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Journal of Bacteriology, May 2000, p. 2668-2671, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Equivalence of Lauric Acid and Glycerol Monolaurate
as Inhibitors of Signal Transduction in Staphylococcus
aureus
Alexey
Ruzin
and
Richard P.
Novick*
New York University Medical Center, Skirball
Institute, New York, New York 10016
Received 8 October 1999/Accepted 7 February 2000
 |
ABSTRACT |
Glycerol monolaurate (GML) inhibits the expression of virulence
factors in Staphylococus aureus and the induction of
vancomycin resistance in Enterococcus faecalis, presumably
by blocking signal transduction. Although GML is rapidly hydrolyzed by
bacteria, one of the products, lauric acid, has identical inhibitory
activity and is metabolized much more slowly. At least four distinct
GML-hydrolyzing activities are identified in S. aureus: the
secreted Geh lipase, residual supernatant activity in a
geh-null mutant strain, a novel membrane-bound esterase,
and a cytoplasmic activity.
| |
TEXT |
Glycerol monolaurate (GML) is a mild
surfactant that is used in the food industry and in cosmetics as a
preservative and emulsifier. At concentrations higher than 20 µg/ml,
it inhibits growth of bacteria. However, at lower concentrations which
do not significantly alter bacterial growth, GML blocks the production
of various exoenzymes and virulence factors, including protein A,
alpha-hemolysin, 
-lactamase, and toxic shock syndrome toxin 1 (TSST-1) (12, 15) in Staphylococcus aureus. The
action of GML is not restricted to S. aureus: it also blocks
the induction of vancomycin resistance in another pathogen, Enterococcus faecalis (13).
The mechanism of GML inhibitory action is not known. It has been shown
that GML affects neither secretion nor intracellular signaling and most
likely acts through the inhibition of signal transduction (12,
13). As shown previously (12), GML does not inhibit
synthesis of RNAIII, a key effector molecule, and therefore does not
act through the agr system (4), which regulates RNAIII production. As proposed previously (12, 13), GML most likely affects exoprotein production through some uncharacterized signal transduction pathway(s). Studies of GML action are aimed toward
identification and characterization of this pathway; however, they are
complicated by the loss of activity in bacterial cultures (12), which has been assumed to be the result of hydrolysis to lauric acid and glycerol. In this paper, we describe a thin-layer chromatographic (TLC) method for monitoring GML and its hydrolysis and
confirm that GML is very rapidly hydrolyzed to lauric acid and glycerol
by staphylococci, with a half-life of ~5 min in a typical culture.
Nevertheless, in earlier studies, it was possible to maintain the
inhibitory effect of GML by hourly additions to a growing culture
(12). Thus, the kinetics of GML hydrolysis does not match
the rate at which activity disappears. This result led us to
demonstrate that lauric acid inhibits the same processes that are
inhibited by GML and that its activity is equimolar with that of the ester.
These results raise the question of whether lauric acid is entirely
responsible for the observed effects of GML. Because this question can
be addressed only in the absence of GML hydrolysis, we have begun to
identify the enzymes responsible for the hydrolysis. We find that there
is GML esterase activity in culture supernatants, in association with
the cell membrane, and in the cytoplasm. We find that the well-known
Geh lipase is responsible for the majority (~80%) of detectable
GML-hydrolyzing activity in culture supernatants, and there is a
previously undescribed membrane-bound esterase that is responsible for
much, possibly all, of the cell-bound activity. Residual (~20%)
hydrolyzing activity in supernatants probably represents a previously
described short-chain esterase, which has detectable activity with
lauric acid esters (8, 16). The role of cytoplasmic
esterases is uncertain and can be evaluated only after elimination of
both extracellular and membrane-bound GML-hydrolyzing activities.
Development of a TLC method to monitor GML and lauric acid.
We
tested solutions of GML and lauric acid in bacterial culture media by
TLC. Samples were centrifuged, and 8 µl of supernatant was applied by
micropipette to Whatman silica gel 60A TLC plates (20 by 20, with
preadsorbent area). Emulsions of GML (40 µg/ml) and lauric acid (30 µg/ml) in CY/GP broth (9) were used as standards. Plates
were air dried and developed with hexane-ethyl ether-methanol
(70:20:10), air dried, baked at 100°C for 10 min, and sprayed with a
0.025% (wt/vol) solution of Coomassie R-250 in 20% (vol/vol) methanol
until lipids were visible as white spots on a blue background. Picture
taking and spot densitometry (not shown) were performed with the
IS-1000 imaging system (Alpha Innotech Corp.). As shown in Fig.
1, we obtained satisfactory separation of
GML (Rf = 0.24) and lauric acid
(Rf = 0.35) from each other and from the
lipid components of culture media (Rf = 0 to
0.07). The detection limit of negative staining was about 15 ng
for GML (not shown). Several other lipid staining procedures (sulfuric acid and rhodamine B) were unsatisfactory.
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FIG. 1.
Degradation of GML monitored by TLC. GML was added to a
growing culture of RN11, and samples (100 µl) were collected at the
indicated time points and applied to a TLC plate. The plate was
developed, stained with Coomassie blue, and photographed. GML and
lauric acid (L.A.) appear as white spots on a dark background.
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Fate of GML in growing bacterial cultures.
The bacterial
strains used in this study are shown in Table
1. The culture medium was CY/GP broth
(9). GML (Personal Products Co., New Brunswick, N.J.) and
lauric acid (Sigma) were prepared at 1% (wt/vol) in 95% ethanol.
Cultures were grown at 37°C with shaking at 240 rpm. To determine the
fate of GML in staphylococcal cultures, we analyzed samples of RN11
culture growing in the presence of GML (20 µg/ml) by TLC. As shown in
Fig. 1, GML disappears with a half-life of about 5 min and is replaced
by lauric acid and, presumably, glycerol (which is not seen on TLC).
Lauric acid persists in the culture for at least 2 h and then
slowly decreases in quantity (Fig. 1).
GML hydrolases.
Previous observations had shown that
elimination of the major secreted esterase, Geh, by 
L54a
prophage-mediated insertional inactivation of its gene (7)
had little if any effect on GML stability (S. Projan, personal
communication), indicating that S. aureus produces one or
more additional esterases capable of hydrolyzing GML.
As we found, both culture supernatant and cells had significant
GML-hydrolyzing activity, (Table
2). Most
of the supernatant
activity could be accounted for by Geh esterase. A
Geh-negative
strain (RN8083), generated by lysogenization with phage
L54a,
which has its attachment site within
geh
(
7), showed less than
22% of normal supernatant activity,
while cell-associated activity
was undiminished (Table
2).
One of the possibilities of cell-associated lipolytic
activity
noncovalent binding of secreted lipases to cell wall
was
excluded
by washing cells in 1 M NaCl and testing for residual
activity.
As shown in Table
2, this treatment did not diminish the
cell-associated
activity.
To localize cell-associated GML-hydrolyzing activity, we fractionated
the cells. Cultures were grown with shaking in 2× Penassay
broth
(Difco), washed in SMM buffer (1 M sucrose, 35 mM sodium
maleate, 85 mM
MgCl
2) plus 1 M NaCl, and treated with lysostaphin
(10-µg/ml final concentration). The resulting protoplasts were
lysed
by osmotic shock and ultrasonication and centrifuged at
15,000 rpm for
10 min. The supernatant was then centrifuged at
70,000 rpm in a Beckman
TLA100.4 rotor. Pellet (membrane fraction)
was washed with 1 M NaCl in
0.1× SMM, recentrifuged, rinsed with
0.1× SMM, and resuspended in
0.1× SMM. We found activity in both
intracellular and membrane
fractions (Table
2). One or more uncharacterized
cytoplasmic esterases
are responsible for GML hydrolysis by the
intracellular fraction; we
suggest that one or more membrane hydrolases
are responsible for the
membrane-associated activity. Based on
these results, we suggest that
S. aureus possesses one or more
novel membrane-bound
lipases, which actively participate in degradation
of
GML.
As a first step toward purification of the membrane-bound enzyme(s),
the membrane fraction was solubilized either with CHAPS
{-3-[(3-cholamidopropyl)-dimethylammonia]-1-propanesulfonate}
(5 mg/ml) or sodium deoxycholate (DOC, 2mg/ml) and subjected to
ultracentrifugation, followed by dialysis of the supernatant against
phosphate-buffered saline-0.1%
-mercaptoethanol. We found that
material solubilized by CHAPS retained GML hydrolase activity,
whereas
that solubilized by DOC did not (not shown). Further purification
of
staphylococcal membrane-bound lipase(s) is currently in
progress.
Inhibitory effects of lauric acid.
Given the observed rapid
degradation of GML and persistence of lauric acid, we tested for the
possibility that lauric acid might be at least partially responsible
for the inhibitory effects of GML. Accordingly, we compared the effects
of lauric acid and GML on the overall production of the exoproteins and
on the induction of two different exoproteins, TSST-1 and
-lactamase, shown previously to be blocked by GML (12,
15). -Lactamase activity was determined in a microplate reader
with nitrocefin essentially as described previously (11).
For analysis of bulk exoprotein production, MN8 cells were grown in
broth, centrifuged and supernatants were separated on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis SDS-PAGE (15%
polyacrylamide) by the method of Laemmli (6). For analysis
of TSST-1 production, separated proteins were transferred electrophoretically to nitrocellulose filters and immunoblotted with
anti-TSST-1 rabbit serum (kindly provided by P. Schlievert) according
to the method of Blake et al. (1). As shown in Fig. 2A, lauric acid and GML
have identical effects on the production of staphylococcal exoproteins:
some proteins are inhibited, while others are overproduced compared to
those of untreated cells. In Fig. 2B and C are shown the results for
-lactamase induction and TSST-1 production, respectively. As can be
seen, lauric acid and GML, at equimolar concentrations, have identical
inhibitory effects.
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FIG. 2.
Effects of GML and lauric acid on production of
exoproteins, induction of -lactamase synthesis, and production of
TSST-1 in S. aureus. (A) Production of exoproteins. MN8
cultures were grown in broth with shaking with either GML, lauric acid,
or no additives until the late-exponential phase (1,000 Klett
units/ml). GML was added to 20 µg/ml at 0 min and to 10 µg/ml at
90, 150, 210 min. Lauric acid was added to 15 µg/ml at 0 min and to
7.5 µg/ml at 90, 150, and 210 min. Supernatants of final cultures
were concentrated 10 times by precipitation with 10% trichloracetic
acid, and proteins were separated by SDS-PAGE (12% polyacrylamide) and
visualized by Coomassie staining. MW, molecular mass markers. (B)
Induction of -lactamase synthesis. Bacterial cultures (RN11) were
grown in broth; equimolar subinhibitory amounts of GML (10 µg/ml) or
lauric acid (7.5 µg/ml) were added at 0, 60, and 120 min; and
-lactamase inducer CBAP (carboxyphenylbenzoyl penicillanic acid) (5 µg/ml) was added at 75 min. Samples (1 ml) were collected at 60, 90, 120, and 180 min and assayed for -lactamase activity. Each data
point represents the average of two experiments.  , no GML or lauric
acid and no CBAP;  , no GML or lauric acid and with CBAP added;
 , GML
and CBAP added;  , lauric acid and CBAP added. (C) Inhibition of
TSST-1 production. Supernatants of MN8 cultures obtained as described
for panel A were separated by SDS-PAGE (15% polyacrylamide). Separated
proteins were transferred to a nitrocellulose filter (BA85; Schleicher
& Schuell) and immunoblotted with anti-TSST-1 rabbit serum (kindly
provided by P. Schlievert) according to the method of Blake et al.
(1).
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Additionally, GML inhibits the production of staphylococcal protein A
(
12), and as shown in Fig.
3,
lauric acid blocks
spa transcription. In this assay,
whole-cell RNA was prepared from
RN6911 and used for Northern blot
hybridization according to the
method of Kornblum et al.
(
5). Blots were hybridized with PCR-labeled
(
32P)
spa-specific DNA probe. PCR was performed
with the 5' ATCTGGTGGCGTAACAC
3' (forward) and 5'
CAGCTTTCGGTGCTTG 3' (reverse) primers and
RN6911 chromosomal DNA
as a template.
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FIG. 3.
Effect of lauric acid on spa transcription.
RN6911 (agr-null mutant in which spa is
overexpressed) was grown in broth either with or without lauric acid.
Lauric acid was added at 0 min (15 µg/ml) and 65 min (7.5 µg/ml)
and samples (10 ml) were collected at 0, 65, 110, and 220 min.
Whole-cell lysates were prepared and used for RNA (Northern) blotting.
The blot was hybridized overnight with spa-specific
PCR-labelled probe, washed, and exposed to Kodak X-Omat AR film.
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|
Conclusions.
In this report, we describe a new TLC method that
has been used to monitor the GML concentration in growing cultures of
S. aureus. We confirmed our earlier hypothesis that GML
degradation is responsible for the transience of its inhibitory effects
(12). Degradation of GML is very rapid
(t1/2 of ~5 min), while inhibitory effects of
GML last for at least an hour. We noticed that lauric acid, a
hydrolysis product of GML, persists in the culture for more than 2 h. We suggested that lauric acid might be responsible for prolonged
inhibitory effects of GML. We found that indeed lauric acid at an
equimolar concentration mimics the inhibitory effect of GML on the
induction of staphylococcal -lactamase activity and, like GML,
blocks expression of protein A and TSST-1 in S. aureus.
Thus, we currently hypothesize that lauric acid might be responsible
for all of the inhibitory effects of GML described so far, although GML
might be active as well.
To test this hypothesis, we first needed to identify the enzyme or
enzymes that are responsible for GML hydrolysis and later
to eliminate
them by mutation. Both supernatant and salt-washed
cells had activity.
That in the supernatant is mainly attributed
to the well-known Geh
lipase. The residual GML-hydrolyzing activity
in supernatant of RN8083
could correspond to an additional secreted
esterase that has recently
been described for
S. aureus NCTC8530
(
8,
16).
Although the preferred substrates for this enzyme
are short-chain
(propionyl- and butyryl-) acyl esters, it has
detectable activity with
lauric acid esters. Cell-associated lipolytic
activity might be
explained by the action of either intracellular
or membrane-bound
lipases. However, intracellular lipases, previously
described in
S. aureus (
2), are not expected to play any
significant
role in GML degradation, since, as mentioned above, GML is
a surfactant
and is considered unlikely to reach the cytoplasm.
Membrane-bound
lipases have been identified in other bacterial species,
e.g.,
Escherichia coli,
Bacillus megaterium,
Mycobacterium phlei, and
members of the genus
Vibrio (
17). These enzymes belong to class
of
acyl hydrolases, functions of which are still poorly understood.
Although they are presumed to play role in bacterial membrane
metabolism, mutants defective in the production of such enzymes
are
viable and have a normal lipid composition, possibly due to
redundancy.
Perhaps lipases and esterases have a role in nutrition
in that their
products can be metabolized by the bacteria. Membrane-bound
lipases
have not been previously described for
S. aureus, and
our
results represent their initial identification in this species.
The
membrane-bound GML hydrolase or hydrolases may or may not
be analogs of
the acyl hydrolases of other species. Our next steps
toward
identification of such enzymes in
S. aureus will include
protein purification, isolation of their genes by reverse genetics,
and
subsequent inactivation. Our goal in this project is to create
a mutant
unable to degrade GML in order to determine whether GML
has any
inhibitory activity of its
own.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grants AI R01-22159 and AI
R01-30138.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: New York
University Medical Center, Skirball Institute, 540 First Ave., New
York, NY 10016. Phone: (212) 263-6290. Fax: (212) 263-8951. E-mail:
novick{at}saturn.med.nyu.edu.
Present address: Wyeth-Ayerst Research, Pearl River, NY 10965.
| |
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Journal of Bacteriology, May 2000, p. 2668-2671, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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