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Journal of Bacteriology, September 1998, p. 4686-4692, Vol. 180, No. 17
Department of Molecular and Cell Biology,
University of California, Berkeley, California 94708-3206
Received 9 April 1998/Accepted 29 June 1998
We found that the previously reported SS-B drug-supersusceptible
mutant of Salmonella typhimurium (S. Sukupolvi, M. Vaara, I. M. Helander, P. Viljanen, and P. H. Mäkelä, J. Bacteriol. 159:704-712, 1984) had a mutation in
the acrAB operon. Comparison of this mutant with its parent
strain and with an AcrAB-overproducing strain showed that the
activity of the AcrAB efflux pump often produced significant resistance
to During the last few years it has
become increasingly clear that multidrug efflux pumps, especially those
containing the resistance nodulation division (RND) family
transporters (33), play a major role in producing both
the intrinsic and the elevated levels of resistance to a very wide
range of noxious compounds in gram-negative bacteria (21, 24,
25). Examples include the AcrAB system of Escherichia
coli (19, 20, 22) and the MexAB-OprM system of
Pseudomonas aeruginosa (17, 30), which increase
in the wild-type cells of these organisms the MICs of agents such as fluoroquinolones, tetracycline, chloramphenicol, novobiocin, and fusidic acid. The former system also contributes significantly to
resistance to erythromycin as well as to various dyes and detergents (21). Inactivation of genes coding for components of these
systems was also shown to decrease the MICs of some Although these results suggested that such systems are capable of
pumping out In this study, we used a close relative of E. coli,
Salmonella typhimurium, to study the efflux-based resistance
to Bacterial strains and growth media.
S.
typhimurium SH5014 (LT2 rfaJ4041 ilv-1178 thr-914 his-6116
metA22 metE511 trpB2 xyl-404 H1-b H2-e,n,x flaA66 rpsL120 cured of
Fels2) and its SS-B-type hypersusceptible mutant SH7616 have been
described previously (39). A P1-sensitive derivative of SH7616, HN1003, was selected from SH7616 by first isolating a rare
P1cml clr 100(ts) lysogen on a chloramphenicol-containing Luria-Bertani (LB) plate (see below) and then curing the phage by
growth at 37°C, as described by Goldberg et al. (9).
S. typhimurium TN716 (apt apeB galE) was
obtained from the Salmonella genetics stock center,
University of Calgary, Calgary, Canada. HN891 was a single-step
multidrug-resistant mutant of SH5014, obtained as a colony growing on
an LB plate containing 8 µg of chloramphenicol per ml inoculated with
approximately 108 cells of unmutagenized SH5014.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multidrug Efflux Pump AcrAB of Salmonella typhimurium
Excretes Only Those
-Lactam Antibiotics Containing Lipophilic
Side Chains
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactam antibiotics in the complete absence of -lactamase.
The effect of AcrAB activity on resistance was more pronounced with
agents containing more lipophilic side chains, suggesting that such
compounds were better substrates for this pump. This correlation is
consistent with the hypothesis that only those molecules that
become at least partially partitioned into the lipid bilayer
of the cytoplasmic membrane are captured by the AcrAB pump.
According to this mechanism, the pump successfully excretes even those
-lactams that fail to traverse the cytoplasmic membrane, because
these compounds are likely to become partitioned into the
outer leaflet of the bilayer. Even the compounds with
lipophilic side chains were shown to penetrate across the outer
membrane relatively rapidly, if the pump was inactivated genetically or
physiologically. The exclusion of such compounds, exemplified by
nafcillin, from cells of the wild-type S. typhimurium
was previously interpreted as the result of poor diffusion across
the outer membrane (H. Nikaido, Biochim. Biophys. Acta 433:118-132,
1976), but it is now recognized as the consequence of
efficient pumping out of entering antibiotics by the active efflux
process.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-lactams.
For example, in P. aeruginosa inactivation of
mexA decreases the MICs of ceftriaxone, cefoperazone,
azlocillin, and carbenicillin by a factor of 8 to 128 (17),
and in E. coli the deletion of acrAB
decreases the MICs of ampicillin and benzylpenicillin
by a factor of 2 to 4 (19).
-lactams and thereby making the cells more resistant to
these antibiotics, convincing evidence has been difficult to obtain.
The presence of chromosomally coded -lactamases in these bacteria
made interpreting data on benzylpenicillin accumulation and MICs quite difficult (16). Furthermore, the observation that some of the -lactams do not diffuse into the cytoplasm during the time span of our experiments yet appear to be excluded from the
cell (16) was rather unexpected for an efflux pump. This increased the skepticism of some workers in the field and led to
attempts to determine whether the outer membrane channel alone could
carry out the -lactam efflux from the periplasm (42) or
at least determine the specificity of the process (36).
-lactams. S. typhimurium does not contain the
structural gene for -lactamase (2) and is therefore a
much simpler system to study. Earlier, a mutant of the S. typhimurium LT2 line, called SS-B, was isolated and shown to map
in the region of the chromosome where acrAB is located
(39). We show that this strain is indeed mutated in the
salmonella homolog of acrAB and use it to demonstrate the function of the AcrAB system in pumping out various -lactam
compounds. Furthermore, we investigated the efflux of various
-lactam compounds to examine the structural requirements for
substrates of AcrAB pump and propose a mechanism that would enable the
cells to pump out various drugs from the cytoplasm and periplasm and
would explain the surprisingly wide substrate specificity of the pump.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
Chemicals. Most antibiotics and other inhibitors were obtained from Sigma. Latamoxef and penicillin N were gifts of Shionogi & Co. Ltd (Osaka, Japan) and Wyeth-Ayerst Laboratories (Philadelphia, Pa.).
Determination of MICs. MICs were determined by twofold serial broth dilution in LB broth. The inoculum was 5 × 104 cells/ml, and the results were read after overnight incubation at 37°C.
Immunoblot analysis. Whole-cell proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein bands were transferred to a nitrocellulose membrane which was stained with a mixture of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate after the application of primary rabbit antibody and the alkaline-phosphatase-conjugated goat anti-rabbit-immunoglobulin G antibody (Bio-Rad) essentially as described earlier (5). The anti-AcrA antibody was generated in rabbits by using as an immunogen the AcrA tagged with hexahistidine at its C terminus, which had been purified by nickel column chromatography (44).
Assay of -lactamase.
Cells were disintegrated in a
Gallenkamp Soniprep 150 sonicator with four cycles of 30-s pulses each,
and enzyme activity was assayed spectrophotometrically at 260 nm with
100 µM cephaloridine as the substrate (26).
Assay of outer membrane permeability.
This assay was carried
out by one of two methods. By the first method, the influx of the
-lactams across the outer membrane caused by spontaneous diffusion
was coupled with subsequent hydrolysis by periplasmic -lactamase as
described earlier (26). Plasmid pBR322, coding for type A
-lactamase, was introduced by transformation (34). Cells
were grown in LB broth to mid-exponential phase, harvested, washed, and
resuspended in an Mg-containing phosphate buffer as described earlier
(26). The hydrolysis of cephalosporins was followed
continuously at 260 nm with 1 mM substrate and a cell with a 1-mm light
path as described previously (26). By the second method, the
entry of the drugs across the outer membrane and then the cytoplasmic
membrane was followed by measurement of the time-dependent decrease of
drug concentration in the external medium in suspensions containing
high concentrations of cells (23). Briefly,
exponential-phase cultures in LB were centrifuged, and cells washed
once with M63 containing 0.5% (wt/vol) glucose were suspended in the
same medium at a concentration of about 0.47 g (wet weight)/ml.
The suspensions were kept at room temperature (24°C) for about 5 min.
Nafcillin (0.3 ml of a 25-mg/ml solution) was then added to 2.1 ml of
cellular suspension at time 0, and portions of the suspensions were
removed and placed at 1-min intervals into heavy-walled glass
centrifuge tubes prechilled in an ice bath to stop further diffusion of
nafcillin through the lipid bilayer regions of the membranes. After
centrifugation, the supernatants were diluted 400-fold, and nafcillin
concentration was determined from optical density at 227 nm
(OD227). The data analysis was performed as described in
reference 23.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Transductional mapping of SS-B mutation. In the original description, conjugational mapping showed the SS-B mutation to be located in the region covering 8 to 12 min of the S. typhimurium chromosome (39). Although E. coli is known to contain three other homologs of acrAB (20), this region of the chromosome contains only acrAB among these homologs. Nevertheless, in order to further confirm the identity of the SS-B mutation, the cotransduction of the apt marker from the donor TN716 into a P1-sensitive derivative of SH7616, HN 1003, was tested. The apt gene is located only 0.14 min away from the acrAB cluster in E. coli (3). Since the arrangement of the mapped genes (such as lon, hupB, apt, and adk) is similar on the chromosomes of E. coli and S. typhimurium in the 9-to-12-min region (3, 35), we expected that acrAB of S. typhimurium would also be located close to the apt gene. When transductants containing the donor acr+ allele were selected on LB agar plates containing 100 µg of cloxacillin (an excellent selective agent for acr+, see below) per ml, 29 transductants out of 40 tested (73%) were shown to have coinherited the mutant apt allele from the donor, as they grew in M63-glucose medium containing 50 µg of an adenine analog, 2,6-diaminopurine (13), per ml (in addition to the required amino acids). Growth of the recipient strain, containing the wild-type apt+ allele, was inhibited even by 2 µg of 2,6-diaminopurine per ml under these conditions. This cotransduction frequency is similar to the predicted frequency (81%) for two markers that are 0.14 min away from each other (43) and confirms that SS-B corresponds to the acrAB homolog of S. typhimurium.
Isolation of a multidrug-resistant mutant, HN891. HN891 was isolated as a spontaneous chloramphenicol-resistant mutant of SH5014 (see Materials and Methods). This strain turned out to be more resistant to a number of antibiotics than the parent strain (Table 1). An examination of the level of AcrA protein by immunoblotting indicated that the strain produced significantly more AcrA than the parent (Fig. 1). (The acr mutant SH7616 produced an apparently normal amount of AcrA of normal size. The mutation therefore may be a missense mutation in acrA or may lie in the acrB gene.) It appears likely, therefore, that HN891 is multidrug resistant owing to the increased production of the AcrAB efflux pump, reminiscent of the situation already encountered in E. coli (28). Although the nature of the mutation is not known, it may be in marR, as efforts to isolate mutants of E. coli with a similar phenotype usually select for marR mutants (8).
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Phenotypes of the acr mutant SH7616 and the Acr
overproducer HN891.
As shown in Table 1, the acr mutant
SH7616 was more susceptible to a wide variety of compounds,
including detergents, dyes, and many antibiotics, confirming the
earlier results obtained with a more limited range of agents
(39). Among antibiotics other than -lactams, SH7616
showed unaltered susceptibility only to aminoglycosides such as
kanamycin and gentamicin and to bacitracin (data not shown). This
hypersusceptibility phenotype is very similar to that of the
E. coli acrAB mutants (19, 20).
-lactamase, known to be present
in most other species of the family Enterobacteriaceae
(2). A -lactamase assay with cephaloridine as the
substrate showed that the activity was <60 pmol mg of
protein
1 min1 in both SH5014 and SH7616.
This activity is less than 1% of the activity in the E. coli K-12 strain (27), which, in turn, is less than
1/1,000 of the activity seen in fully induced cells of
Enterobacter cloacae (7, 41). We could thus
confirm the absence of any detectable -lactamase activity in SH5014
as well as SH7616 cells, yet the presence and the overproduction,
respectively, of the AcrAB pump in SH5014 and HN891 significantly
increased the MICs of penicillin (Table 1) and other -lactams (Table
2) over that of SH7616, clearly
indicating that the AcrAB system does pump out these compounds at
significant rates.
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Entry of nafcillin.
To demonstrate more directly that the
intracellular accumulation of -lactams is affected by AcrAB
activity, we first attempted to examine the entry of
[3H]benzylpenicillin by silicone oil
centrifugation assay (16). However, the R-type
lipopolysaccharide produced by these strains (note the
rfaJ4041 mutation present in SH5014; Materials and Methods) caused strong aggregation of cells, which resulted in a poor and nonreproducible recovery of the cells at the bottom of the tubes.
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in reference
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MICs of various -lactam agents in strains with different levels
of AcrAB pump activity.
In an effort to gain knowledge about the
substrate specificity of the AcrAB pump, we determined the MICs of
various -lactam compounds in the acr mutant SH7616,
the parent SH5014, and the AcrAB overexpressor HN891 (Table 2). For
compounds containing lipophilic side chains, such as nafcillin and
cloxacillin, the acr mutation in SH7616 lowered the MIC by a
factor of more than 100, suggesting that they are good substrates of
the AcrAB pump. Furthermore, MICs of these more lipophilic compounds
became even higher in HN891 than in SH5014. On the other hand, MICs of
compounds containing hydrophilic side chains, such as penicillin N and
cefazolin, were affected little by the presence or absence or the
overproduction of the functional AcrAB system. We will evaluate in the
Discussion section the correlation between the lipophilicity of the
side chains and the magnitude of the change in MIC upon the
introduction of the acr mutation.
Entry rates of several cephalosporins.
MICs will be affected
not only by the rate of excretion of various compounds but also by
their rate of entry through the outer membrane. We therefore determined
the rate of diffusion of three cephalosporins covering a wide range of
lipophilicity, cephalothin, cefazolin, and cephalosporin C, by
measuring the rate of hydrolysis of these compounds with intact cells
of SH7616 and SH5014 containing pBR322, using a method established
earlier (26). The outer membrane of SH7616 had permeability
coefficients of 1.3 × 105, 5.0 × 105, and 1.5 × 105 cm
s1, respectively, for the three compounds mentioned
above. With SH5014, the permeability coefficients obtained were
slightly lower, by about 30% on average, than the values with SH7616;
this result may be due to some efflux of these compounds from the
periplasm through the AcrAB system.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study, we first showed that an SS-B mutant of S. typhimurium, SH7616, had a mutation in the homolog of acrAB of E. coli, based on its phenotype as well as the high frequency of cotransduction observed with the apt marker. This conclusion is consistent with results from other laboratories that were reported during the course of this study. Thus, the S. typhimurium acrB gene was identified by partial sequencing of DNA (15), and insertional inactivation of this gene produced a drug sensitivity phenotype very similar to that of SH7616 (reference 14 and Table 1). Mapping by another laboratory also confirmed the location of acrB on the Salmonella chromosome at a position corresponding to that of the E. coli gene. Thus, a TnphoA insertion into a locus within a few minutes of purE (mutant SM7) was shown to make Salmonella enteritidis noninvasive in tissue culture cells (37), presumably because of its hypersensitivity to detergents (see reference 38). Sequencing of DNA from the insertion site showed that the sequence was similar to that of the E. coli acrB gene (12).
Once nafcillin reaches the periplasm after the rate-limiting diffusion
across the outer membrane, it crosses the cytoplasmic membrane rapidly,
owing to its high lipophilicity (23, 29); this feature makes
the nafcillin accumulation assay much easier to perform than assays
with some -lactam compounds that enter only into the periplasm. The
nafcillin assay showed that the drug entered into the mutant SH7616
cells much more rapidly than into the wild-type parent cells,
suggesting that the hypersusceptibility to -lactams in SH7616 is
caused by either a higher permeability of its outer membrane
or the lack of active efflux of these compounds. The former possibility
can be ruled out because (a) the inhibition of efflux by deenergization
with CCCP also allowed the rapid influx of nafcillin into SH5014 (Fig.
3) and (b) the direct assay of outer membrane permeability with both
moderately lipophilic (cephalothin) and hydrophilic (cefazolin
and cephalosporin C) cephalosporins showed that there is only a
small apparent difference between the two strains (see Results).
Some of the -lactam compounds did not enter the cytoplasm during our
experimental time period (16), and it was unexpected that
active efflux mechanism would be able to capture its substrates without
their entry into the cytoplasm. However, similar observations have been
made repeatedly with the MDR pump of mammalian cells. Thus, Homolya et
al. (11) showed that 3T3 cells expressing the MDR protein
capture and exclude the neutral pentaester of a dye, FURA-2,
from within the membrane bilayer. Furthermore, Ruetz and Gros (32) showed that mouse MDR2, a homolog of MDR, is
a phosphatidylcholine flippase that translocates the lipid molecule
from the inner leaflet to the outer leaflet (Fig.
4A). Thus, both MDR and MDR2 capture substrates from within the bilayer.
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Although mammalian MDR and bacterial RND pumps have no sequence
homology and are energized in different ways, they may have a similar
mechanism for substrate capture. We hypothesize that the RND pump binds
substrates that have already been partitioned into the outer leaflet of
the membrane bilayer and that even -lactam compounds that do not
penetrate into the cytoplasm can be pumped out efficiently in this
manner (Fig. 4B and C). (A similar mechanism involving the capture of
substrates from within the bilayer was independently proposed for a
multidrug efflux pump of a totally different type [4];
in this case, the capture appears to occur exclusively from the
inner leaflet of the bilayer.) This model also explains the unusually
wide specificity of the AcrAB system. The major requirement for any
drug to become a substrate of this pump is an efficient, at least
partial, insertion into the membrane bilayer (25), and this
is precisely what was shown by the correlation between the side-chain
lipophilicity of the -lactams and their ability to become substrates
of the AcrAB efflux pump. Thus, those compounds with strongly
lipophilic side chains that are likely to partition spontaneously into
the bilayer, such as nafcillin and cloxacillin, appear to be good
substrates of the pump because the parent strain producing the
functional AcrAB system was far more resistant to them than the
acr mutant SH7616 (Table 2 and Fig. 5). In contrast,
compounds with hydrophilic side chains, such as penicillin N,
cefazolin, or cefmetazole, were apparently not pumped out efficiently,
as judged by the similar MICs shown by the AcrAB overproducer, the
parent strain, and the acr mutant (Table 2 and Fig.
5). Thus, our hypothesis predicts that
the outer membrane channel alone would obviously be incapable of
pumping out drug molecules from the periplasm, a prediction confirmed by recent studies with P. aeruginosa (36,
42). It must be pointed out, however, that the observed
correlation (Fig. 5) can also be explained by assuming a highly
lipophilic substrate-binding site within the transporter protein but
not that the substrates first enter the lipid bilayer. This hypothesis
is not attractive because it forces the assumption of two
substrate-binding sites, one facing the periplasmic space and the other
facing the cytosol.
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An examination of Table 2 shows that the correlation between lipophilicity and efflux is not perfect. The discrepancy is most prominent with cefsulodin, which showed equal but low activity on the three strains used. Possibly the three charges on this compound hinder even a partial partitioning of the molecule into bilayer. Indeed, the presence of the AcrAB pump has a much weaker effect on the efficacy of compounds with more than one charged group (Fig. 5, triangles). However, this does not alter our conclusion on the correlation of side-chain hydrophobicity and the effectiveness of the efflux process, as monoanionic compounds containing hydrophilic side chains, such as cefazolin and cefmetazole, were apparently poor substrates of the AcrAB pump (Table 2).
The final proof, however, of the correlation between side-chain
lipophilicity and susceptibility to efflux requires the elimination of
another interpretation of the data of Table 2, that the more hydrophilic compounds may not be affected significantly by the presence
or absence of the AcrAB pump simply because they can cross the outer
membrane much more rapidly through porin channels than the hydrophobic
compounds (see reference 26). However, one of the
most lipophilic compounds tested, nafcillin, appears to penetrate
the outer membrane with a permeability coefficient in the range of
2 × 105 cm/s at 37°C. (This result was estimated
from the measured half-equilibration time of 2 to 3 min [Fig. 2 and
3] at 24 to 26°C and from the high-temperature coefficient of the
nafcillin penetration rate [23].) This is comparable
to the coefficients for hydrophilic -lactam molecule permeability
through E. coli porin channels (26) and to
those for the permeability of moderately lipophilic and hydrophilic cephalosporins determined in the S. typhimurium strains
in this study, that is, between 1.3 and 5 × 105
cm/s (see Results). It is thus clear that overall permeation rates
across the outer membrane differed relatively little (less than
fourfold) among various -lactam compounds tested (although the more
lipophilic compounds must have used the lipid bilayer pathway more than
the porin channels for diffusion). This observation, then, rules out
the alternative interpretation described above, which is also
contradicted by the finding that the presence of AcrAB had no effect on
the MIC of cephalosporin C (Table 2), whose permeability coefficient
was one of the lowest among those examined (see Results).
The results presented show that active efflux has a strong influence on
the efficacy and MICs of more lipophilic -lactams. Earlier, one of
the present authors found that considerations of only the permeability
barrier and the kinetic parameters of periplasmic -lactamase allowed
the prediction of the efficacy of various -lactam compounds in
E. coli (27), and this was confirmed by
another laboratory (6). In retrospect, these predictions were successful probably because most of the compounds used were those
with hydrophilic side chains, which were reasonably effective against
the wild-type E. coli. These compounds are now known to be poor substrates of the pump. We now realize that a more complete and
accurate prediction of MICs, especially those of the more lipophilic
compounds, will require consideration of the active efflux.
Furthermore, if an organism has an outer membrane of unusually low
permeability or expresses efflux pump(s) more strongly, models that do
not include efflux processes will be less useful. As shown by Livermore
and Davy (18), this is precisely what happens with P. aeruginosa, whose outer membrane has low permeability.
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ACKNOWLEDGMENTS |
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This study was supported in part by a research grant from the U.S. Public Health Service, AI-09644.
We thank M. Vaara, K. E. Sanderson, and V. L. Miller for bacterial strains and for sharing unpublished information. We also thank W. Pan for his contribution in the early stage of this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular and Cell Biology, 229 Stanley Hall, University of California, Berkeley, CA 94720-3206. Phone: (510) 642-2027. Fax: (510) 643-9290. E-mail: nhiroshi{at}uclink4.berkeley.edu.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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