J. Antibiot.

59(1): 35–43, 2006

THE JOURNAL OF
ORIGINAL ARTICLE ANTIBIOTICS

Inhibition of Lipopolysaccharide Activity by a Bacterial

Cyclic Lipopeptide Surfactin
Taichi Takahashi, Osamu Ohno, Yoko Ikeda, Ryuichi Sawa, Yoshiko Homma,
Masayuki Igarashi, Kazuo Umezawa

Received: November 8, 2005 / Accepted: December 18, 2005

© Japan Antibiotics Research Association

Abstract Compounds that inactivate lipopolysaccharide

(LPS) activity have the potential of being new anti- Introduction
inflammatory agents. Therefore, we searched among
microbial secondary metabolites for compounds that Lipopolysaccharide (LPS) is one of the major constituents
inhibited LPS-stimulated adhesion between human of the outer membrane of Gram-negative bacteria and is
umbilical vein endothelial cells (HUVEC) and HL-60 cells. recognized as a key molecule in the pathogenesis of
By this screening, we found a cyclic lipopeptide surfactin inflammatory syndromes associated with Gram-negative
from the culture broth of Bacillus sp. BML752-121F2 to be bacterial infections. Most of the pathological activities of
inhibitory. The addition of the surfactin prior to the LPS LPS are attributed to its lipid A portion, which consists of a
stimulation decreased HL-60 cell-HUVEC adhesion backbone of 2 phosphorylated glucosamine molecules
without showing any cytotoxicity. We confirmed that acylated with fatty acids [1, 2].
surfactin inhibited LPS-induced expression of ICAM-1 and Recent research has revealed the detailed mechanism by
VCAM-1 in HUVEC. It also inhibited the cellular adhesion which LPS activates mammalian cells. Upon its release
induced by lipid A, the active component of LPS; but it did from bacterial cell walls into the host’s blood circulation,
not inhibit TNF-a or IL-1b -induced cell adhesion. Then, LPS interacts with LPS-binding protein (LBP) present in
surfactin was shown to suppress the interaction of lipid A the blood and transfers LPS to CD14, the primary receptor
with LPS-binding protein (LBP) that mediates the transport for LPS, which exists as a soluble form in the blood or as a
of LPS to its receptors. Finally, surface plasmon resonance glycosylphosphatidylinositol (GPI)-linked molecule on the
(SPR) analysis revealed the surfactin to interact reversibly surface of target cells. Next, LPS binds to Toll-like receptor
with lipid A. Thus, this Bacillus surfactin was shown to be (TLR) 4, the putative transmembrane receptor for LPS that
an inhibitor of LPS-induced signal transduction, directly activates nuclear factor-k B (NF-k B) signaling [3
5]. NF-
interacting with LPS. k B is known to play a critical role in the development of
inflammatory responses by upregulating the expression
Keywords surfactin, lipopolysaccharide, lipid A, human of cell adhesion molecules and many inflammatory
umbilical vein endothelial cells, HL-60 cells, adhesion cytokines, such as tumor necrosis factor-a (TNF-a )
and interleukins (IL). Systemic production of these
proinflammatory mediators causes the sepsis syndrome,
which is clinically characterized by hypotension, fever, and
K. Umezawa (Corresponding author), O. Ohno, T. Takahashi:
respiratory dysfunction, and may lead to multi-organ
Department of Applied Chemistry, Faculty of Science and
Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, failure and death [6, 7].
Yokohama 223-0061, Japan, E-mail: umezawa@applc.keio.ac.jp LPS can be recognized by monocytes, macrophages,
Y. Ikeda, R. Sawa, Y. Homma, M. Igarashi: Microbial neutrophils, and endothelial cells in mammals. Especially
Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, in endothelial cells, LPS induces adhesion molecule
Tokyo 141-0021, Japan expression [8]. The most important of the adhesion
36

molecules involved in this process are intercellular Cell Culture

adhesion molecule-1 (ICAM-1), vascular cell adhesion HUVEC were cultured on type I collagen-coated dishes
molecule-1 (VCAM-1), and E-selectin [8, 9]. Without (Costar, Acton, MA) in MCDB-131 medium (Sigma)
stimulation, only a small amount of ICAM-1 is present supplemented with 10 % heat-inactivated fetal bovine
on the endothelial cell surface [10], and VCAM-1 and serum (FBS; JRH Biosciences, Lenexa, KS) and 10 ng/ml
E-selectin are absent entirely [11, 12], but they are basic fibroblast growth factor (bFGF; Pepro Tech EC Ltd.).
immediately expressed in response to extracellular HL-60 cells, THP-1 cells, and Jurkat cells were cultured in
inflammatory stimulation [13]. ICAM-1 interacts with the RPMI1640 (Nissui, Tokyo, Japan) supplemented with 10%
ligand CD11a/CD18 (LFA-1), which is expressed on heat-inactivated FBS, 100 m g/ml kanamycin, 100 units/ml
neutrophils, lymphocytes, and monocytes; and VCAM-1, penicillin G, 300 m g/ml L-glutamine, and 2.25 mg/ml
with the ligand VLA-4, which is expressed on NaHCO3.
lymphocytes. E-selectin mediates the adherence of
neutrophils, T cells, eosinophils, and monocytes. Cell Adhesion Assay
As described above, LPS has been implicated in the Stimulant-induced adhesion between HUVEC and leukocytes
pathogenesis of Gram-negative bacterial infections and its was measured by the method described previously [18].
attendant vascular complications. Because it triggers HUVEC were seeded at 4104 cells/well in 48-well
the sepsis syndrome [6, 7], inflammation of the bowels collagen-coated plates (Costar) and cultured overnight at
including Crohn’s disease [14, 15], and periodontitis [16, 37°C in 5% CO2. The cells were preincubated with several
17], LPS is considered to be one of the most common concentrations of surfactin for 2 hours, and then treated
and potent pathogenic factors of blood vessels. Therefore, with the stimulant (1 m g/ml LPS, 1 m g/ml lipid A, 10 ng/ml
searching for molecules that can inhibit LPS- TNF-a or 10 ng/ml IL-1b ) for 4 hours. Then, the medium
induced endothelial cell dysfunction should contribute was removed, and the wells were washed with PBS twice.
to the discovery of new anti-inflammatory agents. As Next, leukemic cells (HL-60, THP-1, or Jurkat) were added
microorganisms produce a variety of compounds of at 6104 cells/well to the treated HUVEC monolayers.
low-molecular weight and having unique structures, they After 1 hour, nonadhering leukemic cells were removed by
are ideal sources of novel bioactive secondary metabolites. 3 washes with PBS. Then, the number of adherent cells in 1
Thus, in the present study, we screened microbial microscopic field was counted.
secondary metabolites for compounds that could inhibit the
adhesion of human myelocytic cell line HL-60 cells to Trypan Blue Dye Exclusion
LPS-stimulated human umbilical vein endothelial cells HUVEC were seeded at 1.6105 cells/well in 24-well
(HUVEC). As a result, we isolated a known cyclic collagen-coated plates (Costar) and cultured overnight. The
lipopeptide, surfactin, from the culture broth of Bacillus sp. cells were treated with various concentrations of surfactin
BML752-121F2, and found it to be inhibitory. for 24 hours in the presence of 1 m g/ml LPS. Then, they
were stained with trypan blue, after which the number of
stained cells was counted.
Materials and Methods
Western Blotting Analysis
Materials HUVEC (8105 cells) were treated with LPS for the
HUVEC were purchased from Cell Systems (Lake desired periods. Then, the cells were scraped off, and
Kirkland, WA). HL-60 cells were obtained from the suspended in lysis buffer (20 mM Tris, pH 8.0, containing
Japanese Collection of Research Bioresources. Surfactin 150 mM NaCl, 2 mM EDTA, 100 mM NaF, 400 m M
(surfactin C1; from Bacillus subtilis), LPS (from Na3VO4, 1% NP-40, 1 m g/ml leupeptin, 1 m g/ml antipain,
Escherichia coli Serotype 055:B5), lipid A (1,4
- and 1 mM PMSF). The supernatants were combined
diphosphoryl form, from Escherichia coli F-583), and with loading buffer (150 mM Tris, 30% glycerol,
polymyxin B were purchased from Sigma Chemical Co. 3% bromophenol blue, 3% sodium n-dodecyl sulfate,
(St. Louis, MO). TNF-a was procured from Genzyme- 15% 2-mercaptoethanol), and electrophoresed in 9%
Techne (Cambridge, MA); and IL-1b , from Pepro Tech EC polyacrylamide gels. The gels were electrophoretically
Ltd. (London, UK). Polyclonal antibodies against ICAM-1 transferred to PVDF membranes (Amersham Biosciences,
and VCAM-1 came from Santa Cruz Biotechnology (Santa Piscataway, NJ) at 4°C for 3 hours. The membranes were
Cruz, CA); and monoclonal antibody against LBP (6G3), then blocked with 5% skim milk, and incubated with anti-
from HyCult Biotechnology (Uden, The Netherlands). ICAM-1 or anti-VCAM-1 antibody in TBS buffer (20 mM
 37

Tris-HCl, pH 7.6, and 137 mM NaCl) at room temperature 40 mM of n-octyl b -D-glucoside for 5 minutes at a flow
for 1 hour. The blotted membranes were next washed 6 rate of 5 m l/minute, the lipid A was injected into the flow
times with 0.2% Tween 20 in TN buffer (50 mM Tris-HCl, cell at 1 m l/minute until the saturation level was achieved.
pH 7.5, contains 137 mM NaCl) and incubated with After immobilization, 2 mM NaOH was injected at
horseradish-peroxidase (HRP)-conjugated donkey anti- 20 m l/minute in 1-minute pulses into the flow cell to remove
rabbit IgG (Amersham Biosciences) for 1 hour. Immuno- excess lipid A so that only a monolayer of lipid A
reactive proteins were visualized by using the ECL remained. Washing was continued until the basal SPR
chemiluminescence system (Perkin Elmer Life Sciences, response unit (RU) in the sensorgram stably returned to
Boston, MA) and exposure to Fuji Medical X-ray film HR- the baseline. Typically, around 1000 RU per flow-cell
H (Fuji Photo Film, Tokyo, Japan). surface coating of lipid A was obtained. For the reference
cell, dimyristoylphosphatidylcholine (DMPC; Sigma) at
Assay for the Lipid A/LBP Interaction 0.5 mg/ml in water was used for coating in the same way
Ninety-six-well microtiter plates (Immulon 2H; Dynex as the lipid A. For the binding analysis, samples in the
Technologies, Ashford, U.K.) were coated with lipid A, and running buffer (10 mM Tris-HCl, pH 7.0, 100 mM NaCl)
the interaction of the immobilized-lipid A with LBP was were injected for 2 minutes at a flow rate of 20 m l/minute.
measured as described previously [19, 20]. Briefly, lipid A Association and dissociation curves were obtained from the
at 5 m g/well in 20 m l of ethanol was placed in the ninety- Biacore software. The surface of the sensor chip was
six-well microtiter plates, and the solvent was evaporated in regenerated by the injection of 20 m l of 2 mM NaOH. All
ambient air. After the nonspecific binding had been blocked Biacore experiments were carried out at 25°C, with the
by incubating the wells for 30 minutes at 37°C with 100 m l samples kept on ice before the injection. The value of the
of PBS containing 1% bovine serum albumin (BSA), apparent binding affinity of surfactin or polymyxin B for
100 m l of PBS containing 1, 3, 10, or 30% FBS was added, lipid A was calculated by fitting the sensorgrams of kinetic
and the plates were then incubated for 1 hour at 37°C. They injections to the bivalent binding model with Biacore
were next washed, and 25 nM anti-LBP mAb 6G3 in 50 m l evaluation software version 3.0.
of PBS containing 0.1% BSA was added; and incubation
was then continued for 1 hour at 37°C. The mAb solution
was rinsed out, and 50 m l of HRP-conjugated sheep anti- Results
mouse IgG (Amersham Biosciences) diluted 1,000-fold in
PBS containing 0.1% BSA. Finally, binding of LBP to the Inhibition of Leukemic Cell Adhesion to LPS-
immobilized-lipid A was detected by incubation with stimulated HUVEC by Surfactin
100 m l of 3,3
,5,5
-tetramethylbenzidine (TMB) liquid In the course of our screening of cell adhesion inhibitors,
substrate (Sigma). The reaction was stopped by addition of we found that the filtrate from Bacillus sp. BML752-121F2
100 m l of 0.18 M sulfuric acid, and absorbances at 450 and showed marked inhibitory activity toward HL-60 cell
570 nm were quantified in a micro plate reader. When adhesion to LPS-stimulated HUVEC. From the culture
surfactin was added, the lipid A-coated microtiter plates broth of BML752-121F2 the active principle was isolated
were preincubated for 1 hour at 37°C with 0.3, 1, 3 or through chromatographic separations. Spectroscopic analysis
10 m g/well surfactin in 100 m l of PBS containing 0.1% of the purified compound revealed that the compound was
BSA. After the solution has been removed, 100 m l of PBS identical to a known cyclic lipoheptapeptide, surfactin C1
containing 10% FBS was added; and the plates were (Fig. 1). In the present study, we employed commercially
incubated for 1 hour at 37°C. Binding of LBP was available surfactin (Sigma), which is mainly composed of
determined as described above. surfactin C1.
Treatment of HUVEC with LPS increased the adhesion
SPR Analysis of HL-60 cells to them. The addition of surfactin at 3 m g/ml
Realtime bio-interaction analysis of surfactin or polymyxin prior to LPS stimulation markedly decreased the HL-60
B with lipid A was measured by surface plasmon resonance cell-HUVEC adhesion (Fig. 2A). The IC50 value was
(SPR) using a Biacore X (Biacore AB, Uppsala, Sweden). determined to be 1.10 m g/ml, as shown in Fig. 2B.
The immobilization of lipid A onto the HPA biosensor chip Furthermore surfactin also inhibited the LPS-induced cell
(Biacore AB) was carried out as described previously with adhesion of human monocyte cell line THP-1 cells or
slight modifications [21]. Briefly lipid A at 0.5 mg/ml in human T cell leukemia Jurkat cells to HUVEC (THP-1:
water was sonicated at 37°C for 15 minutes before being IC50 value 1.45 m g/ml; Jurkat: IC50 value 1.43 m g/ml; data
immobilized. After the HPA chip has been washed with not shown). Lipid A, the active component of LPS, also
38

Inhibition of the Interaction of Lipid A/LBP by

Surfactin
Using lipid A-coated plates, we next examined the effects
of surfactin on the binding of lipid A to LBP. When the
lipid A-coated plates were incubated with FBS, serum LBP
bound to the lipid A-coated plates in a serum concentration
dose-dependent manner (Fig. 5A). When the lipid A-coated
plates were pretreated with surfactin the binding of LBP to
the lipid A-coated plates was inhibited in a dose-dependent
manner by surfactin (Fig. 5B). Thus, surfactin is likely to
interact with either lipid A or LBP.

Direct Interaction between Surfactin and Lipid A

Since surfactin did not inhibit TNF-a or IL-1b -induced
Fig. 1 Structure of surfactin C1. signaling, it is likely that surfactin directly acts on LPS or
LBP to inhibit the signal transduction of LPS. So, we
looked into the real-time interaction between surfactin and
induced the adhesion of HL-60 cells to HUVEC, which was lipid A by using surface plasmon resonance (SPR) analysis.
again inhibited by surfactin (Fig. 2B). However, surfactin The increase in resonance units (RU) by binding of
did not inhibit TNF-a - or IL-1b -induced cell adhesions. surfactin to immobilized-lipid A demonstrated the direct
Therefore, surfactin was shown to inhibit LPS-induced cell interaction between surfactin and lipid A (Fig. 6A). We also
adhesion specifically. employed polymyxin B, which is known to bind to lipid A
[23]. Prominent binding of polymyxin B to immobilized-
Effects of Surfactants on LPS-Activity and Cell lipid A also occurred in a dose-dependent manner (Fig.
Viability 6B). However, a rapid and time-dependent decrease in the
Surfactin is an amphiphilic compound and has surface- binding signal for surfactin and lipid A interaction was
activating potential [22]. So, we studied whether the cell detected. Thus, compared with that with polymyxin B, the
adhesion could be inhibited by general surface-activating interaction with surfactin was shown to be reversible. The
compounds. We tested the effects on cell adhesion of association rate (ka), dissociation rate (kd) and the apparent
sodium n-dodecyl sulfate (SDS), which is an anionic dissociation constant (KD) of surfactin binding to lipid A
surfactant like surfactin, cetyltrimethylammonium chloride were calculated to be 2.27104 M1 s1, 2.46103 s1
that is a cationic surfactant; and Triton X-100, Tween-20, and 1.09107 M, respectively. Those parameters of
and n-octyl b -D-glucoside, which are nonionic surfactants. polymyxin B binding to lipid A were calculated
As shown in Fig. 3 and Table 1, all the tested detergents to be 7.86106 M1 s1 (ka), 1.30104 s1 (kd) and
inhibit cell adhesion while exhibiting cytotoxicity at similar 1.651011 M (KD). These kinetic parameters also indicate
concentrations. Only the exception is surfactin. But this can that the binding of surfactin showed slower association and
not exclude the possibility that surfactin inhibits the cell faster dissociation compared with polymyxin B.
adhesion via its general detergent activity. Surfactin did not
influence the viability of HUVEC up to 30 m g/ml after 24
hours, with or without LPS. These results showed that Discussion
inhibition of surfactin on the cell adhesion may not be
attributable to its general detergent activity. In the present study, we looked among microbial secondary
metabolites for compounds that could inhibit HL-60 cell
Inhibition of LPS-Induced Expression of Adhesion adhesion to LPS-stimulated HUVEC. Among several
Molecules by Surfactin thousands of microbial culture filtrates tested, we isolated a
LPS induced the expression of adhesion molecules such as known inhibitory lipopeptide, surfactin, from the culture
ICAM-1 and VCAM-1. Western blotting analysis revealed broth of Bacillus sp. BML752-121F2. The inhibition of
that surfactin inhibited 1 m g/ml LPS-induced expression of LPS signal transduction by surfactin was shown to be due
ICAM-1 and VCAM-1 at 3 m g/ml almost completely (Fig. to the direct interaction between surfactin and lipid A.
4). LPS did not induce E-selectin in our preparation of Surfactin is a lipopeptide antibiotic produced by Bacillus
HUVEC. subtilis, and it was first identified as a potent inhibitor of
 39

Fig. 2 Inhibition of HL-60 cell adhesion to LPS-stimulated HUVEC by surfactin.

(A) HUVEC were preincubated or not with 3 m g/ml surfactin for 2 hours, treated or not with LPS (1 m g/ml) for 4 hours, and, after having
been washed, incubated with HL-60 cells for 1 hour. (B) HUVEC were preincubated with the indicated concentrations of surfactin for 2
hours, then treated (solid columns) or not (open columns) with 1 m g/ml LPS, 1 m g/ml lipid A, 10 ng/ml TNF-a , or 10 ng/ml IL-1b for 4 hours.
After a washing, HL-60 cells were added to the HUVEC. One hour later, the adhesion of HL-60 cells to HUVEC was determined by
measuring the number of adherent cells in 1 microscopic field. Values are the meansS.D. of triplicate determinations.
40

Fig. 3 Effects of various surfactants on cell adhesion and viability of HUVEC.

Columns: HUVEC were preincubated with the indicated concentrations of surfactants for 2 hours, then treated (solid columns) or not
(open columns) with 1 m g/ml LPS for 4 hours. Then, the adhesion of HL-60 cells to HUVEC was determined by measuring the number of
adherent cells as described above. Values are the meansS.D. of triplicate determinations. Circles; HUVEC were treated with the indicated
concentrations of surfactants for 24 hours in the presence of 1 m g/ml LPS, and the cell viability was determined by trypan blue dye
exclusion. Values are the meansS.D. of quadruplicate determinations.

Table 1 Effects of surfactants on LPS-induced HL-60- fibrin clotting [24]. Surfactin contains the structure of a
HUVEC adhesion and viability of HUVEC cyclic heptapeptide and a lipid portion represented by a
mixture of several b -hydroxy fatty acids with chain lengths
IC50 value of IC50 value of of 13
15 carbon atoms [25]. The main component that we
Compounds cell adhesion cell viability isolated was surfactin C1 (Fig. 1). It is an amphiphilic
(m g/ml) (m g/ml)
compound and known to be one of the most powerful
biosurfactants [22, 26]. Surfactin was also reported to
Surfactin 1.38 55.00
inhibit phospholipase A2 (PLA2) [27], and to enhance
Sodium n-dodecyl sulfate 69.20 138.00
Cetyltrimethylammonium chloride 0.29 0.25
plasminogen activation [28]. In addition, it is less toxic than
Triton X-100 18.60 20.90 other surfactants as judged from the results of an acute
Tween-20 70.80 178.00 toxicity study in mice (LD50 value at 100 mg/kg, i.v.)
n-Octyl b -D-glucoside 300.00 300.00 [29]. However, the interaction with LPS has been entirely
not known before. The activity of surfactin observed in the
present research was not due to its surface-activating
potential, since other surfactants were not active in
inhibition of LPS-activity (Fig. 3, Table 1). As mentioned
 41

Fig. 4 Inhibition of adhesion molecule expression in LPS-activated HUVEC by surfactin.

HUVEC were treated with the indicated concentrations of surfactin for 2 hours and then stimulated or not with 1 m g/ml LPS for 4
hours. The cell lysates were analyzed by Western blotting with anti-ICAM-1 and anti-VCAM-1 antibodies.

Fig. 5 Inhibition of interaction of lipid A with LBP by surfactin.

(A) Lipid A/LBP binding was examined by incubating 100 m l of PBS containing 1, 3, 10, or 30% FBS in lipid A-coated 96 well microtiter
plates for 1 hour. After incubation, bound LBP was detected as TMB reaction products obtained with anti-LBP MAb 6G3 and HRP-
conjugated sheep anti-mouse IgG. (B) Lipid A-coated microtiter plates were preincubated with surfactin in 100 m l of PBS for 1 hour. After
the removal of the medium, the plates were incubated with 100 m l of PBS containing 10% FBS for 1 hour. Lipid A/LBP binding was
expressed as a percentage of that obtained by incubation with PBS containing 10% FBS in the absence of surfactin. Values are the
meansS.D. of quadruplicate determinations.

above, surfactin is a known inhibitor of PLA2. Surfactin Since surfactin could inhibit the LBP binding and LPS-
inhibited cytosolic PLA2 purified from bovine platelets induced cellular effects at the concentration necessary to
with an IC50 of 8.5 m M (8.8 m g/ml) [27]. PLA2 was reported interact with lipid A, the reversible interaction may be
to be activated by inflammatory stimuli such as LPS in sufficient to inactivate the lipid A activity.
endothelial cells, and activated PLA2 was shown to lead to Thus, surfactin, a naturally-occurring cyclic lipopeptide,
increased leukemic cell adhesion via the expression of was found to be a selective inhibitor of LPS signal
adhesion molecules [30]. Therefore, the inhibition of PLA2 transduction. Having minimal cytotoxicity, surfactin may
may also contribute to the inhibitory activity of surfactin on have a therapeutic potential for the treatment of LPS-
cell adhesion. triggered syndromes such as Gram-negative bacterial sepsis
Recently, we reported that a novel naturally-occurring and periodontitis.
cyclic heptadepsipeptide, heptadepsin, inhibited LPS
activity [18]. Similar to polymyxin B, heptadepsin was Acknowledgments The authors wish to thank Dr. Y. Akamatsu,
demonstrated to inactivate LPS by its direct interaction Microbial Chemistry Research Center, Tokyo, and Dr. S. Kondo,
with LPS. Compared with polymyxin B and heptadepsin, Bioscience Associates, Tokyo, for their valuable suggestions. This
surfactin showed more reversible interaction with lipid A. work was financially supported in part by grants from the
42

signaling in C3H/HeJ and C57BL/10ScCr mice: mutations

in Tlr4 gene. Science 282: 2085–2088 (1998)
5. Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore
KJ, Gros P, Malo D. Endotoxin-tolerant mice have mutations
in Toll-like receptor 4 (Tlr4). J Exp Med 189: 615–625
(1999)
6. Hardaway RM. Traumatic shock alias posttrauma critical
illness. Am Surg 66: 284–290 (2000)
7. Cohen J. The immunopathogenesis of sepsis. Nature 420:
885–891 (2002)
8. Jersmann HP, Hii CS, Ferrante JV, Ferrante A. Bacterial
lipopolysaccharide and tumor necrosis factor a
synergistically increase expression of human endothelial
adhesion molecules through activation of NF-k B and p38
mitogen-activated protein kinase signaling pathways. Infect
Immun 69: 1273–1279 (2001)
9. Stad RK, Buurman WA. Current views on structure and
function of endothelial adhesion molecules. Cell Adhes
Commun 2: 261–268 (1994)
10. van de Stolpe A, van der Saag PT. Intercellular adhesion
molecule-1. J Mol Med 74: 13–33 (1996)
Fig. 6 SPR analysis for the interaction of surfactin or 11. Rice GE, Munro JM, Bevilacqua MP. Inducible cell
polymyxin B with immobilized-lipid A. adhesion molecule 110 (INCAM-110) is an endothelial
Sensorgrams indicate the association and dissociation phases
receptor for lymphocytes. A CD11/CD18-independent
of the reactions with surfactin (A) or polymyxin B (B). Each adhesion mechanism. J Exp Med 171: 1369–1374 (1990)
chemical was added at 1, 2, 3, 4, 5 m g/ml (from bottom to top) for 2 12. Tedder TF, Steeber DA, Chen A, Engel P. The selectins:
minutes at 20 m l/minute over the monolayer of lipid A immobilized vascular adhesion molecules. FASEB J 9: 866–873 (1995)
on the HPA sensor chip. 13. Risau W. Differentiation of endothelium. FASEB J 9:
926–933 (1995)
14. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF,
programs Grants-in-Aid for Scientific Research on Priority Areas; Ramos R, Britton H, Moran T, Karaliuskas R, Duerr RH,
Grants-in-Aid for the 21st Century Center of Excellence (COE) Achkar JP, Brant SR, Bayless TM, Kirschner BS, Hanauer
Program entitled “Understanding and Control of Life via Systems SB, Nunez G, Cho JH. A frameshift mutation in NOD2
Biology” (to Keio University) of the Ministry of Education, associated with susceptibility to Crohn’s disease. Nature
Science, Culture, and Sports of Japan; and a grant from 411: 603–606 (2001)
Nateglinide Memorial Toyoshima Research and Education Fund 15. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP,
of Keio University. Belaiche J, Almer S, Tysk C, O’Morain CA, Gassull M,
Binder V, Finkel Y, Cortot A, Modigliani R, Laurent-Puig P,
Gower-Rousseau C, Macry J, Colombel JF, Sahbatou M,
Thomas G. Association of NOD2 leucine-rich repeat
References variants with susceptibility to Crohn’s disease. Nature 411:
599–603 (2001)
1. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. 16. Holt SC, Kesavalu L, Walker S, Genco CA. Virulence
Annu Rev Biochem 71: 635–700 (2002) factors of Porphyromonas gingivalis. Periodontol 20:
2. Galanos C, Luderitz O, Rietschel ET, Westphal O, Brade H, 168–238 (2000)
Brade L, Freudenberg M, Schade U, Imoto M, Yoshimura H, 17. Haffajee AD, Socransky SS. Microbial etiological agents of
Kusumoto S, Shiba T. Synthetic and natural Escherichia coli destructive periodontal diseases. Periodontol 5: 78–111
free lipid A express identical endotoxic activities. Eur J (2000)
Biochem 148: 1–5 (1985) 18. Ohno O, Ikeda Y, Sawa R, Igarashi M, Kinoshita N, Suzuki
3. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky Y, Miyake K, Umezawa K. Isolation of heptadepsin, a novel
F. Toll-like receptor-4 mediates lipopolysaccharide-induced bacterial cyclic depsipeptide that inhibits lipopolysaccharide
signal transduction. J Biol Chem 274: 10689–10692 (1999) activity. Chem Biol 11: 1059–1070 (2004)
4. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, 19. Sano H, Sohma H, Muta T, Nomura S, Voelker DR, Kuroki
Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Y. Pulmonary surfactant protein A modulates the cellular
Ricciardi-Castagnoli P, Layton B, Beutler B. Defective LPS response to smooth and rough lipopolysaccharides by
 43

interaction with CD14. J Immunol 163: 387–395 (1999) biochemistry of surfactin. Appl Microbiol Biotechnol 51:
20. Nagaoka I, Hirota S, Niyonsaba F, Hirata M, Adachi Y, 553–563 (1999)
Tamura H, Heumann D. Cathelicidin family of antibacterial 26. Nakano MM, Zuber P. Molecular biology of antibiotic
peptides CAP18 and CAP11 inhibit the expression of TNF- production in Bacillus. Crit Rev Biotechnol 10: 223–240
a by blocking the binding of LPS to CD14 cells. J (1990)
Immunol 167: 3329–3338 (2001) 27. Kim K, Jung SY, Lee DK, Jung JK, Park JK, Kim DK, Lee
21. Zhu Y, Ho B, Ding JL. Sequence and structural diversity in CH. Suppression of inflammatory responses by surfactin, a
endotoxin-binding dodecapeptides. Biochim Biophys Acta selective inhibitor of platelet cytosolic phospholipase A2.
1611: 234–242 (2003) Biochem Pharmacol 55: 975–985 (1998)
22. Moore RA, Bates NC, Hancock RE. Interaction of 28. Kikuchi T, Hasumi K. Enhancement of reciprocal activation
polycationic antibiotics with Pseudomonas aeruginosa of prourokinase and plasminogen by the bacterial
lipopolysaccharide and lipid A studied by using dansyl- lipopeptide surfactins and iturin Cs. J Antibiot 56: 34–37
polymyxin. Antimicrob Agents Chemother 29: 496–500 (2003)
(1986) 29. Kikuchi T, Hasumi K. Enhancement of plasminogen
23. Cooper DG, Zajic JE. Surface active compounds from activation by surfactin C: augmentation of fibrinolysis in
microorganisms. Adv Appl Microbiol 26: 229–253 (1980) vitro and in vivo. Biochim Biophys Acta 1596: 234–245
24. Arima K, Kakinuma A, Tamura G. Surfactin, a crystalline (2002)
peptidelipid surfactant produced by Bacillus subtilis: 30. Yokote K, Morisaki N, Zenibayashi M, Ueda S, Kanzaki T,
isolation, characterization and its inhibition of fibrin clot Saito Y, Yoshida S. The phospholipase-A2 reaction leads to
formation. Biochem Biophys Res Commun 31: 488–494 increased monocyte adhesion of endothelial cells via the
(1968) expression of adhesion molecules. Eur J Biochem 217:
25. Peypoux F, Bonmatin JM, Wallach J. Recent trends in the 723–729 (1993)