these two transporters possess identical binding
site(s) for monovalent substrates. The divalent cation propamidine
non-competitively inhibited QacB-mediated ethidium efflux only at a
substantially higher concentration ( Inhibition of QacA-mediated Transport by Ionophores--
To
investigate which component, i.e. the Inhibition of QacA-mediated Ethidium Transport by Reserpine and
Verapamil--
Sensitivity to reserpine and verapamil has been
observed in an extensive range of multidrug efflux systems, including:
members of the ABC superfamily, e.g. P-glycoprotein (35);
the major facilitator superfamily, e.g. the mammalian VMAT
protein (30), Bmr from B. subtilis (36), and LmrP from
L. lactis (37); and the small multidrug resistance
superfamily, e.g. EmrE from E. coli (38). MIC
analysis demonstrated that QacA does not confer resistance to either
reserpine or verapamil. However, both substances inhibited
QacA-mediated ethidium export at a concentration of 50 µM
(reserpine) or 150 µM (verapamil) (Fig.
4B).
Analysis of Accumulation and Efflux of TMA-DPH--
The
hydrophobic compound TMA-DPH fluoresces when partitioned into the
membrane (27), and has been utilized to identify the sub-cellular
origin of efflux for various multidrug export systems (32, 37, 39).
TMA-DPH was shown to be a QacA substrate by using drug susceptibility
(S. aureus MICs: SK982, 15 µg/ml; pSK1 in SK982, 30 µg/ml), and formate-driven efflux (Fig.
5A) studies. TMA-DPH
accumulation and efflux in cells expressing qacA were indistinguishable from cells lacking qacA, following
treatment with the uncoupler CCCP (Fig. 5, A and
B). It should be noted that CCCP partially quenches TMA-DPH
fluorescence, resulting in the observed lower fluorescence of the
CCCP-treated cells. TMA-DPH has previously been shown to display a
biphasic interaction with bacterial membranes, where the first phase is
represented by the intercalation of this compound into the outer
leaflet of the membrane resulting in a very rapid increase in
fluorescence, and the second phase is represented by the movement of
this compound from the outer leaflet to the inner leaflet of the
membrane resulting in a more gradual increase in fluorescence (24, 39).
Fluorimetric analysis of TMA-DPH accumulation by the staphylococcal
strain SK982, with and without the qacA-containing plasmid
pSK1, indicated that TMA-DPH intercalates into the outer leaflet of the
cytoplasmic membrane in both strains, as represented by a rapid
increase in fluorescence (Fig. 5B). The first phase was
observed as an almost instantaneous increase in fluorescence, occurring
at approximately the 30-s point in Fig. 5B. However, the
second phase, represented by a gradual increase in fluorescence, where
the substrate moves from the outer leaflet to the inner leaflet of
the cytoplasmic membrane, was significantly reduced in the
QacA-containing strain relative to the background strain (Fig.
5B). This suggests that QacA interacts with, and expels
TMA-DPH from, the cell membrane, preventing it from entering the inner
leaflet and hence the cytoplasm.
The staphylococcal multidrug export protein QacA confers
resistance to an extensive range of monovalent and divalent,
antimicrobial, lipophilic compounds (Table I). QacA-mediated
PMF-dependent efflux of one divalent and three monovalent
cations was demonstrated by using fluorescent transport assays (Fig.
1). This suggests that QacA utilizes a common PMF-dependent
efflux mechanism for transport of all of its substrates. QacA-mediated
export of the monovalent cation ethidium and the divalent cation DAPI
was inhibited by the ionophores nigericin and valinomycin (Fig.
4A), which specifically dissipate the Fluorimetric analysis of the kinetic parameters of QacA-mediated export
of DAPI, DiOC3, ethidium, and pyronin Y produced
Km values in the low micromolar range,
i.e. <20 µM (Table II), indicating that QacA
interacts with each structurally dissimilar substrate with high
affinity. Similarly, other multidrug efflux systems have been
demonstrated to possess a high affinity for their substrates, e.g. Smr-mediated efflux of triphenylmethylphosphonium
(Km of 5 µM) (12), and
MexA/B-OprM-mediated efflux of dimethylaminostyryl-1-ethylpyridinium in
P. aeruginosa (Km of 10 µM)
(32). QacA and the closely related QacB share similar binding
affinities for monovalent cations (Table II), and ethidium efflux
mediated by these two proteins was competitively inhibited by other
monovalent cations (Fig. 3, A and B). Taken
collectively, these data are indicative that the QacA and QacB
multidrug efflux proteins potentially utilize a common mechanism for
the recognition of monovalent ligands. However, QacA and QacB differ in
their recognition of divalent cations, reflected by the inability of
QacB to transport DAPI (Fig. 2; Table II). QacA-mediated ethidium
efflux was non-competitively inhibited by divalent cations (Fig.
3C and D), whereas QacB-mediated ethidium efflux
was unaffected by diamidinodiphenylamine and non-competitively inhibited by propamidine, although only at high concentrations. These
data are consistent with the relative resistance profiles conferred by
QacA and QacB (8, 17). Thus, QacA appears to utilize a high affinity
binding site(s) for the recognition of divalent cations, distinct from
the site(s) responsible for recognition of monovalent cations; QacB
lacks this high affinity binding mechanism, but is capable of binding
some divalent cations, albeit with a lower affinity.
Previous studies have shown that a single amino acid, namely Asp-323
located in TMS 10 of QacA, is essential for conferring high levels of
resistance to diamidines and the biguanidine, chlorhexidine, implying that this region may form part of the high affinity binding site(s) for divalent cations in QacA (18). Although QacB can interact
with some divalent cations with a low affinity, mutation of Asp-323 in
QacA to Ala, the residue at this position in QacB, abolished all
resistance to these compounds (18). There are six additional amino acid
differences between QacA and QacB, some of which are likely to be
involved in forming the binding site responsible for low level
resistance to the diamidines mediated by QacB. Previous studies have
supported the notion that QacA evolved from QacB (20). The data
presented here imply that this evolutionary process entailed the
acquisition of a high affinity binding mechanism for divalent cations
and the concomitant loss of a low affinity binding mechanism for
selected divalent cations.
TMA-DPH is a fluorescence probe that specifically interacts with the
cytoplasmic membrane. TMA-DPH was shown to be a substrate of QacA (Fig.
5A), and it was demonstrated that QacA reduces the accumulation of TMA-DPH in the inner leaflet of the cytoplasmic membrane (Fig. 5B), suggesting that QacA is most likely to
interact with this substrate within the membrane. Whether the findings with TMA-DPH can be extrapolated to include all QacA substrates is
unknown, but this is an appealing hypothesis, since the known substrates of QacA are all hydrophobic, lipophilic cations (Table I).
The multidrug transport systems LmrP (37), MexA-MexB-OprM (32), and
P-glycoprotein (41, 42) have been shown to extrude substrates directly
from the cytoplasmic membrane, implying this may be a general feature
of multidrug efflux systems.
The results presented here provide evidence that the multidrug
transport protein QacA interacts directly and specifically with its
substrates and that this interaction may occur, in the first instance,
in the cell membrane as the substrate is entering the cell. Such a
mechanism would prevent toxic antimicrobial agents from reaching
intracellular targets. Intramembranous transporter-substrate interactions imply that critical sites in the protein are accessible directly from within the lipid phase. Residues contained within TMS,
such as Asp-323 in TMS 10 of QacA, are ideal candidates for such interactions.
2) antiport
mechanism. The kinetic parameters, Km and
Vmax, were determined for QacA-mediated export
of four fluorescent substrates, 4',6-diamidino-2-phenylindole,
3',3'-dipropyloxacarbocyanine, ethidium, and pyronin Y. Competition
studies showed that QacA-mediated ethidium export is competitively
inhibited by monovalent cations, e.g. benzalkonium, and
non-competitively inhibited by divalent cations, e.g.
propamidine, which suggests that monovalent and divalent cations bind
at distinct sites on the QacA protein. The quaternary ammonium salt,
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, was used as a
membrane-specific fluorescence probe and demonstrated that the amount
of substrate entering the inner leaflet was significantly reduced in
QacA-containing strains, supporting the notion that the substrate is
extruded directly from the membrane.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
4 dilution of an overnight culture of the strain to
be tested. Plates were incubated for 48 h at 37 °C, and the
MICs were determined by scoring the lowest concentration at which no
growth was observed. For each compound, the MIC analysis was carried
out in triplicate.
RESULTS
Substrates of the QacA transporter
View larger version (32K):
[in a new window]
Fig. 1.
Fluorimetric assays of drug transport in
S. aureus. Efflux of DAPI (10 µM),
DiOC3 (2.5 µM), ethidium (15 µM), and pyronin Y (15 µM) from SK982 cells
harboring plasmid pSK1 (qacA, circles), plasmid
pSK23 (qacB, squares), or no plasmid (background,
triangles). Sodium formate (125 mM) was added at
zero time to energize transport. Efflux of DAPI, DiOC3, and
ethidium is represented by a rapid decrease in fluorescence, whereas
efflux of pyronin Y was observed as a rapid increase in fluorescence,
as described under "Experimental Procedures." Structures of each
substrate are shown at the top of the appropriate graph. All efflux
experiments were performed in at least triplicate.
or the
pH, the result of
which drives the movement of various charged compounds across the cell
membrane (31). To determine if QacA and QacB interact specifically with
structurally dissimilar substrates, kinetic analyses of transport were
performed using various fluorescent substrates. These kinetic analyses,
undertaken in whole cells, were based on the assumptions that after
loading the cells in the presence of CCCP, the intracellular substrate
concentration is effectively equivalent to the extracellular
concentration, and that the use of fluorescence can accurately
represent the amount of substrate inside the cell as has previously
been demonstrated for ethidium, namely the amounts of ethidium retained
by E. coli, calculated utilizing either fluorimetry or
[14C]ethidium produced identical results (22). Consistent
with these assumptions was the observation that the amount of the
fluorescent substrate in loaded cells was proportional to the
concentration used. Similar kinetic studies of multidrug efflux systems
using whole cells have been performed in P. aeruginosa (32)
and Mycobacterium smegmatis (33).
View larger version (16K):
[in a new window]
Fig. 2.
Michaelis-Menten kinetics of ethidium and
DAPI export in S. aureus. Michaelis-Menten kinetics of
export of the monovalent cation ethidium (A) and the
divalent cation DAPI (B) from SK982 cells harboring plasmid
pSK1 (qacA, circles) or plasmid pSK23
(qacB, squares). Efflux experiments were
performed utilizing a range of drug concentrations between 0.1 and 25 µM, as described under "Experimental Procedures."
Initial velocity, represented by fluorescence units per second
(s 1), was plotted as a function of substrate
concentration. All efflux experiments were performed in at least
triplicate.
Comparative kinetic data
View larger version (22K):
[in a new window]
Fig. 3.
Competitive and non-competitive inhibition of
QacA-mediated ethidium transport in S. aureus. Export of the
monovalent cation ethidium from SK982 cells harboring plasmid pSK1
(qacA) in the presence of non-fluorescent substrates, as
described under "Experimental Procedures." Competitive inhibition
is shown as a double-reciprocal plot of the kinetics of ethidium export
in the presence of fixed concentrations (0 µM,
squares; 5 µM, triangles; and 8 µM, circles) of the monovalent cation
benzalkonium (Bc) (panel A) and a
double-reciprocal plot of the kinetics of ethidium export in the
presence of fixed concentrations (0 µM,
squares; 20 µM, triangles; and 40 µM, circles) of the monovalent cation
tetraphenylarsonium (Ta) (panel
B). Non-competitive inhibition is shown as a
double-reciprocal plot of the kinetics of ethidium export in the
presence of fixed concentrations (0 µM,
squares; 2.5 µM, triangles; and 5 µM, circles) of the divalent cation
chlorhexidine (Ch) (panel C) and a
double-reciprocal plot of the kinetics of ethidium export in the
presence of fixed concentrations (0 µM,
squares; 10 µM, triangles; and 60 µM, circles) of the divalent cation
propamidine (Pi) (panel D). The
computer program Cricket Graph (Computer Associates International) was
utilized to obtain a line of best fit, as described under
"Experimental Procedures."
40 µM) compared
with QacA (10 µM; see Fig. 3D), and the
divalent cation diamidinodiphenylamine did not inhibit QacB-mediated
ethidium efflux even at a concentration of 150 µM. This
is consistent with the resistance profile of QacB, which provides a low
degree of resistance to propamidine and no resistance to
diamidinodiphenylamine (8). Thus, despite the high level of similarity
between QacA and QacB, they display completely different binding
characteristics for divalent cations.
pH and/or the
of the
µH+ is involved in energizing
QacA-mediated drug efflux, transport assays were performed in the
presence of the ionophores, valinomycin and nigericin. In
potassium-buffered solutions, the potassium ionophore valinomycin
specifically collapses the
, whereas nigericin, which allows
H+:K+ exchange, specifically collapses the
pH (34). Both nigericin and valinomycin inhibited QacA-mediated
efflux of ethidium (Fig. 4A)
and DAPI (data not shown), indicating that both the
pH and the
are required to energize QacA-mediated efflux of monovalent and
divalent cations. This is consistent with an electrogenic proton/antiport mechanism, e.g. an exchange of two or more
protons for one molecule of substrate in the case of ethidium.
View larger version (17K):
[in a new window]
Fig. 4.
Inhibitors of QacA-mediated
ethidium export. Efflux of 15 µM ethidium from SK982
cells harboring plasmid pSK1 (qacA), in the
presence of the ionophores valinomycin (4 µM,
squares), nigericin (4 µM,
triangles), or no addition (circles)
(panel A) and verapamil (150 µM,
triangles), reserpine (50 µM,
squares), or no addition (circles)
(panel B) as described under "Experimental
Procedures." Sodium formate (125 mM) was added at zero
time to energize transport. All efflux experiments were performed in at
least triplicate.
View larger version (13K):
[in a new window]
Fig. 5.
Efflux and accumulation of the fluorescent
membrane probe TMA-DPH in S. aureus.
A, efflux of 2.5 µM TMA-DPH from S. aureus SK982 cells harboring plasmid pSK1 (qacA,
circles) or no plasmid (background, triangles).
Efflux of 2.5 µM TMA-DPH from the same two strains in the
presence of 10 µM CCCP was identical
(squares). Sodium formate (125 mM) was added at
zero time to energize transport. B, accumulation of 2.5 µM of TMA-DPH in SK982 cells harboring plasmid pSK1
(qacA, circles) or no plasmid (background,
triangles). Accumulation of 2.5 µM TMA-DPH in
the same two strains in the presence of 10 µM CCCP was
identical (squares). The biphasic interaction of the QacA
substrate TMA-DPH with the cytoplasmic membrane can be observed as
follows; intercalation of TMA-DPH into the outer leaflet of the
membrane is represented by the first rapid phase (at 30 s), and
movement of TMA-DPH from the outer leaflet to the inner leaflet is
represented by a relatively slow second phase (30 to 350 s). In
both A and B, the curves obtained in the presence
of CCCP (squares) were substantially lower than the
corresponding curves in the absence of CCCP as CCCP quenches the
fluorescence of TMA-DPH by more than 50%. Accumulation and efflux
experiments were performed in at least triplicate, as described under
"Experimental Procedures."
DISCUSSION
and the
pH,
respectively. This indicates that the PMF-dependent
mechanism utilized by QacA requires both components of the
µH+ to energize transport and implies
for monovalent cations, a stoichiometry of at least two protons being
exchanged for one molecule of substrate. Similar energy requirements
have been shown for some other multidrug efflux systems,
e.g. the mammalian VMAT proteins, which mediate monoamine
antiport in exchange for two protons (40), and LmrP-mediated drug
efflux from L. lactis, which is reliant on both the
and the
pH of the
µH+, suggesting an
electrogenic drug/nH+ (n
2)
antiport mechanism (37).
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr Ray Ritchie for providing statistical analysis programs for Michaelis-Menten kinetics and for valuable discussions. We thank Rhône-Poulenc Rorer for the generous supply of chemicals used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a project grant from the National Health and Medical Research Council (Australia).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of an Australian Postgraduate Award.
§ Recipient of the C. J. Martin Fellowship (National Health and Medical Research Council, Australia).
¶ To whom correspondence should be addressed: School of Biological Sciences, Macleay Bldg. A12, University of Sydney, Sydney, New South Wales 2006, Australia. Tel.: 61-2-9351-2376; Fax: 61-2-9351-4771; E-mail: skurray{at}bio.usyd.edu.au.
The abbreviations used are:
PMF, proton motive
force; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DAPI, 4',6-diamidino-2-phenylindole; DiOC3, 3',3'-dipropyloxacarbocyanine; LB, Luria broth; MIC, minimum inhibitory
concentration; Qacs, quaternary ammonium compounds; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TMS, transmembrane segments; , membrane potential;
pH, pH gradient;
µH+, transmembrane electrochemical proton gradient.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|