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Infection and Immunity, May 2000, p. 2998-3001, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Apoptosis Induced by Staphylococcus
aureus in Epithelial Cells Utilizes a Mechanism Involving Caspases
8 and 3
Carla A.
Wesson,
James
Deringer,
Linda E.
Liou,
Kenneth
W.
Bayles,*
Gregory A.
Bohach, and
William R.
Trumble
Department of Microbiology, Molecular Biology
and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052
Received 7 December 1999/Returned for modification 12 January
2000/Accepted 7 February 2000
 |
ABSTRACT |
In this study, we demonstrate that the mechanism by which
Staphylococcus aureus induces apoptosis in bovine mammary
epithelial (MAC-T) cells involves caspases 8 and 3, two key components
of a proteolytic cascade leading to apoptosis. In addition,
internalized S. aureus induces expression of the
inflammatory cytokines tumor necrosis factor alpha and interleukin-1
by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may
ultimately impact the course of infection.
| |
TEXT |
Staphylococcus aureus is
internalized by a variety of nonprofessional mammalian phagocytes
(1, 8, 19). Work in our laboratories has shown that
internalization requires a specific interaction between fibronectin
binding protein and the host cell, presumably followed by signal
transduction events that ultimately lead to rearrangement of the host
cell cytoskeleton (4). We have also shown that
internalization by bovine mammary epithelial cells induces apoptosis
and that metabolically active intracellular S. aureus are
needed for apoptosis to occur (1, 20).
Assessment of caspase activity.
The induction of apoptosis,
also called programmed cell death, often progresses through an ordered
series of events involving a family of cysteine aspartyl proteases
known as caspases. This family of proteins is divided into two major
groups: initiator caspases, which are involved in triggering the
cascade of events leading to cell death, and effector caspases, which,
when activated by initiator caspases, catalyze the disassembly of cell
structures (17). The caspase cascade can be initiated in
response to the binding of an appropriate ligand to receptors on target
cells or through self-aggregation of receptors in response to their increased density on the cell surface. To determine whether caspases are involved in apoptosis induction, two major caspase pathways were
examined; the first is mediated through the most apical of the
initiator caspases, caspase 8 (10), and the second is
mediated through caspase 1. Either caspase 8 or 1 can activate caspase 3 and result in the cell disassembly associated with apoptosis.
Bovine mammary epithelial cells, designated MAC-T (9), were
infected with S. aureus (wild-type strain RN6390)
essentially as described previously (1, 20). After either 3 or 6 h of infection, MAC-T cell culture supernatants were
discarded, and the monolayers were washed three times with sterile
phosphate-buffered saline (PBS, pH 7.2). The monolayers were harvested
with sterile cell scrapers (Nalge Nunc International, Naperville,
Ill.), and cells were collected by centrifugation at 450 × g for 5 min at 4°C. Each cell pellet was washed once with
sterile PBS and resuspended in 10 ml of sterile PBS. Cells were counted
with a hemacytometer, pelleted again by centrifugation, and resuspended
to a concentration of 108 cells/ml in cell lysis buffer (25 mM HEPES [pH 7.5], 5 mM MgCl2, 5 mM EDTA, 5 mM
dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 10 µg of
leupeptin per ml; all reagents from Sigma, St. Louis, Mo.). Cells were
lysed by freezing and thawing four times, and lysates were clarified by
centrifugation at 16,000 × g and 4°C for 30 min. The
supernatants of the lysates were retained, and the protein
concentration of each was determined with the Bio-Rad protein assay
(Bio-Rad, Hercules, Calif.).
Caspase 8 (Fas-associated death domain-like
interleukin-1
-[IL-1
]-converting enzyme) activity was evaluated
with the caspase-8
fluorometric assay from R & D Systems (Minneapolis,
Minn.) according
to the manufacturer's protocol. Caspase 3 (32-kDa
cysteine protease;
also called CPP32) and caspase 1 (interleukin-1
-converting enzyme)
activities were assayed with the
fluorometric CaspACE assay system
from Promega (Madison, Wis.)
according to the supplier's recommendations.
Briefly, the assays
involved testing the MAC-T cell lysate for
caspase activity by the
addition of a caspase substrate coupled
to a reporter molecule
(7-amino-4-trifluoromethyl coumarin). Caspase
inhibitors were used to
confirm substrate cleavage specificity.
To determine whether the
caspase activity was derived from infected
MAC-T cells and not from a
product expressed by staphylococci,
filter-sterilized supernatants from
overnight
S. aureus cultures
were assayed for caspase
activity. No caspase 8, 1, or 3 activity
was detected (data not
shown).
As shown in Fig.
1A, caspase 8 activity
in MAC-T cells increased 4.4-fold after 6 h of infection (Fig.
1A). As a control,
the
agr virulence mutant RN6911, which
was previously shown to
be internalized but not to induce apoptosis
(
1,
20), did
not stimulate caspase 8 activity (unpublished
data). Based on
the activation of caspase 8 demonstrated in our system,
and because
caspase 8 can cleave caspase 3 but not caspase 1 (
11), we would
predict the activation of caspase 3 but not
caspase 1. Although
caspase 1 has been shown to be the target for the
induction of
apoptosis by
Shigella (
3) and
Salmonella (
7), we found only
minimal stimulation
of caspase 1 activity in MAC-T cells after
6 h of infection (Fig.
1A) and no caspase 1 activity at 3 h after
infection (data not
shown). Thus, unlike the situation in
Shigella and
Salmonella, data from this study indicate that the induction
of apoptosis by
S. aureus in our system does not involve
activation
of caspase 1. In contrast, internalized
S. aureus
caused a 6.6-fold
increase in caspase 3 activity by MAC-T cells
compared with the
uninfected control after 6 h of infection (Fig.
1A). As observed
in the caspase 8 assays, the
agr mutant did
not show increased
caspase 3 activity. It should be noted that the
numbers of intracellular
bacteria remain relatively constant over the
time period in which
caspase activity was assayed (
20),
indicating that the changes
in caspase activity observed are likely not
a function of alterations
in the number of intracellular bacteria
present.
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FIG. 1.
(A) Caspase 1, 8, and 3 activities in MAC-T cells
infected with S. aureus. MAC-T cells were harvested at
6 h after infection with S. aureus and evaluated for
caspase activity. The increase in caspase activity (fold) was
calculated by dividing the relative fluorescence value obtained from
analysis of infected cells by that of the uninfected control. The value
obtained from uninfected cells is represented as 1. In all experiments,
specific caspase inhibitors reduced the fluorescent signal to the level
of the uninfected control cells. Values from four (caspases 1 and 3)
and two (caspase 8) separate experiments were used to calculate the
mean relative activity (± standard errors of the mean). The
differences in caspase 3 and 8 activities observed in control and
infected MAC-T cells were analyzed by Student's t test and
found to be significant (P values of 0.03 and <0.001,
respectively). (B) Cytokine expression by MAC-T cells infected with
S. aureus. The increase in expression of cytokine transcript
levels (fold) was calculated by dividing the normalized S. aureus-induced cytokine value by the normalized cytokine value
from uninfected MAC-T cells. A value of 1 indicates basal transcript
levels. Values from four separate experiments were used to calculate
the mean relative expression (± standard errors of the mean). The
differences in IL-1 and TNF- transcript levels between the
control and infected MAC-T cells were statistically significant
(P = 0.07 and 0.02, respectively), while differences in
IL-6, IL-8, or TGF- transcript levels were not (P = 0.34, 0.27, and 0.54, respectively).
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|
The caspase 8 substrate used in this study has an absolute specificity
for caspase 8 (
18). The caspase 1 and 3 substrates
have
preferential but not absolute specificity (
16,
18). Since
caspase 8 is the most apical caspase in the initiation of a cascade
leading to activation of caspase 3 (
11), these data strongly
suggest that
S. aureus induces apoptosis in MAC-T cells
through
a mechanism involving caspases 8 and 3. This mechanism is
similar
to that induced by Sendai virus (
2) and by
Legionella pneumophila (
6) in
macrophages.
The precise pathways leading from caspase activation through DNA
fragmentation to apoptotic cell death are not fully characterized.
However, in apoptotic events involving caspases, the general sequence
of events involves an initial stimulus followed by activation
of
caspase and finally cleavage by activated caspase of protein
substrates
with apoptotic functions (
10,
15). The time required
for the
various events of apoptosis to occur depends on the type
of initial
stimulus and the type of cell (
10). To determine
the
correlation between the time of measurable caspase activation
and the
earliest detection of apoptotic DNA laddering in MAC-T
cells, we
infected MAC-T cells with
S. aureus RN6390 and assessed
DNA
laddering as described previously (
1). Samples for DNA
extraction were taken at various times postinfection. As shown
in Fig.
2, apoptotic laddering of MAC-T DNA in
increments of 180
bp was evident at 7 h postinfection. Since DNA
laddering is a
terminal event in apoptosis, this time is consistent
with the
observed prior onset of caspase activation (Fig.
1A).
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FIG. 2.
Agarose gel (1.8%) electrophoretic separation of DNA
extracted from infected MAC-T cells. Lane 1, 100-bp DNA marker ladder;
lanes 2 through 10, samples taken 5, 6, 7, 8, 9, 10, 11, 12, and
24 h after infection with S. aureus, respectively; lane
11, uninfected control MAC-T cells harvested after 24 h of
incubation.
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|
Quantitation of cytokine transcripts.
Since the
internalization of bacteria often leads to altered cytokine expression,
which, in turn, can initiate apoptosis, the expression of both pro- and
anti-inflammatory cytokines during the course of infection with
S. aureus in epithelial cell monolayers was also evaluated.
Moreover, the pathway initiated by caspase 8 is also known as "death
receptor-mediated," since binding of tumor necrosis factor (TNF) or
Fas ligand to their cell surface receptors can result in the
proteolytic activation of associated caspase 8 (10). Thus,
the induction of cytokines can play an important role in the induction
of apoptosis.
To examine cytokine expression in MAC-T cells infected with
S. aureus, a quantitative reverse transcriptase-PCR technique
was
used. Trizol reagent (Gibco BRL, Grand Island, N.Y.) was used
to
isolate RNA from MAC-T cell monolayers according to the manufacturer's
instructions. First-strand cDNA was prepared for the PCR with
5 µg of
total RNA and Superscript II reverse transcriptase (Gibco
BRL)
according to the manufacturer's procedures. All reagents
were prepared
using diethylpyrocarbonate (Sigma)-treated water
(0.01%, vol/vol).
Primers, designed from sequences in GenBank,
were used to amplify
bovine transcripts specific for IL-1
, IL-6,
IL-8, transforming
growth factor
1 (TGF-
1), TNF-
, and
-actin
(Table
1). The
-actin transcript was
coamplified and used as
an internal standard. A bulk PCR reagent
mixture was prepared
for each cytokine primer pair. The final
concentrations of reagents
in 50 µl of PCR mixture were 1× PCR
buffer (Gibco BRL), 2.5 mM
MgCl
2, deoxynucleoside
triphosphates (5 mM each A, T, C, and G),
0.3 µM
-actin primer
pair, 0.3 µM cytokine primer pair, and 0.5
U of
Taq DNA
polymerase (Gibco BRL). Forty-nine microliters of
the bulk mixture was
added to 1.0 µl of cDNA solution and overlaid
with mineral oil. PCR
conditions consisted of an initial 95°C
denaturation for 4 min
followed by repeated cycles of 94°C for
30 s, 58°C for 30 s, and 72°C for 30 s, and ended with a terminal
extension step
of 72°C for 4 min. The total number of cycles varied
from 28 to 39 depending on the cytokine transcript being amplified.
Aliquots (10 µl) of each PCR mixture were loaded on a 2% agarose
gel
(Tris-borate-EDTA buffer) containing ethidium bromide (0.3
µg/ml),
and the coamplified cytokine and
-actin products were
separated by
electrophoresis. PCR products were detected by UV
fluorescence, and
fluorescent band intensity (pixel density units)
was measured using the
Multi-Analyst program from Bio-Rad. Cytokine
values were normalized by
dividing by the coamplified
-actin
value.
In this study, internalization of
S. aureus consistently
increased the transcript levels of the proinflammatory cytokines
TNF-
and IL-1
(1.68- and 1.59-fold, respectively) (Fig.
1B)
compared with the levels in the uninfected controls. Viable
staphylococci
were required to induce cytokine expression, because
equivalent
numbers of UV-killed RN6390 cells were unable to elevate any
cytokine
transcript accumulation above basal levels. Although it is
currently
unknown whether activation of caspases 8 and 3 occurs through
signaling by TNF-
, the observation that TNF-
transcripts
accumulate
in cells infected with
S. aureus makes this an
intriguing possibility.
After infection with
S. aureus,
transcripts for the proinflammatory
cytokine IL-6 were slightly
elevated (1.26-fold increase), whereas
levels of transcripts encoding
the anti-inflammatory cytokines
IL-8 and TGF-
were similar to those
in the uninfected controls
(Fig.
1B).
The results of this analysis suggest that infection of MAC-T cells with
S. aureus causes a significant increase in expression
of the
proinflammatory cytokines TNF-
and IL-1
(and possibly
IL-6),
while expression of the anti-inflammatory cytokines IL-8
and TGF-
appears to be unaffected. The effects of other pathogens
on cytokine
expression in response to internalization have been
well documented
and, in some cases, characterized.
Shigella flexneri causes
release of IL-1
through an interaction with caspase 1
to initiate an
inflammatory response (
3). In contrast,
Yersinia spp. inhibit production of TNF-
to suppress inflammation
(
12).
Interestingly, strains of
S. aureus are
variable in their ability
to induce inflammation. For example,
S. aureus is typically considered
to be pyogenic, and many infections
with
S. aureus increase the
level of proinflammatory
cytokines in different cell types (
13,
21). However, the
lack of an inflammatory response frequently
associated with certain
types of
S. aureus infections, such as
subclinical bovine
mastitis (
14) and human toxic shock syndrome
(
5),
might suggest that mechanisms to suppress the inflammatory
response
also exist. Work is currently in progress to determine
whether certain
isolates differ in their ability to induce apoptosis
and inflammatory
cytokines and to characterize the staphylococcal
factor(s) responsible
for the induction of these
events.
| |
ACKNOWLEDGMENTS |
Grant support was received from National Institutes of Health grant
R29-AI38901 (K.W.B.), National Science Foundation-Idaho Experimental
Program to Stimulate Competitive Research grant EPS-9720634 (K.W.B.),
National Research Initiative Competitive Grants Program U.S. Department
of Agriculture grant 9402399 (G.A.B.), Public Health Service grant
AI28401 (G.A.B.), and the United Dairymen of Idaho (G.A.B.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844-3052. Phone: (208) 885-7164. Fax: (208)
885-6518. E-mail: kbayles{at}uidaho.edu.
Editor:
E. I. Tuomanen
| |
REFERENCES |
| 1.
|
Bayles, K. W.,
C. A. Wesson,
L. E. Liou,
L. K. Fox,
G. A. Bohach, and W. R. Trumble.
1998.
Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis.
Infect. Immun.
66:336-342[Abstract/Free Full Text].
|
| 2.
|
Bitzer, M.,
F. Prinz,
M. Bauer,
M. Spiegel,
W. J. Neubert,
M. Gregor,
K. Schulze-Osthoff, and U. Lauer.
1999.
Sendai virus infection induces apoptosis through activation of caspase-8 (FLICE) and caspase-3 (CPP32).
J. Virol.
73:702-708[Abstract/Free Full Text].
|
| 3.
|
Chen, Y.,
M. R. Smith,
K. Thirumalai, and A. Zychlinsky.
1996.
A bacterial invasin induces macrophage apoptosis by binding directly to ICE.
EMBO J.
15:3853-3860[Medline].
|
| 4.
|
Dziewanowska, K.,
J. M. Patti,
C. F. Deobald,
K. W. Bayles,
W. R. Trumble, and G. A. Bohach.
1999.
Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells.
Infect. Immun.
67:4673-4678[Abstract/Free Full Text].
|
| 5.
|
Fast, D. J.,
P. M. Schlievert, and R. D. Nelson.
1988.
Nonpurulent response to toxic shock syndrome toxin 1-producing Staphylococcus aureus. Relationship to toxin-stimulated production of tumor necrosis factor.
J. Immunol.
140:949-953[Abstract].
|
| 6.
|
Gao, L. Y., and Y. Abu Kwaik.
1999.
Activation of caspase 3 during Legionella pneumophila-induced apoptosis.
Infect. Immun.
67:4886-4894[Abstract/Free Full Text].
|
| 7.
|
Hersh, D.,
D. M. Monack,
M. R. Smith,
N. Ghori,
S. Falkow, and A. Zychlinsky.
1999.
The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1.
Proc. Natl. Acad. Sci. USA
96:2396-2401[Abstract/Free Full Text].
|
| 8.
|
Hudson, M. C.,
W. K. Ramp,
N. C. Nicholson,
A. S. Williams, and M. T. Nousiainen.
1995.
Internalization of Staphylococcus aureus by cultured osteoblasts.
Microb. Pathog.
19:409-419[CrossRef][Medline].
|
| 9.
|
Hyunh, H. T.,
G. Robitaille, and J. D. Turner.
1991.
Establishment of bovine mammary epithelial cells (MAC-T): an in vivo model for bovine lactation.
Exp. Cell Res.
197:191-199[CrossRef][Medline].
|
| 10.
|
Kidd, V. J.
1998.
Proteolytic activities that mediate apoptosis.
Annu. Rev. Physiol.
60:533-573[CrossRef][Medline].
|
| 11.
|
Muzio, M.,
G. S. Salvesen, and V. M. Dixit.
1997.
FLICE induced apoptosis in a cell-free system: cleavage of caspase zymogens.
J. Biol. Chem.
272:2952-2956[Abstract/Free Full Text].
|
| 12.
|
Palmer, L. E.,
S. Hobbie,
J. E. Galan, and J. B. Bliska.
1998.
YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNF- production and downregulation of the MAP kinases p38 and JNK.
Mol. Microbiol.
27:953-965[CrossRef][Medline].
|
| 13.
|
Persson-Waller, K.
1997.
Accumulation of leucocytes and cytokines in the lactating ovine udder during mastitis due to Staphylococcus aureus and Escherichia coli.
Res. Vet. Sci.
62:63-66[CrossRef][Medline].
|
| 14.
|
Pyorala, S.
1995.
Staphylococcal and streptococcal mastitis, p. 143-148.
In
M. Sandholm, T. Honkanen-Buzalski, L. Kaartineu, and S. Pyoral (ed.), The bovine udder and mastitis. University of Helsinki, Helsinki, Finland.
|
| 15.
|
Schulze-Osthoff, K.,
D. Ferrari,
M. Los,
S. Wesselborg, and M. E. Peter.
1998.
Apoptosis signaling by death receptors.
Eur. J. Biochem.
254:439-459[Medline].
|
| 16.
|
Talanian, R. V.,
C. Quinlan,
S. Trautz,
M. C. Hackett,
J. A. Mankovich,
D. Banach,
T. Ghayur,
K. D. Brady, and W. W. Wong.
1997.
Substrate specificities of caspase family proteases.
J. Biol. Chem.
272:9677-9682[Abstract/Free Full Text].
|
| 17.
|
Thornberry, N. A., and Y. Lazebnik.
1998.
Caspases: enemies within.
Science
281:1312-1316[Abstract/Free Full Text].
|
| 18.
|
Thornberry, N. A.,
T. A. Rano,
E. P. Peterson,
D. M. Rasper,
T. Timkey,
M. Garcia-Calvo,
V. M. Houtzager,
P. A. Nordstrom,
S. Roy,
J. P. Vaillancourt,
K. T. Chapman, and D. W. Nicholson.
1997.
A combinatorial approach defines specificities of members of the caspase family and granzyme B: functional relationships established for key mediators of apoptosis.
J. Biol. Chem.
272:17907-17911[Abstract/Free Full Text].
|
| 19.
|
Vann, J. M., and R. A. Proctor.
1987.
Ingestion of Staphylococcus aureus by bovine endothelial cells results in time- and dose-dependent damage to endothelial cell monolayers.
Infect. Immun.
55:2155-2163[Abstract/Free Full Text].
|
| 20.
|
Wesson, C. A.,
L. E. Liou,
K. M. Todd,
G. A. Bohach,
W. R. Trumble, and K. W. Bayles.
1998.
The Staphylococcus aureus Agr and Sar global regulators influence internalization and induction of apoptosis.
Infect. Immun.
66:5238-5243[Abstract/Free Full Text].
|
| 21.
|
Yao, L.,
V. Bengualid,
F. D. Lowy,
J. J. Gibbons,
V. B. Hatcher, and J. W. Berman.
1995.
Internalization of Staphylococcus aureus by endothelial cells induces cytokine gene expression.
Infect. Immun.
63:1835-1839[Abstract].
|
Infection and Immunity, May 2000, p. 2998-3001, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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