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J Biol Chem, Vol. 275, Issue 7, 4654-4659, February 18, 2000
From the Enoyl-acyl carrier protein reductase (FabI) plays
a determinant role in completing cycles of elongation in type II fatty
acid synthase systems and is an important target for antibacterial drugs. The FabI component of Staphylococcus aureus (saFabI)
was identified, and its properties were compared with Escherichia coli FabI (ecFabI). ecFabI and saFabI had similar specific
activities, and saFabI expression complemented the E. coli
fabI(Ts) mutant, illustrating that the Gram-positive FabI was
interchangeable with the Gram-negative FabI enzyme. However, ecFabI was
specific for NADH, whereas saFabI exhibited specific and positive
cooperative binding of NADPH. Triclosan and hexachlorophene inhibited
both ecFabI and saFabI. The triclosan-resistant ecFabI(G93V) protein was also refractory to hexachlorophene inhibition, illustrating that
both drugs bind at the FabI active site. Both the introduction of a
plasmid expressing the safabI gene or a missense mutation in the chromosomal safabI gene led to triclosan resistance
in S. aureus; however, these strains did not exhibit
cross-resistance to hexachlorophene. The replacement of the ether
linkage in triclosan by a carbon bridge in hexachlorophene prevented
the formation of a stable FabI-NAD(P)+-drug ternary
complex. Thus, the formation of this ternary complex is a key
determinant of the antibacterial activity of FabI inhibitors.
Bacterial fatty acid biosynthesis is carried out by a universal
series of reactions catalyzed by a collection of enzymes each encoded
by a separate gene (1, 2). Fatty acids are assembled 2 carbon units at
a time in a cyclical sequence of reactions. The
enoyl-ACP1 reductase (FabI)
catalyzes the last step in each cycle and plays a regulatory role in
determining the rate of fatty acid synthesis (3, 4). The FabI enzymes
are receiving increased attention not only because of their regulatory
significance, but also because of the recent discovery that inhibitors
of this step in the pathway are effective antibacterials. The
diazoborines inactivate FabI through the formation of a covalent bond
with NADH in the active site of the enzyme (5, 6). Isoniazid, an
inhibitor of the mycobacterial FabI homolog (termed InhA), is activated
within the cell and forms a covalent complex with NAD+ in
the InhA active site (7, 8). FabI is also a target of the broad
spectrum biocide, triclosan (9-11). Triclosan is a slow-binding inhibitor that inactivates the enzyme via the formation of a stable, non-covalent, FabI-NAD+-triclosan ternary complex (12-14).
Triclosan is used as an antibacterial additive in a wide range of
consumer goods, including cutting boards, mattress pads, facial
cleansers, and hand soaps (15). Triclosan has a broad spectrum of
antibacterial activity and is effective against both Gram-negative and
Gram-positive organisms. Prior to the identification of FabI as the
intracellular target of triclosan, it was widely believed that its
antimicrobial action resulted from nonspecific disruption of cellular
membranes (16-18).
Hexachlorophene is another antimicrobial compound used in disinfectants
and surgical scrubs (19, 20). Hexachlorophene has a more limited
spectrum of biological activity than triclosan, being most active
against Gram-positive bacteria, especially strains of
Staphylococcus, but less effective against Gram-negative
organisms (21). Hexachlorophene is thought to act by disrupting
bacterial membranes, although it is unclear as to whether this is the
primary mode of action (22-24). Hexachlorophene and triclosan are
strikingly similar compounds (Fig. 1). In
particular, hexachlorophene contains the hydroxyphenyl moiety that is
essential for the activity of triclosan and related
hydroxydiphenylethers (10, 12). The similarities in their structures
and their proposed mechanisms of action prompted us to determine
whether hexachlorophene was also a FabI inhibitor. Since
hexachlorophene is selectively effective against Gram-positive
organisms and FabI enzymes have not been characterized from this group
of bacteria, we cloned and expressed the FabI protein of
Staphylococcus aureus to investigate whether a unique FabI
protein may account for the differential sensitivity of
Escherichia coli and S. aureus to
hexachlorophene, or whether hexachlorophene interacts with a different
target in Gram-positive bacteria. Both S. aureus FabI
(saFabI) and E. coli FabI (ecFabI), are inhibited by
hexachlorophene and triclosan; however, FabI is unlikely to be a
biologically relevant target for the antibacterial activity of
hexachlorophene due to the inability of this drug to form a ternary
complex with FabI and the nucleotide cofactor.
Materials--
His-tag FabI and FabI(G93V) from E. coli were purified as described previously (3, 12). The
trans-2-octenoyl-N-acetylcysteamine, trans-2-butenoyl-N-acetylcysteamine, and
2,2'-dihyroxydiphenylether were the generous gifts from Rocco Gogglioti
and John Domagala at Parke-Davis Pharmaceuticals. KIC (Aromonk, NJ)
supplied the triclosan while hexachlorophene,
2,2'-dihydroxydiphenylmethane, and
2,2'-dihydroxy-4,4'-dichlorodiphenylmethane were purchased from TCI
America. Sigma provided NADH and NADPH. American Radiolabeled Chemicals, Inc. supplied the
[2,8-3H-adenine]NAD+ (specific activity: 25 Ci/mmol). All other reagents were of the highest available grade.
Cloning of the safabI Gene--
The fabI gene from
S. aureus was identified in the unfinished microbial genomic
database by comparison with ecFabI. The fabI gene was cloned
from the genome of S. aureus strain RN4220 using the
primers RJH101 (bp 1-32) (5'-CATATGTTAAATCTTGAAAACAAAACGTATGTCAT) and
RJH102 (bp 771-745) (5'-GGATCCTTATTTAATTGCGTGGAATCCGCTATC) and
ligated into the TA vector pCR2.1 (Invitrogen). The gene was sequenced
and then excised with NdeI and BamHI and ligated
into the expression vector pET15b digested with the same enzymes. This construct, pETsaI, was transformed into E. coli strain
BL21(DE3) for expression following isopropylthiogalactoside
induction. The NH2-terminal His-tagged protein was purified
by Ni2+ chelation chromatography. Following purification,
the protein was exchanged into 20 mM Tris, pH 7.6, 1 mM dithiothreitol, 100 mM EDTA by dialysis
overnight. Omission of the EDTA led to precipitation of saFabI in the
dialysis tubing. The protein was homogeneous as judged by SDS-gel
electrophoresis. The yields and purity were similar to those reported
previously for ecFabI (3, 4).
Plasmid pETsaI was digested with XbaI and EcoRV,
and the 0.8-kilobase pair fragment was ligated into similarly digested
pBluescript KSII(+) to create pAN2 to express saFabI in E. coli. pAN2 was transformed into strain RJH13 (fabI(Ts))
and plated at 30 °C. Individual colonies were then tested for growth
at 42 °C by spotting onto LB agar plates. Controls were RJH13
(fabI(Ts)) transformed with either pFabI (10) or the empty vector.
Spectrophotometric Assay of FabI--
Reduction of
trans-2-enoyl-N-acetylcysteamine substrates was
measured spectrophotometrically as described previously (10). Briefly,
a standard reaction contained 0.1 M sodium phosphate, pH
7.5, 100 µM
trans-2-octenoyl-N-acetylcysteamine, 200 µM NADH or NADPH, and 6 µg of FabI in a final volume of
300 µl. Decrease in absorbance at 340 nm was measured at 25 °C for
the linear period of the assay (usually the first 1-2 min).
Modifications to this assay are noted in the text and figure legends
where applicable. The rate of substrate-independent hydrolysis of
NAD(P)H was monitored and subtracted from each point from at least two
independent experiments. Kinetic parameters were obtained by varying
the concentration of cofactor or substrate as indicated. Drugs were
added as serial dilutions from a stock in dimethyl sulfoxide, and the
final volume of carrier was kept constant (1.7%) through the entire
assay. In one series of experiments, the enzyme and drug were
preincubated for 20 min at room temperature prior to addition to the
assay, and the change in absorbance was followed with time.
[3H]NAD+ Binding Assay--
Assays
were performed as described (12) in 0.1 M sodium phosphate,
pH 7.6, containing 4 µM NAD+ (specific
activity, 4 Ci/mmol), 1 µg/ml triclosan, and 8 µg of protein in a
total volume of 50 µl and were incubated for 20 min at room
temperature, then placed into an Ultrafree-Probind centrifugal filtration unit containing a 0.2-cm2, 0.45-µm
polyvinylidene difluoride membrane (Millipore Corp.). These units were
then centrifuged, and the filter was washed with 0.1 M
sodium phosphate buffer and then counted in 5 ml of ScintSafe scintillation fluid.
MIC Determinations--
The MIC values were determined by the
microbroth dilution assay according to the National Committee for
Clinical Laboratory Standards methods for antimicrobial susceptibility
tests for aerobically growing bacteria (Approved Standard M7-A2, NCCLS,
Villanova, PA, 1990). All of the determinations were performed twice
with comparable results.
Isolation of Triclosan-resistant S. aureus--
Approximately
6.5 × 108 cells of strain RN4220 (25) were plated on
to brain-heart infusion agar medium (Becton-Dickinson) containing 0.5 µg/ml triclosan. The plates were incubated at 37 °C with periodic
monitoring. After 48 h, resistant clones were picked and their
phenotype confirmed by regrowth on the original selective concentration
of triclosan. The S. aureus fabI gene from
triclosan-resistant mutants was characterized by double-strand nucleotide sequencing of PCR products amplified from chromosomal DNA
(26). All PCR reactions utilized PCR SuperMix (Life Technologies, Rockville, MD). The reactions contained 100 µM of the
primer GER68 (5'-ATGTTAAATCTTGAAAACAAAAC; complementary to bp 1-23)
and primer GER71 (5'-TTATTTAATTGCGTGGAATCCGC; complementary to bp
771-749). Sequencing was performed on an Applied Biosystems model 377 DNA sequencing system using a PRISM Sequenase® dye
terminator sequencing kit using GER68, GER71, and two additional internal fabI primers (5'-GCGGGACGCTTTTCTGAAACTTCACG and
5'-CGCCACCTAAATATGTTGTTGCAACAATGC) to ensure complete coverage.
Isolation of saFabI Clones--
A chromosomal DNA plasmid
library derived from strain RN4220 was constructed by ligating sucrose
gradient-purified Sau3A1 fragments (3-7 kilobase pairs)
into BamHI-restricted plasmid pSK265 (27). After
electroporation into strain RN4220, approximately 2,000 clones were
pooled and frozen at Cloning of safabI--
The gene for the
trans-2-enoyl-ACP reductase from S. aureus was
identified by a BLAST search of the preliminary sequence data of
unfinished microbial genomes obtained from the Institute for Genomic
Research web site. The predicted protein sequence was 48% identical to
ecFabI (Fig. 2) and included the
YX6K sequence motif containing the key tyrosine
and lysine active site
residues.2 The gene was
amplified by PCR from the chromosome of S. aureus strain
RN4220 and cloned into the expression vector pET15b. Homogeneous protein was obtained by Ni2+ affinity chromatography as
described under "Experimental Procedures" (data not shown).
Activity of saFabI--
We utilized the spectrophotometric assay
described under "Experimental Procedures" to analyze the nucleotide
dependence of ecFabI and saFabI (Fig. 3).
The ecFabI exhibited a marked preference for NADH over NADPH (Fig.
3A). The apparent Km of ecFabI for NADH
was 25 ± 4 µM (Fig. 3A,
inset). We were not able to get a reliable estimate for the
NADPH Km for ecFabI due to the low reaction rates,
even at high enzyme concentrations. However, it was clear that the
apparent Km was >2 mM. Whereas saFabI
utilized NADH as a cofactor, the specific activity was very low;
however, when NADPH was employed, a robust rate was observed (Fig.
3B). The saFabI displayed non-linear kinetics with NADPH,
and a distinct upward curvature of the double-reciprocal plot indicated
positive cooperative NADPH binding (Fig. 3B,
inset). The data fitted almost perfectly to a second order
polynomial equation (r2 = 0.9997), and analysis
by a Hill plot revealed that NADPH binding to saFabI was highly
cooperative with a Hill coefficient, nH, of 2.2 (Fig. 3C). The cofactor concentration that yielded 50% of
the maximal rate was 65 µM. The very low rate of reaction
with NADH precluded a detailed analysis of NADH binding to saFabI, but
the apparent Km was >1 mM. Analysis of
the ecFabI and saFabI with crotonyl-N-acetylcysteamine and
NADH or NADPH, respectively, revealed that both enzymes also had
similar affinities for this substrate analog (ecFabI,
Km = 5 ± 0.3 mM and Vmax = 2 ± 0.5 nmol/min/µg; saFabI,
Km = 8 ± 0.3 mM and Vmax = 0.3 ± 0.1 nmol/min/µg).
The gene for saFabI was expressed in E. coli using plasmid
pAN2 as described under "Experimental Procedures." The introduction of plasmid pAN2 into strain RJH13 (fabI(Ts)) (3)
complemented the temperature-sensitive growth defect of this strain at
42 °C. Analysis of the fatty acid composition of the transformed
strains by gas-liquid chromatography show a normal E. coli
profile that was the same as the parent strain (data not shown).
Additionally, saFabI was able to substitute for ecFabI in the type II
fatty acid synthase system reconstituted in vitro with
purified enzymes of E. coli (data not shown). These
experiments illustrated that saFabI was functionally equivalent to
ecFabI and was capable of catalyzing all of the reactions required for
the synthesis of straight chain saturated and unsaturated fatty acids.
Inhibition of ecFabI and saFabI by Triclosan and
Hexachlorophene--
The efficacy of hexachlorophene as a FabI
inhibitor was compared with triclosan in a series of kinetic
experiments where the reactions were initiated by the addition of
enzyme and initial rates were measured over the first 90 s of the
reaction (Fig. 4). ecFabI was inhibited
by triclosan with an IC50 of 2 µM in this
assay (Fig. 4A), which was essentially identical to the
result obtained previously (12). The saFabI was also inhibited by
triclosan with an IC50 of 3 µM (Fig.
4A), demonstrating that saFabI was also a target for
triclosan. As expected, the ecFabI(G93V) active site mutant was
more resistant to triclosan inhibition (Fig. 4A and Ref.
12). Hexachlorophene also inhibited ecFabI and saFabI in this in
vitro assay (Fig. 4B). Both enzymes exhibited an
IC50 of approximately 4 µM, making
hexachlorophene almost as potent a FabI inhibitor in this kinetic assay
as triclosan. The ecFabI(G93V) protein was resistant to hexachlorophene
inhibition (Fig. 4B), indicating that hexachlorophene, like
triclosan, functioned by interacting with the FabI active site.
Triclosan is a particularly effective FabI inhibitor due to the slow
formation of a stable, ternary FabI-NAD+-triclosan ternary
complex, and this property of triclosan is responsible for its
antibacterial activity (12). Two methods were used to determine if
hexachlorophene also formed a similar ternary complex with FabI. First,
a binding assay with [3H]NAD+ and ecFabI was
performed (Fig. 5). ecFabI forms a stable
ternary complex with NAD+ and triclosan, resulting in the
isolation of radiolabeled ecFabI in the presence of triclosan. In
contrast, hexachlorophene did not trigger an increase in
[3H]NAD+ binding, indicating that a ternary
ecFabI-NAD+-hexachlorophene complex did not form. ecFabI
did not bind detectable amounts of [3H]NAD+
in the absence of triclosan. We were unable to demonstrate ternary complex formation with saFabI using this assay. However, since saFabI
has a low affinity for NADH (Fig. 3), this negative result was likely
due to the poor interaction of the radiolabeled ligand with the enzyme.
Labeled NADP+ was not available, precluding similar
experiments with this cofactor.
The second method used to detect ternary complex formation was to
examine the effect of preincubation of the enzymes with drug on the
kinetics of the reaction (Fig. 6).
Slow-binding inhibitors form time-dependent, stable
complexes during the preincubation period, resulting in little to no
reaction rate following initiation of the reaction by the addition of
substrate. In contrast, inhibitors that do not form tight binding
complexes exhibit kinetic behavior in reactions initiated with
substrate that is indistinguishable from initiating the reaction with
the addition of enzyme (28). Triclosan exhibits the hallmarks of a
slow-binding inhibitor. The FabI-NAD+-triclosan ternary
complex takes several minutes to form and eventually results in
complete inhibition (12). This experiment clearly showed that triclosan
was a slow-binding, dead end inhibitor with either ecFabI (Fig.
6A) or saFabI (Fig. 6B). Furthermore, triclosan inactivated saFabI with essentially the same time course as we observed
previously with ecFabI (data not shown) (12). In contrast, hexachlorophene did not irreversibly inhibit either of the FabI enzymes
(Fig. 6), but rather the inhibition pattern was similar to that
observed when enzyme was used to initiate the reaction (Fig. 5). Thus,
triclosan formed a stable inhibitory complex with ecFabI and saFabI;
however, hexachlorophene did not form a slowly dissociating ternary
complex with either FabI enzyme.
Structural Features That Determine Inhibitory
Activity--
Triclosan and hexachlorophene have two structural
differences that could account for the different behavior of the
compounds as FabI inhibitors. A carbon bond in hexachlorophene replaces the ether bond in triclosan, and the oxygen bridge may be critical to
the formation of a ternary complex. Alternately, hexachlorophene has
two hydroxyl groups and three additional bulky chlorine atoms that may
interfere with the proper positioning of the molecule within the FabI
active site. Molecular modeling of the energy-minimized hexachlorophene
structure overlaid on the FabI-NAD+-triclosan structure
indicated that neither the extra hydroxyl group nor the chlorine
substituents would interfere with drug binding. We further investigated
the structural basis for the differences in activity by testing three
additional compounds as FabI inhibitors. Dihydroxydiphenylether was a
potent ecFabI inhibitor; however, dihydroxydiphenylmethane was not
(Fig. 7). The only difference in these
two compounds is the substitution of a carbon for the oxygen bridging
the two phenyl rings. We also examined a
dihydroxydichlorodiphenylmethane (Fig. 7). This compound was not a very
potent ecFabI inhibitor, although it was more effective than the
compound without the chlorine substituents. The chlorine on the
4-position of triclosan promotes tight binding of the drug to the FabI
active site by forming a hydrogen bond interaction with the backbone
amide of Ala-95 (12). Increased opportunities for hydrogen bond
formation between the chlorines and the protein is one possible
explanation for understanding the potency of hexachlorophene as a FabI
inhibitor compared with analogs that lack chlorine substitutions. These
data indicated that the ether linkage was an essential feature for
ternary complex formation, and that the presence of the chlorine atoms
on hexachlorophene promoted, rather than attenuated, its interaction
with FabI.
Differential Sensitivity of S. aureus and E. coli to Growth
Inhibition by Triclosan and Hexachlorophene--
The MIC for triclosan
and hexachlorophene were determined on a panel of S. aureus
and E. coli strains (Table I).
As expected from previously published work (21), E. coli was
resistant to hexachlorophene, whereas S. aureus was
sensitive to the compound. E. coli and S. aureus
were both sensitive to triclosan (16, 21). Both the presence of
multiple plasmid-borne copies of the fabI gene and missense
mutations confer triclosan resistance to E. coli (10), and
neither of these had any effect on the already high level of resistance
of this organism to hexachlorophene (Table I). However, FabI could be
the hexachlorophene target in S. aureus if resistance to
this compound in E. coli was due to the inability of the
drug to penetrate the cell or to the presence of an active mechanism to
efflux hexachlorophene. Therefore, we generated triclosan-resistant S. aureus mutants and tested them for cross-resistance to
hexachlorophene. First, we selected triclosan-resistant S. aureus on 0.5 µg/ml triclosan and then sequenced the
safabI gene to determine if it was altered. A single
missense mutation in the safabI gene that converted saFabI
to saFabI(G23S) increased the resistance of S. aureus to
triclosan by an order of magnitude (Table I). Second, we introduced the
safabI gene on plasmid pTri-1 into S. aureus and
observed increased resistance to triclosan. However, neither of these
manipulations affected the sensitivity of the cells to hexachlorophene.
These data are consistent with triclosan targeting the FabI in S. aureus, and suggest that FabI inhibition is not a component of the
biocidal activity of hexachlorophene against this organism.
The Gram-negative (ecFabI) and Gram-positive (saFabI) enoyl-ACP
reductases have many properties in common, but are distinguished by
their cofactor selectivities. The cofactor specificity of ecFabI has
been the subject of some debate. The initial report of enoyl-ACP reductase activity in cell extracts suggested that two enzymes were
present in E. coli, one requiring NADH and the other
utilizing NADPH (29). The pH optima of the two enzymes were slightly
different, with the NADH activity being maximal at pH 7.5 and the NADPH
activity preferring pH 6.5. Subsequent genetic and biochemical
experiments lead to the conclusion that there is only a single
NADH-dependent enoyl-ACP reductase gene in E. coli (3, 30). However, Bergler et al. (31) recently
reported that ecFabI has both NADH- and NADPH-dependent
activities. The NADPH-dependent activity was only revealed
when ecFabI was purified at acidic pH, and this activity was
irreversibly lost when the pH was raised to 7.0 or above (31). We have
attempted to exactly repeat these experiments without success, and
conclude from our present study that ecFabI is highly selective for
NADH. In contrast, saFabI is clearly a NADPH-dependent reductase that exhibits a high degree of positive cooperativity. This
property cannot be considered characteristic of Gram-positive FabI
enzymes because the enoyl-ACP reductase from Bacillus
subtilis is a NADH-dependent enzyme.2
Otherwise, saFabI has specific activities that are very similar to
ecFabI for synthetic substrates and is able to complement E. coli strains that are defective in ecFabI. S. aureus
produces branched-chain fatty acids (32), and its ability to substitute for ecFabI in an organism that produces straight-chain saturated and
unsaturated fatty acids illustrates the broad substrate specificity of
enoyl-ACP reductases.
The structural similarities between triclosan and hexachlorophene
suggest that they have a common target, and both drugs inhibit enoyl-ACP reductases in vitro. The resistance of FabI(G93V)
to both triclosan and hexachlorophene support the concept that both drugs interact with FabI in the vicinity of the substrate binding site.
However, the resistance of E. coli to hexachlorophene and the genetic analysis of S. aureus argues that FabI is not a
biologically relevant target for the cellular action of
hexachlorophene. The ability of FabI inhibitors to form stable ternary
complexes with the enzyme is the critical feature required for
antibacterial activity (Fig. 8). This
class of agents are called slow-binding inhibitors because inhibitor
complexes with the enzyme take a long time (seconds to minutes) to form
relative to the catalytic rate (28). This is due to a slow
conformational isomerization of the enzyme-drug complex from a state
where the enzyme and drug are in rapid equilibrium to a state where the
enzyme-drug complex undergoes very slow dissociation (Fig. 8).
Triclosan exhibits all the hallmarks of a classic slow-binding
inhibitor. The inhibition of FabI by triclosan becomes progressively
stronger with time and is essentially irreversible after several
minutes (12). This irreversible inhibition correlates with the
formation of a stable FabI-NAD+-triclosan ternary complex
(12, 13) that is accompanied by a conformational change in a flexible
loop in FabI including residues 194 to 210 (14). The diazaborines are
another class of potent FabI inhibitors that act via the formation of a
tight binding bisubstrate complex (6). The formation of the
FabI-NAD+-diazaborine inhibitory complex is also
accompanied by an analogous conformational change in this flexible loop
(5). In contrast, hexachlorophene neither exhibits the kinetic
properties of a slow-binding inhibitor nor forms a ternary complex.
Our results point to the ether linkage in triclosan as a structural
feature that is essential to the formation of the inhibitory ternary
complex. This conclusion is drawn from the fact that
dihydroxydiphenylether is a FabI inhibitor, whereas
dihydroxydiphenylmethane is not. Triclosan is sandwiched between the
protein and the cofactor and a hydrogen bond network and stacking
interactions form the bridge that connects the drug, protein and
NAD+ (12-14). Clearly, the substitution of a carbon bridge
in hexachlorophene for the ether oxygen in triclosan results in a
compound that cannot orient itself to optimally participate in this
network. This is most likely attributed to the different angle of the
carbon bridge compared with the ether bridge, preventing
hexachlorophene from forming hydrogen bond connections and the stacking
interactions with NAD+. Stewart et al. (14)
propose that the ether oxygen of triclosan is part of a hydrogen bond
network that also includes the hydroxyl groups of Tyr-156, triclosan,
and the NAD+ ribose (14). However, the distance between
these atoms is about 4 Å in the various published structures (12-14),
and it seems unlikely that this weak hydrogen bond acceptor will
significantly contribute to the binding affinity at that distance. The
substitution of a thioether for the ether in hydroxydiphenyl compounds
also significantly reduces antibacterial activity (10), indicating that
the bulkier sulfur atom also prevents the proper alignment of the
aromatic rings and hydroxyl group in the FabI active site. Thus, the
presence of the ether oxygen is clearly important in promoting tight
drug binding and the ensuing conformational change that leads to
essentially irreversible FabI inhibition and potent antibacterial activity.
We thank Amy Sullivan and Magda Kaminska for
their excellent technical assistance, Charles Ross for molecular
modeling, Rocco Gogliotti and John Domagala for the substrate analogs,
Martin Shapiro for the MIC determinations, and Eric Olson for
evaluation of the manuscript.
*
This work was supported by National Institutes of Health
Grants GM 34496, Cancer Center (CORE) Support Grant CA 21765, and the
American Lebanese Syrian Associated Charities.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF197058 (fabI gene from S. aureus strain RN4220).
2
R. J. Heath and C. O. Rock,
unpublished observations.
The abbreviations used are:
ACP, acyl carrier
protein;
ecFabI, enoyl-ACP reductase from E. coli;
saFabI, enoyl-ACP reductase from S. aureus;
triclosan, 2,4,4'-trichloro-2'-hydroxydiphenylether;
hexachlorophene, 2,2'-dihydroxy-3,3',5,5',6,6'-hexachlorodiphenylmethane;
MIC, minimum
inhibitory concentration;
PCR, polymerase chain reaction;
bp, base pair(s).
Inhibition of the Staphylococcus aureus
NADPH-dependent Enoyl-Acyl Carrier Protein Reductase by
Triclosan and Hexachlorophene*
,¶
Department of Biochemistry, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105, the
§ Department of Infectious Diseases, Parke-Davis
Pharmaceutical Research, Ann Arbor, Michigan 48105, and the
¶ Department of Biochemistry, University of Tennessee,
Memphis, Tennessee 38163
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 1.
Structures of triclosan and
hexachlorophene. A, triclosan; B,
hexachlorophene.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. A 1:10 or 1:100 dilution of an
overnight culture of this library incubated in tryptic soy broth plus
chloramphenicol (7 µg/ml) at 37 °C was plated onto brain-heart
infusion medium containing 7 µg/ml chloramphenicol and 0.5 µg/ml
triclosan. After 48 h of incubation at 37 °C, resistant clones
were confirmed by regrowth on the same media. To confirm the presence
of safabI on the plasmid of the all the resistant clones,
two PCR reactions were performed per isolate. Each reaction contained safabI-specific primer GER71 and either primer
GER23 (5'-GTTTTATGCCTAAAAACCTACAG) or GER24
(5'-GAATTAGAATATATTTATTTGGCTC), which flank the multiple cloning
site of plasmid pSK265. In all cases, the isolated plasmids harbored
the safabI gene.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
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Fig. 2.
Alignment of ecFabI and saFabI proteins.
The amino acid sequence of ecFabI is aligned with saFabI. Identical
residues present in both proteins are highlighted.
View larger version (14K):
[in a new window]
Fig. 3.
Cofactor utilization of the FabI
proteins. E. coli (panel A) and S. aureus (panel B) FabIs were assayed using varying
concentrations of either NADH ( 
) or NADPH (
) in the
spectrophotometric assay described under "Experimental Procedures."
Insets show the double-reciprocal plots of the data.
Panel C, Hill plot of the data from saFabI using NADPH as
the cofactor. Reactions were initiated by the addition of FabI, and the
initial rates were determined from data collected during the first
90 s.
View larger version (20K):
[in a new window]
Fig. 4.
Inhibition of enoyl reductase activities by
triclosan and hexachlorophene. Panel A, inhibition of
the FabI proteins from E. coli ( ), S. aureus
() or the triclosan-resistant mutant ecFabI(G93V) (
) by
triclosan. Panel B, inhibition of the FabI proteins from
E. coli (), S. aureus (), or the
triclosan-resistant mutant ecFabI(G93V) () by hexachlorophene.
Spectrophotometric assays were performed with the indicated
concentration of drug as described under "Experimental Procedures."
The reactions were initiated by the addition of protein, and initial
rates were calculated from the data obtained in the first 90 s.
View larger version (13K):
[in a new window]
Fig. 5.
Ternary complex formation by triclosan and
hexachlorophene. [3H]NAD+ binding to
wild-type ecFabI was measured in the presence of the indicated
concentrations of triclosan ( ) or hexachlorophene () and was
performed as described under "Experimental Procedures."
View larger version (15K):
[in a new window]
Fig. 6.
Hexachlorophene was not a slow binding
inhibitor of enoyl-ACP reductases. FabI proteins from either
E. coli (panel A) or S. aureus
(panel B) were assayed for reductase activity after
preincubation of the enzyme with 5 µM triclosan
(TCL), 5 µM hexachlorophene (HEX)
or carrier alone ( DRUG). The reaction rates were then
measured as described under "Experimental Procedures."
View larger version (12K):
[in a new window]
Fig. 7.
Inhibition of ecFabI by a
hydroxydiphenylether and two hydroxydiphenylmethanes. ecFabI was
assayed in the presence of the indicated concentrations of three
diphenyl compounds using
trans-2-octenoyl-N-acetylcysteamine as the
substrate as described under "Experimental Procedures." The
compounds evaluated were: 2,2'-dihydroxydiphenylether ( ),
2,2'-dihydroxy-5,5'-dichlorodiphenylmethane (), and
2,2'-dihydroxydiphenylmethane ().
Triclosan-resistant bacteria were not cross-resistant to
hexachlorophene
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 8.
Model for the inhibition of FabI by triclosan
and hexachlorophene. The FabI reaction sequence is depicted as an
ordered sequential mechanism, although the order of substrate addition
may be random. Triclosan and hexachlorophene bind to the
substrate/product site on FabI and are capable of reversible inhibition
by this mechanism. However, triclosan differs from hexachlorophene in
that the FabI-NAD+-triclosan complex undergoes a slow
conformational change to a state where the drug is more tightly bound
and the slow rate of dissociation results in essentially irreversible
inhibition.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3491; Fax:
901-525-8025; E-mail: charles.rock@stjude.org.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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