U.S. patent application number 11/248085 was filed with the patent office on 2006-04-27 for compositions and methods for activating toll-like receptor 4.
Invention is credited to Michael Karin, Jin Mo Park, Richard Rest.
Application Number | 20060089305 11/248085 |
Document ID | / |
Family ID | 36206888 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060089305 |
Kind Code |
A1 |
Rest; Richard ; et
al. |
April 27, 2006 |
Compositions and methods for activating toll-like receptor 4
Abstract
Methods for activating Toll-like receptor 4 via
cholesterol-dependent cytolysins isolated from a Gram-positive
bacteria are provided. In addition compositions containing an
isolated cholesterol-dependent cytolysin or a fragment thereof or a
mimetic of the cytolysin or fragment thereof and methods for use of
such composition in inhibiting binding and/or interaction of
Toll-like receptor 4 with endotoxin are provided. Methods for
identifying modulators of Toll-like receptor 4 activation by a
cholesterol-dependent cytolysin and use of such modulators in
treatment of septicemia and/or septic shock are also provided.
Inventors: |
Rest; Richard; (Rosemont,
PA) ; Karin; Michael; (La Jolla, CA) ; Park;
Jin Mo; (Charlestown, MA) |
Correspondence
Address: |
LICATLA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
36206888 |
Appl. No.: |
11/248085 |
Filed: |
October 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60617894 |
Oct 12, 2004 |
|
|
|
Current U.S.
Class: |
514/1.4 ;
514/2.4 |
Current CPC
Class: |
A61K 38/164 20130101;
A61P 31/00 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/17 20060101 A61K038/17 |
Goverment Interests
[0002] This invention was supported in part by funds from the U.S.
government (NIH.Grant No. AI61712 and U54 AI57168). The U.S.
government may therefore have certain rights in the invention.
Claims
1. A method for activating Toll-like receptor 4 comprises
contacting Toll-like receptor 4 with a cholesterol-dependent
cytolysin isolated from a Gram-positive bacteria.
2. The method of claim 1 wherein the cholesterol-dependent
cytolysin is Listeriolysin O, Streptolysin O, perfringolysin O, or
anthrolysin O.
3. A composition comprising an isolated cholesterol-dependent
cytolysin from a Gram-positive bacteria or a fragment thereof or a
mimetic of the isolated cholesterol-dependent cytolysin from a
Gram-positive bacteria or the fragment thereof which inhibits
binding of Toll-like receptor 4 and endotoxin.
4. The composition of claim 3 further comprising an acceptable
carrier or vehicle for administration of a subject.
5. A method of inhibiting binding and/or interaction of Toll-like
receptor 4 with endotoxin in a subject comprising administering to
the subject the composition of claim 3.
6. A method of inhibiting binding and/or interaction of Toll-like
receptor 4 with endotoxin in a subject comprising administering to
the subject the composition of claim 4.
7. A method for treating septicemia or septic shock in a subject
comprising administering to the subject the composition of claim
3.
8. A method for treating septicemia or septic shock in a subject
comprising administering to the subject the composition of claim
4.
9. A method for identifying a modulator of Toll-like receptor 4
activation comprising: (a) measuring activation of Toll-like
receptor 4 by a cholesterol-dependent cytolysin in the presence and
absence of a test agent; and (b) comparing the measured activation
of Toll-like receptor 4 by the cholesterol-dependent cytolysin in
the absence of the test agent with the measured activation of
Toll-like receptor 4 by the cholesterol-dependent cytolysin in the
presence of a test agent, wherein any change in activation of
Toll-like receptor 4 activity by the cholesterol-dependent
cytolysin in the presence of the test agent is indicative of the
test agent being a modulator of Toll-like receptor 4
activation.
10. A composition for treatment of septicemia or septic shock
comprising an agent which inhibits activation of Toll-like receptor
4 by a cholesterol-dependent cytolysin.
11. A method for treating septicemia or septic shock in a subject
comprising administering to the subject the composition of claim
10.
Description
INTRODUCTION
[0001] This application claims the benefit of priority from U.S.
Provisional Application Ser. No. 60/617,894 filed Oct. 12, 2004,
which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions comprising a
member of the cholesterol-dependent cytolysin (CDC) family and
methods of using these compositions to activate Toll-like receptor
4 and to identify modulators of Toll-like receptor 4 activation.
Activation of Toll-like receptor 4 by endotoxin or
lipopolysaccharide (LPS) can lead to septic shock or septicemia.
Compounds identified as modulators, and more particularly
inhibitors, of Toll-like receptor 4 activation by a member of the
cholesterol-dependent cytolysin (CDC) family are expected to useful
in the treatment of septic shock and/or septicemia. It is believed
that isolated cholesterol-dependent cytolysins or fragments or
mimetics thereof may inhibit the interaction of Toll-like receptor
4 and endotoxin or LPS as well.
BACKGROUND OF THE INVENTION
[0004] Toll-like receptors play pivotal roles in recognizing and
resisting microbial infection (Kopp, E. and Medzhitov, R. Curr.
Opin. Immunol. 2003 15:396-401; Beutler, B. and Rietschel, E. T.
Nat. Rev. Immunol. 2003 3:169-176). Among the immediate outcomes of
the TLR-dependent immune response is the production of cytokines by
inflammatory cells such as macrophages. The production and release
of such cytokines is responsible for the inflammatory response that
accompanies bacterial infection. Toll-like receptor 4 (TLR4)
activation by endotoxin or lipolysaccharide (LPS) produced by
gram-negative bacteria leads to septic shock (Cohen, J. Nature 2002
420:885-891).
[0005] Infection of bone marrow derived macrophages with live B.
anthracis (Sterne strain) has also been demonstrated to result in
extensive apoptosis dependant on signaling from the LPS-responsive
Toll-like receptor TLR4, which activates the proapoptotic double
stranded (ds) RNA-dependent protein kinase PKR (Hsu et al. Nature
2004 428:341-345). However, B. anthracis is a Gram-positive
bacterium that does not produce LPS.
[0006] In addition, the interaction of TLR4 with the
cholesterol-dependent cytolysin pneumolysin was recently reported
to be critically involved in the innate immune response to
gram-positive pneumococcus (Malley et al. Proc. Natl Acad. Sci 2003
100(4):1966-1971). In particular, experiments were performed
demonstrating that the inflammatory response of macrophages to
pneumolysin was dependent on TLR4. Further, experiments with mutant
mice lacking functional TLR4 revealed the mutant mice to be
significantly more susceptible to invasive disease and death after
exposure to pneumolysin-producing pneumococci when compared to
control mice. Malley et al. thus concluded that the interaction of
the pneumolysin with TLR4 is an important protective component of
the host response to pneumococcus (Malley et al. Proc. Natl Acad.
Sci 2003 100(4):1966-1971).
[0007] Cholesterol-dependent cytolysins (CDCs) are known to be
major virulence factors in Gram-positive bacterial infections
(Portnoy et al. Infect. Immun. 1992 60:1263-1267; Paton, J. C.
Trends Microbiol. 1996 4:103-106; Rood, J. I. Annu. Rev. Microbiol.
1998 52:333-360). The mechanisms by which CDCs contribute to
pathogenesis in Gram-positive infections have so far been
attributed to their cytolytic activity and their role in regulating
intracellular compartmentalization of pathogenic bacteria (Decatur,
A. L. and Portnoy, D. A. Science 2000 290:992-995). For example,
Listeriolysin O (LLO) from Listeria monocytogenes and Streptolysin
O (SLO) from Streptococcus pyogenes have also been shown to
activate macrophages and mast cells to produce proinflammatory
cytokines and chemokines (Tsukada et al. Cell. Immunol. 1992
140:21-30; Stassen et al. Infect. Immun. 2003 71:6171-6177). In
addition, Streptolysin O (SLO) from Streptococcus pyogenes has been
shown to mediate vectorial transport of streptococcal proteins into
the cytoplasm of host cells (Madden et al. Cell 2001 104:143-152;
Meehl, M. A. and Caparon, M. G. Mol. Microbiol. 2004
52:1665-1676).
SUMMARY OF THE INVENTION
[0008] An aspect of the present invention relates to a method for
activating Toll-like receptor 4 which comprises contacting
Toll-like receptor 4 with a cholesterol-dependent cytolysin
isolated from a Gram-positive bacteria.
[0009] Another aspect of the present invention relates to a method
for identifying modulators of Toll-like receptor 4 activation which
comprises measuring activation of Toll-like receptor 4 by a
cholesterol-dependent cytolysin in the presence and absence of a
test agent, wherein a change in activation of Toll-like receptor 4
in the presence of the test agent is indicative of the test agent
being a modulator of Toll-like receptor 4 activation.
[0010] Another aspect of the present invention relates to
compositions comprising an isolated cholesterol-dependent cytolysin
or a fragment or mimetic thereof which inhibits binding and/or
interaction of Toll-like receptor 4 with endotoxin.
[0011] Another aspect of the present invention relates to a method
for treating septicemia or septic shock in a subject comprising
administering to the subject a composition comprising an isolated
cholesterol-dependent cytolysin or a fragment or mimetic thereof
which inhibits binding and/or interaction of Toll-like receptor 4
with endotoxin.
[0012] Another aspect of the present invention relates to a method
for treating septicemia or septic shock in a subject comprising
administering to the subject an agent which inhibits activation of
Toll-like receptor 4 activity by a cholesterol-dependent
cytolysin.
[0013] Yet another aspect of the present invention relates to
compositions for treatment of septicemia or septic shock comprising
an agent which inhibits Toll-like receptor 4 activation by a
cholesterol-dependent cytolysin.
DETAILED DESCRIPTION OF THE INVENTION
[0014] It has now been found that cholesterol-dependent cytolysins
induce inflammatory and apoptotic responses in host immune cells
via Toll-like receptor 4 (TLR4), a major contributor to
Gram-positive induced septic shock.
[0015] Identification of a cholesterol-dependent cytolysin as a
TLR4 agonist was first performed in B. anthracis.
[0016] In these experiments, B. anthracis cell wall preparations
and culture supernatants were first tested for their ability to
stimulate TNF-alpha gene expression and induce apoptosis of bone
marrow-derived macrophages (BMDMs) in the presence of the p38
inhibitor SB202190. Treatment of BMDMs with a crude, commercially
available, B. anthracis cell wall preparation did not strongly
induce TNF-alpha mRNA expression or apoptosis. In contrast, the B.
anthracis culture supernatant induced both TNF-alpha mRNA and
apoptosis under the same conditions. The TNF-alpha- and
apoptosis-inducing activity in the culture supernatant was
sensitive to proteinase K digestion, indicating that a
proteinaceous component is responsible for both activities. As only
TLR4 agonists, but not agonists for other TLRs, can strongly
potentiate macrophage apoptosis in the presence of SB202190 (Park
et al. Science 2002 297:2048-2051; Hsu et al. Nature 2004
428:341-345), this protein component was expected to act as a TLR4
agonist.
[0017] To identify this protein, the B. anthracis culture
supernatant was sequentially purified through DEAE-Sepharose, Mono
S, and phenyl-Sepharose chromatography columns. On the
phenyl-Sepharose column, the TNF-alpha- and apoptosis-inducing
activities cofractionated as a single peak centered at fraction 26.
Analysis of the protein composition of the different column
fractions revealed that a 63-kDa polypeptide copurified with both
activities. Among the numerous secreted proteins predicted by the
B. anthracis genome sequence to be present was anthrolysin O, a
cholesterol-dependent cytolysin (CDC) encoded by the BA3355 gene
(Shannon et al. Infect. Immun. 2003 71:3183-3189). The anthrolysin
O polypeptide consists of 512 amino acids with the N-terminal 35
residues coding for a signal peptide, a size consistent with the
63-kDa band described above.
[0018] The phenyl-Sepharose fractions were thus analyzed by
immunoblotting with anti-anthrolysin O antibody. It was found that
anthrolysin O indeed co-purified with the 63-kDa protein, as well
as the macrophage-stimulating and apoptosis-inducing
activities.
[0019] To directly test whether anthrolysin O stimulated
macrophages in a TLR4-dependent manner, pure recombinant
anthrolysin O was prepared by expression in E. coli. Treatment of
BMDMs with recombinant anthrolysin O or other TLR agonists with
different receptor specificities resulted in a strong induction of
TNF-alpha mRNA. To compare the gene induction specificity of
anthrolysin O to those of other TLR agonists, the mRNA levels of
other cytokines including interleukin (IL)-1alpha, IL-1.beta., and
IL-6 were examined. Whereas TNF-alpha was induced to similar
extents by anthrolysin O and the different TLR agonists, the
IL-1alpha and IL-6 genes were most strongly induced by anthrolysin
O and LPS, but were less responsive to other TLR agonists.
Furthermore, treatment of BMDMs with either anthrolysin O or the
different TLR agonists in conjunction with SB202190 revealed that
only anthrolysin O and LPS were able to cause a robust apoptotic
response.
[0020] To specifically examine the role of TLR4 in the response to
anthrolysin O, BMDMs were prepared from wild type (C3H/OuJ) and
TLR4 mutant (C3H/HeJ) mice and their responses to anthrolysin O
treatment were compared. Anthrolysin O induced activation of p38
MAPK and degradation of I.kappa.B.alpha. in TLR4 wild type, but not
in TLR4 mutant BMDMs. The TLR4 mutant BMDMs showed no defect in
their response to the TLR2 agonist synthetic bacterial lipopeptide.
Anthrolysin O also failed to induce TNF-alpha and IL-6 gene
expression in TLR mutant BMDMs. These observations indicate that
anthrolysin O activates macrophages via TLR4.
[0021] To rule out the possibility that TLR4 activation by
recombinant anthrolysin O is due to the presence of contaminating
LPS in the preparation, BMDMs were treated with anthrolysin O, LPS,
and taxol, another TLR4 agonist, in the presence of polymyxin B,
which blocks LPS binding to TLR4. Activation of p38 MAPK by LPS,
but not by anthrolysin O or taxol, was inhibited by polymyxin B.
Polymyxin B treatment neutralized the activity of LPS at
concentrations as high as 0.5 .mu.g/ml.
[0022] Serum requirements for biological activity and the
proteinase K sensitivity of anthrolysin C and LPS were also
examined. TLR4 activation by LPS in vitro depends strictly on
factors such as soluble CD14 and LPS-binding protein (LBP) that are
provided by inclusion of serum (Ulevitch et al. Annu. Rev. Immunol.
1995 13:437-457). It was found that p38 MAPK activation in
macrophages by recombinant anthrolysin O can occur in serum-free
medium, in which LPS fails to activate p38 MAPK. The converse was
observed after proteinase K digestion. Whereas proteinase K
treatment completely abolished the ability of anthrolysin O to
activate p38 MAPK, the activity of LPS was largely unaffected.
Taken together, these experiments demonstrate that TLR4 activation
by anthrolysin O is not due to contamination with LPS, whose
activity is proteinase K-resistant and serum-dependent.
[0023] Anthrolysin O is closely related to other CDCs produced by
Gram-positive pathogens. Accordingly, the ability of other CDCs
produced by Gram-positive bacteria to activate TLR4 was examined.
Listeriolysin O from Listeria monocytogenes and Streptolysin O from
Streptococcus pyogenes, as well as perfringolysin O from
Clostridium perfringens and anthrolysin O were produced by in vitro
translation in reticulocyte lysates and added to BMDMs. All of the
in vitro translated CDCs were found to activate iNOS expression by
BMDMs, whereas a control lysate programmed with an empty backbone
plasmid was inactive. Hence, all four CDCs are capable of
activating macrophages.
[0024] Recombinant CDCs produced in and purified from E. coli were
also examined for their dependence on TLR4 for macrophage
activation. Treatment of TLR4 wild type BMDMs with increasing
amounts of anthrolysin O, Listeriolysin O, Streptolysin O and
perfringolysin O resulted in induction of TNF-alpha and IL-6 mRNAs.
In most cases, the gene induction response reached its maximum at
100 ng/ml of CDC (or approximately 1.7 nM for an approximately 60
kDa protein). By contrast, TLR4 mutant BMDMs did not respond with
cytokine gene expression to the same concentration of any of the
CDCs. These results are indicative of the ability to activate TLR4
being a general property shared by CDCs from Gram-positive
bacteria.
[0025] Thus, the present invention provides methods for activating
Toll-like receptor 4 by contacting Toll-like receptor 4 with a
cholesterol-dependent cytolysin isolated from a Gram-positive
bacteria. In a preferred embodiment, the cholesterol-dependent
cytolysin is Listeriolysin O (LLO) from Listeria monocytogenes,
Streptolysin O (SLO) from Streptococcus pyogenes, perfringolysin O
(PFO) from Clostridium perfringens, or anthrolysin O (ALO) from B.
anthracis.
[0026] TLR4 is also known to bind to and/or interact with
endotoxin. The ability of cholesterol-dependent cytolysins to bind
with TLR4 is indicative of the potential utility of these proteins
or fragments thereof or mimetics of these proteins or fragments
thereof to inhibit binding and/or the interaction of TLR4 with
endotoxin. Thus, it is expected that compositions comprising an
isolated cholesterol-dependent cytolysin or a fragment thereof or a
mimetic of these proteins or fragments thereof will be useful in
treating septicemia and/or septic shock. Preferred
cholesterol-dependent cytolysins or fragments thereof for use in
these compositions are anthrolysin O, Listeriolysin O, Streptolysin
O and perfringolysin O. Such compositions may further comprise
acceptable carriers or vehicles for administration to a
subject.
[0027] By "mimetic" it is meant to include both peptidomimetics and
small organic molecules that bind to TLR4 in similar fashion to a
cholesterol-dependent cytolysin, thereby inhibiting the ability of
TLR4 to bind to and/or interact with endotoxin.
[0028] The present invention also provides methods for identifying
modulators of a Toll-like receptor 4 activity which comprises
measuring activation of Toll-like receptor 4 by a
cholesterol-dependent cytolysin in the presence and absence of a
test agent. In these methods, a change in activation of Toll-like
receptor 4 activity in the presence of the test agent is indicative
of the test agent being a modulator of a Toll-like receptor 4
activity.
[0029] Various assays for measuring TLR4 activation and/or
identifying modulator of TLR4 activation can be used.
[0030] For example, a screening assay for TLR4 stimulation has been
described wherein cells in culture are transfected with two
plasmids, one carrying the gene for human TLR4 and the other, a
detector plasmid, carrying a promoter that binds to NFkappa B
upstream of a luciferase gene (Vogel, S. J. Biol. Chem. 2003
278:222506).
[0031] Alternatively a yeast two-hybrid system can be used for
screening for TLR4 activation. With this system, two or three
plasmids are transformed into a single yeast cell. In the positive
control, one plasmid contains all or part of a gene for a
cholesterol-dependent cytolysin such as anthrolysin O, and a second
plasmid contains all or part of the TLR4 gene. These genes are
transcribed and translated within the yeast cell. Binding of the
cholesterol-dependent cytolysin to TLR4 is measured by a change in
growth or color of the yeast. Inhibitors of this interaction can be
identified by introduction of a third plasmid or screening of a
library of small molecules encoded by DNA within a plasmid that
bind to either the cholesterol-dependent cytolysin or to TLR4.
[0032] Another alternate or additional screening assay involves
measuring the ability of an agent to bind to either recombinant
TLR4 or a cholesterol-dependent cytolysin bound to a multi-well
plate. Agents identified as having the ability to bind to either
recombinant TLR4 or a cholesterol-dependent cytolysin can then be
further tested for their ability to inhibit binding of TLR4 and
cholesterol-dependent cytolysin.
[0033] Given the important role of activated TLR4 in endotoxin
related septicemia and septic shock, it is expected that test
agents identified as inhibitors of TLR4 activation in the above
described methods of the present invention will be useful in
preventing or treating endotoxin-related septicemia and/or septic
shock.
[0034] Thus the present invention also provides methods and
compositions for treating septicemia and/or septic shock in a
subject by administering to the subject an agent which inhibits
activation of Toll-like receptor 4 activity by a
cholesterol-dependent cytolysin. Such methods and compositions may
be used in subjects exhibiting symptoms of septicemia and/or septic
shock. Such compositions and methods can also be used
prophylactically in subjects at high risk for development of
septicemia or septic shock including, but not limited to patients
undergoing major surgery, and in particular operations in the gut
area, as well as immunosuppressed subjects undergoing surgical
procedures.
[0035] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
Mice and Macrophages
[0036] C57BL/6J, C3H/OuJ, C3H/HeJ, B6.MRL-Tnfrsf6.sup.1pr/J
(Faslpr/1pr), and C57BL/6-Tnfrsfla.sup.tm1Imx (TNFR1.sup.-/-) mice
were obtained from the Jackson Laboratory. IFNR1.sup.-/- mice in
the 129/SvEv background were obtained from Dr. E. Raz (University
of Calif., San Diego). BMDMs were prepared in accordance with
procedures described by Park et al Science 2002 297:2048-2051.
Example 2
Reagents
[0037] B. anthracis cell walls were purchased from List Biological
Laboratories, Inc. Other reagents used for treatment of BMDMs
included: LPS (E. coli; Sigma), peptidoglycan (Fluka), poly(I-C)
(Amersham Biosciences), CpG oligodeoxynucleotide (TIB MOLBIOL),
Pam3CSK4 (EMC Microcollections), and R-848 (GLS Synthesis).
SB202190 was from Calbiochem.
Example 3
Bacterial Strains, Culture and Infection
[0038] B. anthracis Sterne strain 7702 and its derivatives have
been described by Shannon et al. infect. Immun. 2003 71:3183-3189).
Bacteria were grown in brain heart infusion broth (BHI; Difco),
without added bicarbonate, with shaking (200 rpm) at 37.degree. C.
in an air shaker incubator or on BHI agar in a humidified
incubator. Bacterial infection of macrophage cultures was performed
in accordance with procedures set forth by Hsu et al. (Nature 2004
428:341-345).
Example 4
Purification of Anthrolysin O from Bacterial Culture
Supernatants
[0039] All buffers used in dialysis and column chromatography
contained protease inhibitors (10 .mu.M phenylmethylsulfonyl
fluoride, 20 nM pepstatin A, 6 nM leupeptin, and 20 .mu.M
bisbenzamidine). To purify macrophage-stimulating activity from B.
anthracis culture supernatants, bacteria were grown in BHI broth
until OD595 reached 1.0. After removing bacteria by centrifugation,
the supernatant (2 liters) was filtered through a 0.2 .mu.m-pore
Nylon filter set (Nalgene), concentrated up to 80-fold on a
Centricon Plus-20 Filter Device (Millipore), and then dialyzed in
buffer D100 (20 mM Tris-Cl [pH 7.0], 100 mM sodium chloride, and
0.1 mM EDTA). Proteins (84 mg) in the culture concentrate were
applied to a DEAE-Sepharose column (10 ml) equilibrated with buffer
D100. The macrophage-stimulating activity was found to pass through
this column under this particular loading condition. Proteins in
the flow-through fraction (61 mg) were equilibrated in buffer S50
(20 mM HEPES-KOH [pH7.0], 50 mM sodium chloride, and 0.1 mM EDTA)
by dialysis and applied to a Mono S column (1 ml per 20 mg protein)
equilibrated with buffer S50. After washing with buffer S50, bound
proteins were eluted with a linear gradient of 50 to 1000 mM sodium
chloride. The major peak fractions of macrophage-stimulating
activity (3.1 mg) were pooled and mixed with an equal volume of 100
mM Tris-Cl (pH 7.0) and 3 M ammonium sulfate, and applied to a
phenyl-Sepharose column (0.5 ml) equilibrated with buffer P1500 (50
mM sodium phosphate [pH 7.0], 1.5 M ammonium sulfate, and 0.1 mM
EDTA). After washing with buffer P1500, bound proteins were eluted
with an inverse linear gradient of 1.5 to 0 M ammonium sulfate. The
phenyl-Sepharose fractions active in macrophage stimulation were
stored at 80.degree. C.
Example 5
Preparation of Recombinant Proteins
[0040] Recombinant LF, PA and CDCs were expressed in and purified
from the E. coli strain BL21 (DE3) bearing the appropriate plasmid
construct as described previously (Shannon et al. Infect. Immun.
2003 71: 3183-3189; Cunningham et al. Biochemistry 1998
37:15737-15746; Shepard et al. Biochemistry 1998 37:14563-14574).
Purified LLO and LLO expression vector were provided by Dr. D.
Portnoy (University of California, Berkeley, Calif.), and PFO and
SLO expression vectors by Dr. R. Tweten (The University of Oklahoma
Health Sciences Center, Oklahoma City, Okla.).
Example 6
Protein Analysis
[0041] Whole-cell extracts for immunoblot analysis were prepared
with lysis buffer (20 mM HEPES-KOH at pH 7.6, 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 25 mM .beta.-glycerophosphate, 2 mM
EDTA, and protease inhibitors) and then subjected to SDS-PAGE.
Proteins transferred to nitrocellulose membrane were probed with
rabbit antiserum against recombinant anthrolysin O (Shannon et al.
Infect. Immun. 2003 71: 3183-3189), and antibodies directed against
actin (Sigma), iNOS, phospho-p38a, p38a, and I.Ba (all from Santa
Cruz Biotechnology), and the immune complexes were visualized with
the ECL Western blot reagent (Pierce).
Example 7
RNA Analysis
[0042] Total RNA was isolated from BMDMs and RAW264.7 cells using
the RNAwiz reagent (Ambion). For real-time PCR analysis, cDNAs were
synthesized with the Superscript II reverse transcriptase system
(Invitrogen). An amount of cDNA equivalent to 0.2 .mu.g of total
RNA was subjected to 40 cycles of PCR amplification consisting of a
15-second incubation at 95.degree. C. and a 1-minute incubation at
60.degree. C. Output was monitored using SYBR Green core reagents
and the ABI Prism 7700 System (PE Applied Biosystems). The results
were normalized to the level of cyclophilin mRNA.
Example 8
Measurement of Cell Viability
[0043] The TUNEL assay and Hoechst staining were performed as
described (Park et al. Science 2002 297:2048-2051). The MTT assay
was carried out using an MTT kit (Roche), according to the
manufacturer's directions.
* * * * *