From the Department of Molecular Life Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara 259-1193, Japan
Received for publication, November 26, 2002
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ABSTRACT |
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Infection of Pseudomonas aeruginosa
in cystic fibrosis patients is a major cause of mortality. This
organism shows wide ranging antibiotic resistance that is largely
attributable to the expression of xenobiotic efflux pump(s). Here, we
show a novel mechanism by which the
resistance-nodulation-division-type xenobiotic transporter expels
potential hazards and protects the interior of the cells. The
xenobiotic transporters MexB and MexY preferentially export Pseudomonas aeruginosa shows intrinsic and mutational
resistance to a broad spectrum of antibiotics and this resistance is mainly attributable to a synergy of expression of the xenobiotic efflux
transporters and a tight outer membrane barrier (1-4). Multiantibiotic
resistance in this organism is a major cause of mortality of hospital
patients whose immune activity was lowered by some other factors.
P. aeruginosa encodes several
RND1 family efflux pumps
including MexAB-OprM and MexXY-OprM (3). These transporters consist of
the inner membrane spanning the proton-antibiotic antiporter (MexB or
MexY), the membrane fusion proteins that are assumed to connect the
inner and outer membranes (MexA or MexX) and the outer
membrane-associated lipoprotein OprM common to both transporters (3).
The inner membrane spanning subunit (RND transporter) has been found to
traverse the cytoplasmic membrane 12 times leaving amino and carboxyl
termini in the cytoplasm and the TMSs connected by extra membrane loops
(5-7). Although most loops are short, the loops connecting TMS-1 and
TMS-2 (loop-1/2) and TMS-7 and TMS-8 (loop-7/8) were found to consist
of over 300 amino acid residues largely protruding toward the
periplasmic space. Of five charged amino acid residues in the TMSs of
MexB, three in TMS-4 and TMS-10 were shown to be essential for the
transporter function probably forming a proton pathway (8). MexB and
MexY preferentially export Most active transporter proteins recognize the specific stereochemical
structure of substrates, while many xenobiotic efflux pumps export
structurally dissimilar molecules. Analysis of mutant transporters that
altered the substrate selectivity suggested that the mutations were
mainly located in TMS. We assessed the question for the substrate
recognition site in the RND-type xenobiotic efflux pump by means of
domain swapping experiments, and the results let us to propose a novel
mechanism of bacterial self-protection against xenobiotics.
Bacterial Strains, Plasmids, and Primers--
Bacterial strains
and plasmids used are listed in Table I.
Primers are listed in Supplemental Fig. 2.
Construction of Plasmids, Deletion of Chromosomal mexY or mexXY
by Gene Replacement, and Other Methods--
These are provided in
Supplementary Figs. 3 and 4.
Swapping of Extramembrane Loops--
Taking the advantage of
sequence similarity, clear substrate selectivity, and the shared outer
membrane subunit between MexB and MexY, we carried out domain-swapping
experiments. We took first the MexY transporter, deleted loop-1/2 and
replaced it with loop-1/2 of MexB. Similarly, loop-7/8 of MexY was
replaced with that of MexB. The wild-type MexB conferred resistance
against
Next, we replaced both loop-1/2 and loop-7/8 of MexY with the
corresponding loops derived from MexB. Cells expressing a mosaic protein showed the aztreonam MIC of 3.13 µg ml
Since the above domain swapping experiment was successful in the
presence of MexA, the question of whether or not two large periplasmic
domains cooperate with membrane fusion protein in the xenobiotic export
can be investigated. We have carried out such an experiment by
expressing the above-mentioned MexB(loop1/2+loop7/8)/MexY(TMSs) hybrid
protein in the host producing MexX, but lacking MexA, MexB, and MexY.
As seen in Fig. 1b, this mosaic protein failed to transport any substrate so far tested, and it became evident that loop-1/2 and
loop-7/8 were unable to cooperate with MexX, but did cooperate with
MexA. Therefore, it is highly likely that two periplasmic domains
interact with the partner membrane fusion protein.
To ascertain whether or not lack of antibiotic transport activity in
the constructs carrying either one of two large periplasmic domains is
due to low level protein expression, we tested the presence of
histidine-tagged MexB or MexY in cells harboring the respective
plasmids. As seen in Fig. 1, all the cells harboring the plasmid
encoding mexBHis,
mexYHis, mexB(loop1/2)/mexY(TMSs)His,
mexB(loop7/8)/mexY(TMSs)His, and mexB(loop-1/2+loop-7/8)/mexY(TMSs)His
showed comparable expression of proteins reactive with
anti-hexahistidine andibody. These results clearly indicated that the
undetectable function of MexB(loop-1/2)/MexY(TMSs) and
MexB(loop-7/8)/MexY(TMSs) was not attributable to low level expression
of the hybrid protein.
Swapping of Transmembrane Segments--
Although the above
experiment demonstrated that loop-1/2 and loop-7/8 were responsible for
substrate recognition, this does not rule out the possibility that
these loops select substrates by coupling with TMS(s). If the TMS of
MexB participates in substrate recognition, replacement of the TMS of
MexB with that of MexY must result in hybrid protein dysfunction in the
transport of the MexB substrate. Thus, we replaced the TMS of the MexB
transporter with that of MexY such that TMS-1 of MexB was replaced by
TMS-1 of MexY and so on. The results revealed that all MexY-TMSs were fully cooperative with loop-1/2 and loop-7/8 of MexB in the transporter function and showed that aztreonam transport activity was comparable with that of intact MexB. There were considerable variations in the
activity of the hybrid transporter whose proteins having TMS-5, -6, and
-7 showed low transport activity, yet all the proteins exhibited
MexB-type substrate selectivity (Fig. 2).
The hybrid proteins were unable to transport the MexY substrate,
gentamicin. Therefore, it became evident that the TMS of MexB was not
directly involved in the selection of either
To verify the proper expression of the hybrid proteins, crude membrane
fractions were subjected to Western blotting analysis using antibody
against MexB (loop-1/2). The results showed that all the constructs
expressed the hybrid protein in an amount comparable with intact MexB,
except for the protein containing TMS-6 from MexY. However, the
substrate selectivity and transport activity of this hybrid protein was
comparable with that of the others. Therefore, we concluded that
variable transport activity of the hybrid proteins containing TMS from
MexY was not attributable to the low level expression.
Studies on the substrate recognition site of xenobiotic
transporters in mammalian and prokaryote cells showed the following results. Mutations, which altered the substrate specificity of P-glycoprotein and multidrug resistant proteins, were scattered in TMSs
and cytoplasmic loops. Thus, it has been assumed that these
transporters select substrates from either cytoplasmic and exoplasmic
leaflets of the membrane, which are mechanisms proposed as the
"vacuum cleaner model" and "flippase model," respectively (10-12). Substrate recognition in the xenobiotic transporters of the
prokaryote have been investigated in Bmr, LmrP, MfdA, QuacA, and EmrE,
revealing that most, if not all, mutations affecting substrate
recognition have been identified in the TMS(s) (13-16).
Recently, the x-ray crystallographic structure of AcrB, an
Escherichia coli homologue of MexB, has been solved
showing the presence of three major domains: a cluster of 12 TMSs, a
pore-forming domain, and a TolC-docking domain (5). The pore domains
formed a central cavity, designated as a vestibule, that connected with three channel openings toward the periplasm, and a funnel-like TolC-docking domain. The authors predicted that these vestibules are
likely candidates for the substrate-collecting ducts. Since the amino
acid identity of AcrB and MexB appeared to be about 70%, it is likely
that the three-dimensional structure of MexB was much like that of
AcrB. In fact, the x-ray crystallographic analysis of AcrB confirmed
our previous results that TMS-4 and -10 of MexB are close to each other
(8).
This raises the question of which part of the RND transporters collects
the substrates. It has been proposed that the substrate-collecting site(s) of the RND transporters are located at both the inner membrane
and the cytoplasm (2, 17). Since the results presented in this paper
clearly demonstrated that membrane insoluble substrates were selected
in the periplasmic domains, it is highly likely that the
substrate-collecting ducts in MexB and MexY are located in the
periplasmic domain. If this proposal is valid, one can answer the
following interesting questions: (i) how do the cells protect the
cytoplasmic membrane from membrane deteriorating surfactants, such as
sodium dodecyl sulfate and bile salts? (ii) How are lipid-insoluble antibiotics such as The following model plausibly explains the mechanism by
which the RND transporters might collect, recognize, and transport xenobiotics. Substrates that have diffused into the periplasmic space
across the outer membrane are sucked up into the periplasmic pore
inlets before diffusing into the inner membrane. They are then
subjected to filtration by the substrate-recognizing site probably
located in the pore domains and subsequently transported to the
extracellular milieu (Fig. 3). The
xenobiotics may not penetrate the cytoplasm of cells with a tight outer
membrane barrier and powerful xenobiotic transporters, which protect
the interior of the cells from potential hazards. This is an elegant
and efficient means of safeguard. However, this new model is not
mutually exclusive of the hypothesis that the transporters collect
substrates from the inner membrane. During the course of this
manuscript preparation, we learned that MexD mutations with altered
substrate selectivity were mapped in the large periplasmic domains (18)
and that domain-swapping experiments have been carried out with
different combinations of the transporters (19). The results are
partially consistent with the present results and, therefore, can be
regarded as supporting the new model proposed in this report.
-lactam
and aminoglycoside antibiotics, respectively. When two large
extramembrane loops of MexY were replaced by the corresponding loops of
MexB, the hybrid protein exhibited
-lactam selectivity (MexB-type),
but failed to recognize aminoglycoside. As the transmembrane segment of
MexB was replaced with a corresponding transmembrane segment of MexY,
one-by-one for all 12 segments, all the hybrid proteins showed
MexB-type antibiotic selectivity. These results clearly demonstrated
that the resistance-nodulation-division-type efflux pump in
P. aeruginosa selects and transports substrates via
the domains that largely protrude over the cytoplasmic membrane. The
transmembrane segments were unlikely to have been involved in substrate
selectivity. These observations led us to propose a novel mechanism by
which the xenobiotic transporters in Gram-negative bacteria select and
expel substrates from the periplasmic space before potential hazards
penetrate into the cytoplasmic membrane.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactams and aminoglycosides,
respectively, although both transporters export agents such as
chloramphenicol, fluoroquinolones, and tetracycline as well (9). The
amino acid sequences of these transporters appeared to be about 47%
identical (see Supplemental Fig. 1).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids used
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactam antibiotics (aztreonam), nalidixic acid, and
chloramphenicol to an MIC level of 3.13, 50, and 50 µg
ml
1, respectively, but not against gentamicin
(aminoglycoside) (0.1 µg ml
1) (Fig.
1a). The MexY transporter
conferred resistance against gentamicin, naldixic acid, and
chloramphenicol to an MIC level of 0.78, 12.5, and 6.25 µg
ml
1, respectively, but not against
-lactam (0.2 µg
ml
1) (Fig. 1b). If loop-1/2 or loop-7/8 alone
was responsible for substrate selectivity, one would expect that cells
expressing such a hybrid protein would exhibit antibiotic
susceptibility similar to that in cells producing the MexAB-OprM efflux
pump. Alternatively, if the TMSs were solely responsible for selecting antibiotics, the hybrid protein might exhibit MexY-type antibiotic selectivity. Cells expressing the hybrid protein with loop-1/2 or
loop-7/8 of MexB and the rest of the domains from MexY appeared totally
non-functional (Fig. 1, a and b). The results
suggested that MexY-TMSs alone could not select the substrate or that
the transporter might require both loop-1/2 and loop-7/8 from the same
source. It is also possible that the construction of the loop-TMS joint
site might not have been appropriate.
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Fig. 1.
Substrate selectivity of the MexB-MexY hybrid
protein. MICs of selected antibiotics were expressed as relative
values to bare MICs in the hosts harboring only the vector that was
shown at the top of the column. Host cells: TNP088
(A+, B , X
, Y
)
(a) and TNP089 (A
, B
,
X+, Y
) (b). Expression of protein
was probed by antibody against hexahistidine by the Western blotting
method. Solid and gray lines in the transporter
model represent domains from MexB and MexY, respectively. Methods of
MIC determination and Western blotting were described earlier
(8).
1, which
was identical to the MIC in cells expressing the wild type MexB (Fig.
1a). The cells exhibited the gentamicin MIC that was
identical to that of MexY-negative cells, which was 0.1 µg ml
1 (Fig. 1a). One may argue that substrate
selectivity by the periplasmic domains may be limited to very
hydrophilic substrates, such as
-lactams and aminoglycosides.
However, this might not be the case, because selectivity of this
MexB-MexY mosaic protein toward more hydrophobic substrates was also
changed from the MexY-type to the MexB-type whose naldixic acid and
chloramphenicol MICs both were 50 µg ml
1 (Fig.
1a). These results clearly indicated that the RND xenobiotic transporters in P. aeruginosa select substrates by means of
two large periplasmic domains.
-lactam or
aminoglycoside antibiotics. Substrate selectivity of these hybrid
proteins for other antibiotics such as nalidixic acid and
chloramphenicol appeared to be the MexB-type without exception.
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Fig. 2.
Substrate selectivity of the MexB-MexY hybrid
protein with replaced TMS. Only one each of MexB-TMS was replaced
with the corresponding TMS of MexY. For details, see the legend to Fig.
1. Antibody used to probe protein was raised against MexB (loop-1/2).
The host cell used was TNP088 (A+, B ,
X
, Y
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactams and aminoglycosides efficiently transported? (iii) How do these low substrate-selective transporters prevent leakage of cytoplasmic materials?
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Fig. 3.
Proposed model for xenobiotic export by the
RND efflux pump and mechanism to protect the interior of the
cells. Arrows indicate possible routes of xenobiotic
flows. The substrates may be collected in the periplasmic space via the
collecting duct, filtered in the large periplasmic domains, and
exported outside the cell. Substrate flows from the inner membrane and
cytoplasm were not supported by the present report.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education, Culture, Sport, Science and Technology; the Japan Society for Promotion of Science; the Ministry of Health, Labor and Welfare; Tokai University (Project Research Grant); and Tokai University School of Medicine (Research Project Grant).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.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figs. 1-4.
To whom correspondence should be addressed. Tel.: 81-463-93-5436;
Fax: 81-463-93-5437; E-mail:
nakae@is.icc.u-tokai.ac.jp.
Published, JBC Papers in Press, December 1, 2002, DOI 10.1074/jbc.C200661200
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ABBREVIATIONS |
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The abbreviations used are: RND, resistance-nodulation-division; TMS, transmembrane segment; MIC, minimum growth inhibitory concentration of antibiotic.
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