Department of 1Microbiology and 2 First Department of Internal Medicine, Kumamoto University School of Medicine,. Kumamoto, Kumamoto 860, Japan.
Microbiol.Immunol.,40(6), 415-423, 1996
Bradykinin Proteases Circulation
Generation Triggered by Pseudomonas Facilitates Invasion of the Systemic by Pseudomonas aeruginosa
Yoshifumi Sakata1,2, Takaaki and Hiroshi Maeda*1
Akaike1,
Moritaka
Suga2,
Sumiko
Ijiri1, Masayuki
Ando2,
Department of 1Microbiologyand 2First Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto,Kumamoto 860, Japan Received
January
25, 1996.
Accepted
March
13, 1996
Abstract: To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo. Key words:
Pseudomonas
protease,
Bradykinin,
Septicemia
seems possiblethat bacterial proteases facilitatebacterial invasion of the vascular system through potent tissue destruction(9, 17, 20, 25). However,the detailedmechanism of bacterial invasion of the vasculature and entry into the blood circulation remains unclear. We have previously demonstrated that a number of microbial proteases from pathogenic bacteria and fungi activate the bradykinin-generating cascade at one or more steps, i.e., Hageman factor, prekallikrein, and/or high-molecular-weight kininogen (11, 12, 17-19, 21, 24). Thus, bradykinin generation is stimulatedby these bacterial proteases during the infection and acts as a universal mediator in an inflammatory reaction, producing pain, edema formation, and relaxationof vascu-
Pseudomonas aeruginosa is now widely recognized as an important opportunistic pathogen often causing infections complicated by septicemia and sepsis syndrome (3), a serious medical condition in which the host's immune system overreacts to the infection (6). Septicemia is one of the most critical consequences of systemic bacterial infection (27). Most frequently, gram-negative bacilli gain access to the blood from extravascular septic foci via the lymphatics or by the direct invasion of small blood vessels within a local site of infection (16). P. aeruginosa produces a number of extracellular products: exotoxin A, exoenzyme S, and extracellular proteases such as pseudomonal elastase and alkaline protease (5). Accumulating evidence indicates that bacterial extracellular proteases play a critical role in the pathogenesis of various bacterial infections (17). It
Abbreviations: DTPA,
*Address correspondence to Dr. H. Maeda, Department of Microbiology, Kumamoto University School of Medicine, 2-21, Honjo, Kumamoto, Kumamoto 860, Japan. 415
BK, bradykinin;
CFU,
diethylenetriaminepentaacetic
aminetetraacetic
acid;
charide;
ovoM,
ovomacroglobulin;
buffered
saline,
colony
acid;
LD,0, 50% lethal
dose; LPS, PBS,
pH. 7.3; PE, pseudomonal
forming
EDTA,
unit;
ethylenedilipopolysac-
10 mm phosphateelastase.
416
Y. SAKATA
lar smooth muscle. Recent investigations revealed that bradykinin generated by either pseudomonal elastase or lipopolysaccharide (LPS; endotoxin) plays an important role in the pathogenesis of septic shock (13, 14). In this experiment, we investigated the role of the pseudomonal elastase and alkaline protease in triggering septicemia caused by P aeruginosa, in view of the in vivo action of bradykinin. The present results indicate that pseudomonal extracellular protease facilitates septicemia as well as toxemia through activation of the bradykinin-generating cascade. Materials
and Methods
ET AL
previously (30). Briefly, various concentrations of Pseudomonas proteases such as Pseudomonas elastase and alkalineproteasewas incubatedin the reactionmixture containing2.5 mg/mlof azocasein(AE:Z=30.1) in 40 mm sodium phosphate buffer (pH 7.0) at 37 C for 30 min. Each protease activity was expressed as unit defined as the activity which hydrolyzes 1.0 mg of azocasein/hr. In some experiments, the proteases were preincubated with two types of inhibitors for Pseudomonasproteases,e.g., ovoM and Zinkovinhibitor, at molar ratios of 1:2 to 1:10 (protease vs. inhibitors)at 37 C for 1 hr, followed by protease assay as just described. Bacteria
Animals. Male ddY mice (SPF grade, 6 weeks old, Japan SLC, Shizuoka) were used throughout the study. Pseudomonal elastase and alkaline protease and pseudomonal toxins. Purified P. aeruginosa elastase (39 kDa) and alkalineprotease (48 kDa) were purchased from Nagase Biochemicals, Osaka, Japan. LPS from P aeruginosa serotype 10 (Habs) was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. P aeruginosa exotoxin A was a kind gift from Dr. Hiroshi No2uchi, Sumitomo Seiyaku Co., Ltd., Osaka, Japan. Oth(7rrea8ents.
Bradykinin
was purchased
from Pep-
tide Institute Inc., Osaka. Captopril and Glu-Trp-ProArg-Pro-Glu-Ile-Pro-OH(SQ20881) were a gift from SankyoCo., Ltd.,Tokyo,and Squibb Institute,Princeton, N.J., U.S.A., respectively. DL-2-Mercaptomethy1-3guanidinoethylthiopropanoicacid (Plummer's inhibitor) was purchasedfrom Calbiochem,La Jolla, Calif.,U.S.A. Diethylenetriaminepentaaceticacid (DTPA) anhydride was obtained from Dojindo Laboratories, Kumamoto. 5℃rCl
3waspurchasedfrom
ICN Radiochemicals,
Costa
Mesa, Calif., U.S.A. Chicken egg white ovomacroglobulin(ovoM) was a gift from Otsuka Pharmaceutical Co., Tokushima. 2-(N-Hydroxycarboxamido)-4methylpentanoyl-L-alanyl-glycine amide (Zincov inhibitor) was kindly supplied by Dr. NorikazuNishino, Kyushu Instituteof Technology,Kitakyushu. The Toxicolor limulus test, a sensitive quantitative assay kit for bacterial endotoxin, was purchased from Seikagaku Kogyo, Tokyo. An enzyme immunoassay kit for bradykinin, MARKIT M, which was developed recently by Prof.Makoto Katori,Kitasato UniversitySchool of Medicine, Kanagawa, and is 100 times more sensitive than a previous immunoassay kit (MARKIT A), was suppliedfrom DainipponPharmaceuticals,Osaka,Japan. All other chemicals were purchased from local commercial sources. Quantification of protease activity. Protease activity of Pseudomonas proteases was measured by using azocasein (Sigma Chemicals) as a substrate as was reported
nosa
and
were
culture.
used.
isolated
from
Two
One an
within
3
days
large
amount
tease).
PA
nosa
that
was
septic
from
the
of
produces
very
at 33
washed
suspended (i.p.)
in
to
to
kaguma
supernatant
tered
with
volume
by
Danver,
Mass.,
centrated based
on
its
used
0.58
in
Zincov
was inhibitor
The the
which
result
indicated
concentrated
to
in
not that culture
at
81%
size).
be
582.6
of
supernatant
con-
azocasein and
as
alkaline units/rig
respectively.
concentrated
presence
or
culture absence
of
(almost
ratio
of
protease of
90%)
1:10
alkaline the
Inc., the
units/ml
1.25
inhibited a molar
The
one-fifth
of
activity,
Pseudomonas
fil-
(Amicon
showed
the
cen-
was
to
elastase
the
The
by and
pore
against
in
was
621.
activity
found
strongly
elastase but
4 C)
membrane protease
activity
PA
obtained
at
caseinolytic
quantified
pseudomonal
tease:inhibitor)
10
strain strain
concentrated
experiment
of
of
was min
activity
protease
supernatant
that
pseudomonal
this
culture
pseudomon-
kaguma
(0.22-vm
was
units/ƒÊg
Furthermore,
30
The
The
of
kaguma
strain
then
YM
proteolytic
above.
protease
the
a
U.S.A.).
supernatant
described
and
using
bacteria
to obtain
amount
to
for
was
col-
at 4 C)
phosphateThe
P. aeruginosa
filter
supernatant
were
intraperitoneally
aeruginosa
this
a Millipore
resultant
621
Detroit,
min
M
7.3).
mainly
similar
g
PA
below.
P
of
Strain
dissemination.
used
of
(20,000 •~
P. aerugi-
30
0.01
given
described
supernatant
trifugation
a
of
bacteria
(pH
a high
a manner
death
pro-
(Difco,
for
in
was
activity.
in
The
then
supernatant
cultured
hr.
intravascular
contained
its protease
culture
were
as
Culture
broth
(PBS)
strain
al exoproteases
and
16
times
allow
that
exoprotease.
(20,000 •~g
PBS
mice,
The
C for
M saline
in produces
strain
infusion
three
buffered/0.15
septicemia,
strain
isolated
little
centrifugation
had
(elastase/alkaline
in brain-heart
by
and
exoproteases
was
who
resulted
This
aerugi-
strain,
severe
that
onset.
is a clinically
U.S.A.)
lected
with
shock
621
cultured
Mich.,
and
of P
kaguma patient
pneumonia
emboli,
strains
the
immunocompetent
community-acquired septic
different
strain,
(pro-
protease. activity
kaguma
of strain
BACTERIAL
was
attributed
residual
to
fraction
dent
on
the
of
i.p.
with
P.
P.
aliquot
ing
(100 ƒÊg
kininase bacteria,
ture,
and
was
bers
in
es,
after
used
of
a
the that
selective
medium
ide
(NAC
The growth
of
pseudomonal
elastase
on
of P
aeruginosa
Specifically, with
or
into
without
observed In
24
a separate
aeruginosa
2.0 ture with the the
mg/ml
for
with
elastase)
supernatant these spread
of
both
suppressed
P
of
bacteria
elastase almost
Zinkov alkaline
or
of of
at 5.0 and
1 hr
aeruginosa then
into
the
in
P.
the
inhibitor
culture
protease
and
its
only
treated
supernatant
of by
on
partially
621. kagu-
treatment
However,
ovoM (at
maxi-
of
the
to
in
this
were
assay
quantitated
M (19).
but
not
thus, kinin
analysis.
punc-
min
after
and
concen-
centrifugation supernatant
kinins
with to
was
the
the
enzyme
instructions and
kallidin
des-Arg-bradykinin,
the
the
depro-
(final
bradykinin
to
only
inhibitor
immediate
according Both
lavage
ethylenedi-
cardiac
15
resulting
immunoaffinity
in our
Statistical
by
samples
MARKIT
kit;
by and
acid
of
of
Peritoneal
0, 5,
quantitation
assay
administration
obtained
The
blood
a kininase
followed
of
enzyme
1 mM
and
stimulation
Generation circulating
mice.
(EDTA)
the
manufacturer
similar
and
containing
min).
(Lys-bradykinin), quite
and
PA
inhibited ovoM.
cul-
circulation
subjected
10
by
i.p.
to
determined counter.
vivo.
trichloroacetic
to
for
Fifteen
sensitive
lavage,
"Cr-
physiological
a gamma
after
elastase,
6%) g
corn-
A was
in
(50 ƒÊg)
adding
immunoassay at
effect
of
(10,000 •~ then
both
The
strain
by
sodium
(200
cavity
samples
of
generated
PBS
peritoneal
teinization
on
radioactive
exotoxin
a highly
acid
of
band
A
200 ƒÊl
with
M)
with
Blood
tration
inhibitors for
aeruginosa
completely
was
of
using
elastase
without
furThe
electrophoresis
bradykinin
(MARKIT
administration
inhibitor
for
systemic
ture,
of P
C in PBS.
tested
cocktail.
was
chelation.
the
in
peritoneal
by
performed
or
with
bradykinin
the
pseudomonal
P
bacteria.
mg/ml
kaguma
was
by
the
in
kinins
i.p.
exoproteases
examined
separated Uppsala,
exotoxin
radioactivity of
by
8.5),
detection.
amount
the
then
conjugate
gel
without the
aminetetraacetic
injected mice
the
Zincov
at 37
of
or
later,
with (pH
a homogeneous
original
(540 ƒÊg)
pseudomonal
was
0.5 •~
supernatant with
i.p.
LIDO
was the
A
Quantitation
for
inves-
or
0.4 •~
LD50)
culture
(ovoM
induced
was
CFU,
rate
treated
elastase, for
inhibitors
lethality
strain
was
by also
supernatant [El,
0.2 •~
the
proteases and
was
administration
strain
protease
10
survival
after
kaguma
Pseudomonas ma
hr
(4 •~ culture
(50 ƒÊg, The
621
injected
bradykinin
were
the
were
counting
was
showed
A
labeled
buffer
"Cr
autoradiographic
with
with
purified
produced
PA
(100
experiment,
of pseudomonal alkaline
621
either
mice.
for
agar
and
lethality
strain
elastase the
the
PA
kaguma
pseudomonal i.p.
effective
NAC
proteases
administration
aeruginosa
the
by
(7).
of
exotoxin
minutes
was
pseudomonal
LD,o)
cetrim-
Tokyo)
showed
in
saline a
aeruginosa.
effect
tigated.
that
appearance
acid
Co.,
with
Mice
agar
that
A
was
Pharmacia,
sulfate-polyacrylamide
labeled
onto
exotoxin
serum.
conjugated
A
radioactive
the exotoxin
was
DTPA-exotoxin
after assay
in
A
A conjugate
with
to
bined
that
soy
colony
nalidixic
agar
dodecyl
dish-
The
LPS
bicarbonate
G-25,
the
min
a commercial of
A
into
30
pseudomonal
(Sephadex
kaguma
administered
exotoxin
mM
DTPA-exotoxin filtration
-labeled
similar
of
10
of
aeruginosa,
was
by
exotoxin in
P
obtained
quantitation
spread
protease
toxins from
(200 ƒÊg),
circulation,
Briefly,
labeled
and
to
tryptic
Chemical
soy
Petri
the gel
rub-
Species
isolated
aeruginosa,
tryptic
in similar
on
the
(Eiken
and
performed.
colonies
P
a typical
as P
The
for
on
and
counted
was
inoculating
agar)
colonies
saline
soy
rinsing
and
ther "Cr
the
anhydride
Sweden).
the
mesh
"Cr.
DTPA
by
was
tryptic
by
sterile
assay
sample
formed by
using
followed
colony-forming
of punc-
blood
experiments,
physiological
blood
identified
agar
assay
with a
for systemic
bacterial
analyzed
for
was
the
treatment.
obtained
samples
were
inhibitor
of
supernatant
of
blood
test)
vs.
10%
inhibitor
bradykinin
LPS
test the
culture Zinkov
(1.0 ƒÊg)
and
of
To
as
injection
in the
without mice,
protease
about
dissemination
(Toxicolor
into
Plum-
cardiac
some
with
kit
into
to
after by
bacteria
surgically
and
referred
taken
In
sterile
which
bacteria
were
viable
grinding
i.p.
or to
thus
the
and
LPS
injection or
(kinin-degrad-
hours
was
with
purified
injected
for
of the
and
in
ovoM
Assay
ratio
1:10,
remains after
i.p.
if the
to
bloodstream.
of
PBS),
SQ20881,
a colony-forming
by
with
of
of
removed
1 ml
were
Three
Laboratories).
homogenization
200 ƒÊl),
henceforth
sample
107 exo-
together
200 ƒÊl
even up
strain
supernatant
kininase
captopril,
cocktail.
by
(Difco
spleen
of
number
quantitated agar
PBS)
a mixture
a blood the
or in
in 200 ƒÊl
(5 •~ little
bacteria
(100
(100 ƒÊg),
inhibitor
the
20%)
increased
inject-
417
SEPTICEMIA
mum,
depen-
were 621
culture
(50 ƒÊg
inhibitors:
inhibitor
other
be
produced
strain
strain
or without
enzyme)
mer's
621
elastase
with
PA
which
concentrated
kaguma
bradykinin mice
the
Mice
strain
PBS), PA
of
aeruginosa
the
to
AND
activity
aeruginosa
pseudomonal
and
thought
dissemination.
Similarly,
an
was
protease.
200 ƒÊl
protease.
elastase
activity
bacterial
CFU/mouse;
with
the
alkaline
Analysis ed
Pseudomonas of
PROTEASE
show
antikinin
antibody
B2-receptor
used
agonists
are
assay. All
values
±SE
of three
or four samples.
mice
treated
with
P.
are
expressed
as
The survival
aeruginosa
pseudomonal proteases were evaluated Fisher's exact probability test.
with
means
rates of
or without
statistically
by
418
SAKATA
Fig.
1.
Effect
of
culture
supernatant
protease-producing mouse), P
strain
which
ture;
spleen
use
not
treated
the
colony-forming
of
was
also
proteases
P.
aeruginosa
PA
obtained
i.p. and
and
protease
to
mice.
then
621.
Mice
was The
with
Three
hours The
soy
with
of
agar.
viable
PI,
of
aeruginosa i.p.
200111
of
culture 621
in in
for
the
aeruginosa
PA
the
and
the
the
taken
by
treated
text
for
10
nonCFU/ of
cardiac
with
punc-
inhibitors
of
homogenate
See
the
supernatant
administration
spleen
proteases.
was
strain
to
(5 •~
culture
sample
kaguma
of
621
concentrated
similar
blood
dissemination
a blood
of
pseudomonal
intravascular
10-fold
bacteria,
a manner
the
P.
of the
supernatant
PA
on
with
sample
injection
bacteria
inhibitors
P
injected
or
after
administered numbers
tryptic
strain
were
a 100-
homogenized.
ovoM)
inhibitor.
assay
kaguma
Simultaneously,
given
(Zincov
with
protease-producing
exoprotease.
strain
was
pseudomonal natant
little
kaguma
the
of
produced
aeruginosa
of the
ET AL
culture
of super-
were
quantitated
on
aeruginosa
by
details.
Results
Enhancement of Bacteremia by Pseudomonal Proteases and Involvement of Bradykinin P.
aeruginosa
increase of
in
culture
With
the
200-ƒÊl
was
of
supernatant
of
P
a
dose-dependent
the
after
kaguma
in
the
into
the
blood
and
of
the
supernatant
10-fold
effect
strain PA
1).
dissemi-
and
This
kaguma
aeruginosa
(Fig.
such
blood
controls.
injection
strain
supernatant,
100-fold with of
of cavity
the
injection
compared
semination toneal
showed dissemination
increased
spleen
ture
621
supernatant
the
nation
PA intravascular
of
in
the
cul-
in promoting
621
(from
spleen)
was
dis-
the
peri-
abrogated
by
Fig.
2.
621
bacteremia.
Effect
purified
preincubation of
pseudomonal
the
proteases
values
without
respectively
(Fig.
the
the
substantial
due
event,
these in
facilitated
the to the
the
confirm on
culture
621
the
was in
the
of
the
in Fig.
effectively
bacterial
protease.
of
the
enhancing
effect
i.p. manner
colony
formation
puncture
of
is In
any
protease kaguma
together as
in
P 1.
(50 ƒÊg) (KI)
aeruginosa
after text
cells with
was in
in
P.
PA with
presence
or absence
same
manner
quantitated
blood
of
P
administered the
the
were
the
injection for
(PE)
621
cocktail
sample
as in
by
use
the
Fig.
of
obtained
by
aeruginosa
and
pseudomonal
exhibited
potent
the
cardiac
details.
2, pseudomonal
In
pro-
shown
the
addition,
test
this
ment
inhibitors)
from in
was
the
2),
generated
by
cavity i.p.
injection
inhibitor
vs.
by
cocktail by
elastase
indicating
into
P the
of
the
blood
involveelastase.
aeruginosa
bradykinin
a t-
with
pseudomonal of
blood.
enhanced
determined
alone
strongly
augmeninto
was
0.025
dissemination
peritoneal
increased
aeruginosa
a kininase
(PC
elastase
(Fig.
bradykinin
of
elastase data,
P
dissemination
injection
the
unpaired
of
of
increased
by with
for
elastase
dissemination
Furthermore,
aeruginosa As
of
significantly
pseudomon-
with Fig.
PA
elastase
assay
3 hr
together
strain
pseudomonal
purified
P
See
tation
ninase
bacteremia,
inhibitor
viable
Fig.
inhibitors
pseudomonal
supernatant
pseudomonal
1. The
elastase
aeruginosa
inhibit
translocation
alkaline
P
a kininase
elastase.
Therefore,
protease
that
1,
primarily
above.
the
spleen,
dissemination.
injected same
and shown
as
of
one-seventh
inhibited not
in
suggest
Pseudomonas
al elastase
did
pseudomonal
results
blood
described
presence
bacterial
To
but as
inhibitors
and
experiment
increment
in
mostly
the
the
used
protease
observed
tease
In
elastase,
alkaline
PA
1).
specific
one-tenth in
inhibitors
the
activity
to
inhibitors
protease
Pseudomonas
with
of pseudomonal
PA621 and
spleen (100 ƒÊg)
ki-
BACTERIAL
PROTEASE
AND
419
SEPTICEMIA
A
Fig.
4.
Kinin
generation
pseudomonal
B
blood
elastase
was
measured
at various
time
PBS)
given
was
Fig.
3.
Mice
Acceleration were
absence
of an
aeruginosa
with
authentic
assay of
in
the
was
the
the
bacteria
of and
of P
to
kininase
the
kininase
the
inhibitor
Effect
as
in
cocktail.
The
A,
in
See
the
text
and
with
the
time
3).
This
promoting
simultaneous cocktail,
which
resulted
in
peritoneal In i.p.
of
the
details.
cavity addition,
kinins.
kinins
by
kinin blood
was
contrast,
lavage In
fluid
a separate
ty was
in
found
circulation.
4, an the
However, that
were enzyme
experiment, peritoneal
vivo
by not
mice
within
15
endogenous in
the (•ƒ0.1
of
the
of
given
profile
of in
min
the
after
kininases.
kinin-degrading
cavity
mice
generated
immunoassay strong
after
to
time
kinin
detected
the
amount
of the
the
in
immunoassay
blood
rapidly,
kinins
in
exoprotease
appreciable
possibly
in the
blood
and
kinins
bradykinin
enzyme
decomposed
our
of
Fig. in
by
bradykinin
a specific
showed
injection,
the
peritoneal ng/ml). activi-
mice
PBS, for
(50 ƒÊg
of
a vehicle
was the
mice,
given
i.p.
in
for
and
elastase-treated
kinins 100
Ill the
kinins
in
group.
See
of
pseudomonal i.p.
amount
of
quantitated
pseudomonal
by the counter.
LPS
to
LPS use
mice and of
text
with A
of for
by
'Cr-labeled
in
commercial
radioactivity See
or
together exotoxin
a
toxemia
'Cr-labeled
bradykinin
exotoxin bradykinin
the
blood
assay
kit
A
was
(200 ƒÊg). circulation
for
exotoxin
LPS
was and
A
with
by a
details.
by
inhibitor
action
pseudomonal
elastase.
was
in of
produced
generation
elastase
even
using
shown
pseudomonal
In
and
of
As
of
biological
generation
enhanced
kininase
Enhancement Either
counting
further
the
degradation
prolonged
examined
for
was of
inhibited
administration
was
effect
administration
ill
for
elastase
the
details.
administered The
bradykinin
absence
(BK).
gamma
(Fig.
immunoassay
group
as
in
on
(KI)
or
5.
100
quantitated
of
generated
for-
examined
on
bacteria
Fig.
control
administration
injec-
inhibitors
presence for
colony
was
bradykinin
for
enzyme
pseudomonal
In the
elastase, were
an
i.p. of kinins
of P
3 hr after
cocktail
of
text
using
after
after
amount
or
number
kininase
blood
i.p.
mice
The
(BK). presence
the
at
bacteremia
bacteremia.
mice,
of
1,
inhibitor
B.
the
The
use
Fig.
of of
bradykinin.
aeruginosa
given
effect
potentiation
administration
in
in
A.
by as
bradykinin
621
(100 ƒÊg).
manner The
by
PA
quantitated
same
bacteria.
by
profile
induced
aeruginosa
bradykinin
bradykinin-induced
were
P
organisms
mation tion
of bacteremia
injected
the
by
points
pseudomonal
in
(PE).
(data
not
shown). Moreover,as described below, various materials such as bacterial toxin and LPS as well as bacterial cells seem to be deliveredquicklyto the blood circulation after simultaneous i.p. administration of bradykinin. Rapid clearance appears to occur in the case of kinins generated de novo in the peritoneal cavity of the mice. Thus, failureto detect kinins in peritoneallavagefluid of mice injected with the pseudomonal elastase is most likely due to the rapid degradation and clearance of kinins from the peritoneal cavity. All these results indicate that Pseudomonas exoproteases significantlyenhancebacterialdisseminationfrom local bacterialfoci to the systemic circulation,at least in part through bradykinin-generatingactivity. In fact, the lethal effect of PA 621 was markedly augmentedwhen this strain was administeredi.p. to the mice together with either the culture supernatantof the
420
Y. SAKATA
Table 1. Lethality natant
Fig. 6. A schematic sible involvement
induced
of P aeruginosa
representation
by P aeruginosa
kaguma
of the mechanism
of the bradykinin-generating
kaguma strain or the purified Pseudomonas (Table 1).
PA 621 in mice enhanced
by culture
super-
strain
of bacteremia
cascade
ET AL
activated
elastase
Enhancement of Pseudomonal Toxemia by Bradykinin To detail the action of bradykinin in Pseudomonas bacteremia, the effect of bradykinin on the movement of
and bacterial by bacterial
toxemia proteases
mediated
by bacterial
in the spread
proteases.
of bacterial
Note the pos-
cells and toxins.
pseudomonal endotoxin and exotoxin, such as LPS and Pseudomonas exotoxin A, from the peritoneal cavity into the blood circulationwas investigated.The delivery of LPS and 'Cr-labeled exotoxin A was significantly enhanced when these toxins were given to the mice with the bradykinin (Fig. 5); the blood concentration
BACTERIAL of LPS was
increased
4-fold,
and that
showed an almost 2-fold increase. These data suggest that bradykinin in the septic foci, which
could
of exotoxin
generated
be stimulated
al proteases, enhanced not only bacteremia toxemia of P aeruginosa.
PROTEASE A
de novo
by bacteribut also the
Discussion
It is now well known that the virulent P. aeruginosa produces a number of extracellular toxic factors, e.g., exotoxinA, exoenzymeS, elastase,and alkalineprotease (5). Much attention has been paid to the important role of pseudomonal proteases (elastase and alkaline protease) in the pathogenesisof P aeruginosa infection (2, 9, 10, 14, 15, 17, 22-25). In particular, we have been studying the pathogenic potential of pseudomonal proteases and focusing on their potent stimulatory activity for bradykinin generation (17, 24). We previously reported that extracellular proteases from various microbes includingP aeruginosa activate the bradykinin-generatingcascade at several steps in vitro (12, 17, 21) and cause increased vascular permeabilityin vivo(10, 11, 17, 24). Both pseudomonal elastase and alkalineprotease proteolyticallyactivate Hageman factor and also act directly on high-molecularweight kininogens, which is followed by generation of bradykinin (17, 24). Thus, generation of kinins triggered by bacterialproteasesin the septic foci may be one of the most critical reactions in the host infected with pathogenicbacteria. The results obtained in this experiment make it clear that extracellular proteases of P. aeruginosa and bradykinin, which is produced endogenously via activation of the kinin-generatingcascade by pseudomonal protease or exogenously administered to mice, facilitated the bacteremia as well as toxemia caused by P aeruginosa. In addition, kinin-potentiating kininase inhibitors augmentedsuch effects, putatively involving activation of the kinin-generatingcascade by virtue of their inhibiting kinin degradation and enhancing local kinin concentration. This is the first definite demonstrationindicatingthe possibleinvolvementof the kiningenerating cascade in the mechanism of P aeruginosa invasionof the systemiccirculationsystem,which causes septicemia. Although the mechanism of enhanced intravascular dissemination of bacterial cells and toxic products as causedby bradykininis still incompletelyunderstood, it may be that the invasionof P aeruginosa and passageof bacterial toxins toward the intravascular lumen are amplified by bradykinin through its potent action in causing the intracellularjunctions of endotheliumin the
AND SEPTICEMIA
421
vasculature to open up (1). Enhanced vascular permeability inducedby bradykininwill result in buildupof the tissue hydrostatic pressure, which will then facilitate bacterial translocation from local septic tissue to the inside of the vascular lumens. Furthermore, it is possible that exacerbationof bacteremiaand toxemiamightbe caused by increased lymphatic flow that is accessible to the circulation system. Specifically,bacterial cells and toxins could enter the blood circulation system via the increased lymphatic flow. In any event, a novel pharmacologicalaction of kinin, i.e., enhancement of intrusion of bacterial cells and products, is clarified, and this effect may have implications for the pathogenesis of septicemiacaused not only by P aeruginosa but also by other species of pathogenic bacteria. A number of reports have demonstratedthe strong tissue-destructivepotency of pseudomonal elastase in various animal models of P aeruginosa infection, such as pulmonary and corneal infections (20, 23) and dermal infection of burned sites (10). This potent action of pseudomonalproteasesis based on the proteolyticdegradation of matrixproteins,e.g., elastin,denaturedcollagen (gelatin), and fibronectin (25). In addition, our recent investigationindicatesthat pseudomonalelastase strongly activatedmatrixmetalloproteasessuch as progelatinase and procollagenase,which were purifiedto homogeneity from human neutrophils, through limited proteolysis of the zymogens by the bacterial protease (unpublished observation).A similar observationin a model of corneal infection with P aeruginosa in rats was obtained by Matsumotoet al (20). Further,it has also been reported that pseudomonalelastase destroys a wide range of substances of the host used for defense, such as immunoglobulinA (8), complement (28), and a plasma serine protease inhibitor (26), and that it suppresses neutrophil function (15). Therefore, in addition to the direct destruction by pseudomonalproteasesof host tissues and defense mechanisms, activation of the bradykinin-generatingcascade by the Pseudomonas exoproteases might accelerate the triggeringof septicemiaof pathogenic bacteria showing strong tropism for blood vessels. It was recently reported that pseudomonal proteases are the most important factor promoting P aeruginosa septicemiain a mouse model of gastrointestinalinfection (4). Moreover, Holder et al (9) and Snell et al (29) found that P aeruginosa virulence is maximally expressedwhen both Pseudomonas exotoxinA and exoproteases are secreted by the pathogenic strain of P aeruginosa. This synergistic effect of proteases and exotoxin may be explained by the effect of bradykinin, generatedby stimulationwith the proteases at the infectious foci, on delivery of pseudomonal toxins to the
422
Y. SAKATA
systemic circulation, as shown in Fig. 5. Yamamotoand coworkersrecently showed the important role of kininsin the pathogenesisof septic shock analyzed in a guinea pig model by using purifiedPseudomonas elastase (14). Their experiment suggested that kinin, of which generation is stimulated by the pseudomonal elastase in the systemic circulation, is the responsible mediator in the induction of hypotension and shock in P aeruginosa infection. Therefore,kinins induced by Pseudomonas exoproteases may play the most critical role not only in bacterial invasion of the blood circulation,triggering bacteremia,but also in septicemia, as in shock and multiple organ failure. In conclusion, as summarized in Fig. 6, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are crucial principles in the pathogenesisof Pseudomonassepticemia,in that they facilitatebacterialdisseminationand exacerbate septicemia. Septicemia and sepsis syndrome are often encountered as serious complications of opportunistic infections caused by gram-negative bacilli (such as P aeruginosa) with multiple drug resistance. Therefore, attempts to treat septicemia by focusing on the host's derived factors such as kinins and the endogenous protease cascade now become essential.
ET AL
Bendinelli, as
6)
Glauser,
H.
(eds),
Pseudomonas
pathogen,
7)
8)
Goto,
S.,
agar.
Jpn.
Heck,
aeru-
Plenum
Press,
by 144:
I.A.,
the
New
10)
and
and
in
by
in
tem
by
of
elastase.
Experimental
due
to Pseudomonas
J.
as in
studies aeru-
vivo
virulence
593-599. 1989.
Pseudomonas
burned
of
Yamamoto,
serratial
hosts
the
T.,
protease of
pig.
Kaminishi, Y
K.,
Degradation
1983.
elastase
25:
factor
activation
hara,
Morihara,
1990.
host
by
elastase
Hageman
kinin
factor-
cascade.
Infect.
3345-3348.
R.,
guinea
and
A.N.
activation
A
tion
C.G.
Neely,
a virulence
57:
J.
cetrimide
aeruginosa
protease
J. Microbiol.
Kamata,
acid
R.M.,
J.F.
infections
I.A.,
1985.
12)
of
Can.
Immun. 11)
Kulhavy,
Haidaris,
Holder,
dependent
Cohen,
732-736.
2253-2257.
extracellular
as
Nalidixic
Pseudomonas
factors.
acts
1970.
and
338:
65-72.
Mestecky,
pathogenesis
ginosa:
J.D., Lancet
RG.,
and
proteins
Holder,
S. 14:
Alarcon,
Immunol.
of
Enomoto,
M.W.,
Baumgartner,
pathogenesis.
J. Microbiol.
L.W.,
IgA
G.,
shock: and
Russell,
9)
Zanetti,
Septic
the
Infect.
H.,
Hageman
Candida
albicans
Maeda,
T.,
the
H. reac-
pathway
747-753.
Cho,
of
and
permeability
factor-dependent 48:
M.,
Activation
K.,
vascular
Immun.
Tanaka,
1990.
Matsumoto,
causes
Maeda,
plasma
H.,
and
Hagi-
kallikrein-kinin
proteinase.
Infect.
sys-
Immun.
58:
2139-2143. 13)
Katori,
M.,
plasma
Y.
by
Khan,
in
References
A.,
and
vitro.
In
A.I.,
diseases
and
Company,
18)
H.,
K.,
Akaike,
Maeda,
H.
1993.
and
high
K.,
Ono,
vascular
A.
pro-
leukocytes
and
Fierer,
microbiology,
1989.
W.B.
Pathogenic
Acta
T.,
185:
Inada,
Effect
of
molecular
Akaike,
T.,
p.
1177-
J.
(eds),
Saunders
role
of
bacteri-
357-368.
Y.,
Ohkubo,
microbial
and
weight
Arao,
T., T.,
I., mite
kininogens.
on,
K.,
K.R.
1993.
metalloproteases Invest. Matsumoto,
Shams,
Ono,
T.,
and
proteases J.
Biol.
by 1074:
on Chem.
house
K.,
Yamamoto,
Sci.
dust
Hanninen, activation
Pseudomonas Vis.
S.,
Inada,
Triggering
of the
of the
Hageman
mite
protease.
62-68.
and
Ophthalmol.
Kohmoto,
1991.
activation
N.B.K.,
Cleavage by
H. by
system Acta
Y,
Maeda,
reaction
Biophys.
Matsumoto,
Matsumura,
and
permeability
Biochim.
21)
N.H.
alkaline
bacteremia, C.E.,
medical
Chim.
factor-prekallikrein
20)
Valerius,
17711-17715.
Maruo, Y.,
and
161-165.
Davis,
Molla,
Clin.
Maruo,
268: 19)
and
proteases.
low
model.
Philadelphia.
Maeda, al
N.,
aeruginosa
Gram-negative
Braude,
Infectious
shock
polymorphonuclear
43:
and
119-126.
human
1986.
Y.,
factor/kallikrein-kinin
G., HƒÓiby,
Immun.
W.R.
a
1383-1391.
Shibuya,
elastase-induced
with
N., of
hypotension
98: H.,
Hageman
of Pseudomonas
Infect.
McCabe,
immediate
Araki,
1157:
Doring,
elastase
Sunahara,
involvement
J. Pharmacol.
of
Acta
Interaction
1183.
17)
Role
Biophys.
Kharazmi,
in 16)
1993.
the
the
T.,
pseudomonal
Biochim.
in
Br.
R.,
for
system
Yamamoto,
T.
system
Odoi-Adome,
Evidence
endotoxin.
M.M.H.,
Kambara,
15)
M.,
1989.
kallikrein-kinin
produced 14)
Majima,
Uchida,
tease
ginosa as an opportunistic pathogen, Plenum Press, New York. 4) Furuya, N., Hirakata, Y., Tomono, K., Matsumoto, T., Tateda, K., Kaku, M., and Yamaguchi, K. 1993. Mortality rates amongst mice with endogenous septicemia caused by Pseudomonas aeruginosa isolates from various clinical sources. J. Med. Microbiol. 39: 141-146. 5) Galloway, D.R. 1993. Role of exotoxin in the pathogenesis of P. aeruginosa infections, p. 107-127. In Campa, M.,
Friedman,
opportunistic
M.P.,
1991.
1984.
1) Bhoola, K.D., Figueroa, C.D., and Worthy, K. 1992. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol. Rev. 44: 1-80. 2) Blackwood, L.L., Stone, R.M., Iglewski, B.H., and Pennington, J.E. 1983. Evaluation of Pseudomonas aeruginosa exotoxin A and elastase as virulence factors in acute lung infection. Infect. Immun. 39: 198-201. 3) Botzenhart, K., and Doring, G. 1993. Ecology and epidemiology of P. aeruginosa, p. 1-18. In Campa, M., Bendinelli, M., and Friedman, H. (eds), Pseudomonas aeru-
and
an
York.
and
We thank Ms. Judith Gandy for editorial review, and Ms. Rie Yoshimoto for typing the manuscript. Thanks are also due to Prof. Makoto Katori for his generosity in providing the enzyme immunoassay kit MARKIT M. This work was supported by a Grant-in-Aid for Scientific Research from the Monbusho (Ministry of Education, Science and Culture), Japan, and by a grant from Yakult Honsha Co., Tokyo.
M.,
ginosa
L.A., of
corneal
aeruginosa 34:
T.,
and
Kenymatrix
proteases.
1945-1953. Kamata,
R.,
and
Maeda,
H.
BACTERIAL
22)
23)
24)
25)
PROTEASE
1984. Pathogenesis of serratial infection: activation of Hageman factor-prekallikrein cascade by serratial protease. J. Biochem. 976: 739-749. Miyagawa, S., Nishino, R., Okamura, R., and Maeda, H. 1991. Effects of protease inhibitors on growth of Serratia marcescens and Pseudomonas aeruginosa: chick ovomacroglobulinis a potent growth suppressor.Microb. Pathog. 11: 137-141. Molla, A., Matsumura, Y., Yamamoto, T., Okamura, R., and Maeda, H. 1987. Pathogenic capacity of protease from Serratia marcescens and Pseudomonas aeruginosa and their suppression by chicken egg white ovomacroglobulin. Infect. Immun. 55: 2509-2517. Molla, A., Yamamoto, T., Akaike, T., Miyoshi, S., and Maeda, H. 1989. Activation of Hageman factor and prekallikrein and generation of kinin by various microbial proteinases. J. Biol. Chem. 264: 10589-10594. Morihara, K., and Homma, J.Y. 1985. Pseudomonas proteases, p. 42-79. In Holder, I.A. (ed), Bacterial enzymes and virulence, CRC Press, Inc., Boca Raton, FL.
AND
SEPTICEMIA
423
26) Morihara, K., Tsuzuki, H., and Oda, K. 1979. Protease and elastase of Pseudomonas aeruginosa: inactivation of human plasma a1-proteinase inhibitor. Infect. Immun. 24: 188-193. 27) Roberts, F.J., Greere, I.W., and Coldman, A. 1991. A threeyear study of positive blood culture, with emphasis on prognosis. Rev. Infect. Dis. 13: 34-46. 28) Schultz, D.R., and Miller, K.D. 1974. Elastase of Pseudomonas aeruginosa: inactivation of complement components and complement-derived chemotactic and phagocytic factors. Infect. Immun. 10: 128-135. 29) Snell, K., Holder, I.A., Leppla, S.A., and Sealinger, C.B. 1978. Role of exotoxin and protease as possible virulence factors in experimental infections with Pseudomonas aeruginosa. J. Clin. Microbiol. 9: 538-540. 30) Starkey, P M. 1977. Elastase and cathepsin G: the serine proteinases of human neutrophil leukocytes and spleen, p. 5889. In Barrett, A. J. (ed), Proteinases in mammalian cells and tissues, Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands.
