Previous Article | Next Article 
Infection and Immunity, October 1998, p. 4965-4970, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Two Laminin-Binding Fimbriae, the Type 1 Fimbria of Salmonella enterica Serovar Typhimurium and the G
Fimbria of Escherichia coli, as Plasminogen
Receptors
Maini
Kukkonen,1
Sirkku
Saarela,1
Kaarina
Lähteenmäki,1
Ulla
Hynönen,1
Benita
Westerlund-Wikström,1
Mikael
Rhen,2 and
Timo K.
Korhonen1,*
Division of General Microbiology, Department
of Biosciences, FIN-00014 University of Helsinki,
Finland,1 and
Microbiology and Tumor
Biology Center, Karolinska Institute, 17177 Stockholm,
Sweden2
Received 9 March 1998/Returned for modification 23 April
1998/Accepted 12 July 1998
 |
ABSTRACT |
Escherichia coli strains carrying recombinant plasmids
encoding either the type 1 fimbria of Salmonella enterica
serovar Typhimurium or the G fimbria of E. coli exhibited
binding of human 125I-Glu-plasminogen and enhanced the
tissue-type plasminogen activator-catalyzed conversion of plasminogen
to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen
and enhanced its activation. The binding of plasminogen did not
involve the characteristic carbohydrate-binding property of the
fimbriae but was inhibited at low concentrations by the lysine analog
-aminocaproic acid. Because these fimbrial types bind to laminin of
basement membranes (M. Kukkonen et al., Mol. Microbiol.
7:229-237, 1993; S. Saarela et al., Infect. Immun. 64:2857-2860,
1996), the results demonstrate a structural unity in the creation and
targeting of bacterium-bound proteolytic plasmin activity to basement
membranes.
| |
INTRODUCTION |
Plasminogen is the precursor of the
human protease plasmin and is abundant in human plasma and
extracellular fluids. It is converted to the active, proteolytic form
plasmin by eukaryotic activators such as tissue-type plasminogen
activator (tPA) and urokinase. tPA is the principal activator in plasma
and intercellular fluid, and its action is due to proteolytic cleavage
of plasminogen. Plasminogen activation by tPA proceeds poorly in
solution but is dramatically enhanced by immobilization of plasminogen
on fibrin, eukaryotic tissue surfaces, or certain bacterial cells.
Immobilization of plasminogen is a key determinant in the control of
plasminogen activation as well as of the formed plasmin activity
(reviewed in reference 37). The immobilization is
mediated by five kringle domains of plasminogen which bind to
lysine-containing domains on the target proteins; immobilization
on receptor proteins is associated with dramatic conformational
changes in the plasminogen molecule (27). The bound
plasminogen is more susceptible to tPA-mediated activation and, on
the other hand, is more resistant to the physiological inhibitors of
activation and of the formed plasmin activity. Plasmin is a
trypsin-like serine protease with a broad substrate specificity and
functions in, e.g., the degradation of fibrin (fibrinolysis) and of
noncollagenous proteins of the extracellular matrix (ECM), in
activation of latent procollagenases, and in penetration of basement
membranes (BM) by metastatic cancer cells (for reviews, see references
4, 30 and 37).
Several gram-positive and gram-negative invasive bacterial pathogens
have been found to express a plasminogen receptor (PlgR) function
(1, 8, 19, 20, 32, 41-43). These bacteria immobilize
plasminogen on their cell surfaces and enhance the tPA-catalyzed plasminogen activation. In essence, the
bacterial PlgRs function to generate proteolytic activity
on the bacterial surface by utilizing a host-derived proteolytic
system. To date, only a few bacterial PlgR molecules have been
identified. The S fimbriae in Escherichia coli and the
curli, or thin aggregative fimbriae, in Salmonella enterica
have been identified as PlgRs (32, 39). The M53 protein
of group A streptococci and flagellar antigens of E. coli
have been found to have plasminogen-binding characteristics
(1, 23). An outer cell surface lipoprotein functions
as a PlgR in Borrelia burgdorferi (8). In
addition, a plasmin receptor of group A streptococci has been
identified as a surface-bound glyceraldehyde 3-phosphate
dehydrogenase (24, 31).
Bacterial enzymes acting directly on mammalian ECM or activating latent
procollagenases exist but are not frequently expressed by invasive,
pathogenic species (9, 11). Because plasmin degrades
noncollagenous proteins of ECM, such as laminin, and activates latent
procollagenases, it has been proposed that one function of bacterial
PlgRs is to potentiate bacterial damage to and bacterial spread
through tissue barriers, such as BM. This hypothesis (17)
was based on findings that adhesiveness to BM (reviewed in reference
45) and expression of the PlgR function (reviewed in references 2 and 25)
are shared by a number of invasive bacterial pathogens. Furthermore, it
is supported by experimental evidence recently obtained in vitro
with S. enterica (21) and with
Haemophilus influenzae (44). These two
bacteria penetrate in vitro through a reconstituted BM with the help of bacterium-bound plasmin activity. Plasminogen has also been shown to
potentiate bacterial transcytosis across an epithelial cell monolayer
(47) and, in experimental infections, to enhance
spirochetemia by B. burgdorferi in mice (3).
S. enterica serovar Typhimurium SH401, used in this study,
adheres to the high-mannose chains of laminin as well as to
reconstituted BM, and the adherence is mediated by the type 1 fimbriae
(18). Typhimurium SH401 also expresses PlgR
activity, and it has been demonstrated that plasmin bound on the
surfaces of SH401 cells degrades laminin and potentiates bacterial
penetration through BM (21). Other studies have described the G (34) or F17 (6) fimbriae on uropathogenic
and septicemic enterotoxigenic E. coli that bind to
terminal N-acetyl-D-glucosamine residues on glycoproteins, such as laminin of BM
(36). We report here that both fimbrial types also exhibit
the PlgR function.
| |
MATERIALS AND METHODS |
Bacteria.
S. enterica serovar Typhimurium SH401
(18, 21) and the nonfimbriated recipient E. coli
strain LE392 (5) were available from previous work. E. coli IHE11088(pRR-5), expressing the G fimbria gene cluster from a
uropathogenic E. coli O2 isolate, and IHE11088(pHUB110),
expressing a mutated gene cluster, have been described (34,
36). pHUB110 contains a 6-bp deletion within the
coding region of the gafD gene, resulting in
G-fimbrial filaments lacking the GlcNAc-binding property.
The G fimbriae were expressed in the IHE11088 background because in
K-12 derivatives the lectin activity of the G fimbria causes
autoaggregation of E. coli cells (34). The
strains were cultivated in Luria broth or on Luria agar plates, which
were supplemented with antibiotics in the case of the recombinant
strains.
DNA techniques.
Construction of a cosmid library in plasmid
pHC79 from partially Sau3AI-digested Typhimurium genomic
DNA, DNA hybridization, subcloning of the fim gene cluster,
and DNA manipulations were performed by standard procedures
(38).
Plasminogen binding and activation assays.
The
binding of plasminogen to purified fimbriae was measured by
time-resolved fluorometry (essentially as described in references 19, 21, and 23). The type 1 fimbriae were purified from S. enterica SH401 and the
recombinant E. coli and the G fimbriae were purified
from E. coli IHE11088(pRR-5) by using deoxycholate and
concentrated urea (16). We used the fimbriae at a
concentration of 10 µg per ml, human Glu-plasminogen (Biopool, Umeå,
Sweden) at 10 or 50 µg/ml, and, in inhibition tests,
-aminocaproic acid (EACA) (Sigma) at 10 mM. To assess the
binding of plasminogen to bacterial cells, Glu-plasminogen was
labeled with 125I (Amersham, Little Chalfont,
Buckinghamshire, United Kingdom) by the Iodogen method (28).
The activities obtained were 6 × 105 cpm/µg of
plasminogen (the preparation used with the type 1 fimbriae) and 2 × 106 cpm/µg (the preparation used with the G fimbriae).
Binding of 125I-plasminogen to bacterial cells was assessed
as described previously (19, 23). We used 2 × 109 cells and 0.2 µg of plasminogen in the assay; the
inhibition assays were performed with 4 mM EACA.
The ability of the purified fimbriae to enhance plasmin formation by
tPA was measured as detailed earlier (23). Purified fimbriae
were tested at 10 to 100 µg/ml; bovine serum albumin (BSA), a
negative control, was tested at 100 µg/ml; and laminin (Upstate
Biotechnology, Lake Placid, N.Y.), a positive control, was tested at 10 µg/ml. Control assays included tests from which plasminogen or
tPA was omitted. The ability of bacterial cells to enhance plasmin
formation was measured as detailed elsewhere (21, 23, 32).
We used a bacterial density of 4 × 108 per ml,
plasminogen was tested at 20 µg per ml, tPA was tested at 50 ng per
ml, the chromogenic substrate S-2251 was tested at 0.45 mM, and EACA
was tested at 0.4 mM, in a test volume of 200 µl. Results from the
activation assays are given as data from a representative assay with
duplicate independent samples; the range of individual test results was
within ±10% of the mean.
Other tests.
Mannose-sensitive yeast cell agglutination
(14), agglutination tests with an antiserum raised against
purified type 1 fimbriae of S. enterica serovar Typhimurium
(15), and electron microscopy of negatively stained
recombinant cells (16) were used to assess expression of the
type 1 fimbriae of S. enterica SH401. Bacterial agglutination in an antiserum raised against the type 1 fimbriae of
E. coli (16) was assessed as a negative
control. The expression of the G fimbriae was confirmed by
hemagglutination of endo-
-galactosidase-treated human erythrocytes
and by bacterial agglutination in an antiserum raised against the
purified G fimbriae (34).
| |
RESULTS |
Binding of plasminogen to the type 1 fimbriae of S. enterica.
It has been shown that strain SH401 of S. enterica serovar Typhimurium expresses PlgR activity
(21). A recombinant strain harboring a cosmid with a 40-kb
fragment of SH401 DNA and strongly enhancing tPA-mediated
plasminogen activation was isolated and named E. coli
LE392(pMK1). The strain agglutinated yeast cells in a
mannose-reversible manner, expressed fimbriae as determined by electron
microscopy, and was agglutinated in an antiserum raised against the
type 1 fimbriae of S. enterica but not in an antiserum against the type 1 fimbriae of E. coli (data not
shown), indicating that the recombinant strain LE392(pMK1)
expressed the type 1 fimbriae of Typhimurium SH401. Furthermore,
the restriction map of pMK1 (data not shown) showed the presence
of a 12.5-kb SphI fragment similar to that identified by
Purcell et al. (33) and shown to carry the fim
cluster of Typhimurium. To test the function of the type 1 fimbria of
S. enterica as a PlgR, we subcloned the SphI
fragment from pMK1 into plasmid pUC19, obtaining plasmid pMK25, and
expressed the SH401 fimbriae for binding and activation experiments
in the nonfimbriated strain E. coli LE392.
Binding of plasminogen to
E. coli LE392(pMK25) is
shown in Fig.
1. The strain
LE392(pMK25) bound plasminogen three times more
efficiently than
did
E. coli LE392(pUC19) carrying the vector
plasmid alone (Fig.
1). It has been shown (
23) that the
flagella
of
E. coli LE392 bind plasminogen, and it is
likely that the activity
seen with the nonfimbriated strain
LE392(pUC19) resulted from
the binding of plasminogen to
the flagella. Binding of plasminogen
to both strains was inhibited
by 4 mM EACA to the background level
seen in tests performed without
added bacteria; EACA is a lysine
analog and a well-characterized
binding inhibitor of the kringle
domains of plasminogen
(
37). Plasmin has a higher affinity than
plasminogen for
lysine-containing target proteins (
19). To exclude
the
possibility that the observed binding to LE392(pMK25) cells
was induced by plasmin possibly present in the plasminogen preparation
or formed during the assay, we also performed the binding
experiments
in the presence of 150 kIU of aprotinin, an inhibitor
of plasmin
activity (
25). The presence of aprotinin did not
decrease the
binding of plasminogen to LE392(pMK25) or
LE392(pUC19) cells;
on the contrary, a slight increase was
observed (Fig.
1).
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 1.
Binding of 125I-plasminogen
(125I-plg) onto recombinant E. coli
expressing the type 1 fimbriae from S. enterica. Bars a to d
show the nonfimbriated strain E. coli LE392(pUC19);
bars e to h show E. coli LE392(pMK25) cells
expressing the type 1 fimbriae. The background binding to
BSA-coated plastic is shown in bars i to l. The binding in the
absence of EACA and aprotinin is shown by bars a, e, and i, and the
binding in the presence of 4 mM EACA is shown by bars b, f, and j.
Bars c, g, and k show the binding in the presence of 150 kIU of
aprotinin, and bars d, h, and l show the binding in the presence of
both EACA and aprotinin.
|
|
We next assessed whether the fimbriated recombinant
E. coli strain enhanced tPA-catalyzed plasminogen
activation. In different
experiments, strain LE392(pMK25) caused a
5- to 10-fold enhancement
in plasmin formation by tPA, compared to the
activation seen in
buffer alone, whereas the strain LE392(pUC19)
caused only a 2-fold
increase in the activation (Fig.
2). With both bacterial strains,
EACA
decreased plasmin formation close to the level seen in buffer
without
bacteria. Control experiments in the presence of LE392(pMK25)
or
LE392(pUC19) cells showed that no plasmin was formed if either
plasminogen or tPA was omitted from the suspensions (Fig.
2).
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 2.
Enhancement of tPA-catalyzed plasmin formation in
the presence of recombinant E. coli expressing the type
1 fimbriae of S. enterica. Kinetic measurements of
tPA-catalyzed plasmin formation in the presence of LE392(pUC19)
cells ( ), LE392(pUC19) cells and 0.4 mM EACA ( ),
LE392(pMK25) cells ( ), and LE392(pMK25) cells and 0.4 mM
EACA ( ) and in the absence of bacterial cells ( ) are shown.  ,
plasmin formation when tPA or plasminogen was omitted in the tests
performed with strain LE392(pMK25).
|
|
To confirm the function of the type 1 fimbria of
S. enterica
SH401 as a PlgR, we tested the capacity of purified fimbriae
to
bind plasminogen. The purified fimbriae efficiently bound plasminogen
(Fig.
3). The binding was dependent
on the concentration of plasminogen
used in the assay and was inhibited
by EACA to a level close to
the background level seen with immobilized
BSA. At 10 µg/ml, the
type 1 fimbriae caused a 100-fold
enhancement of the tPA-catalyzed
activation compared to the
activation in phosphate-buffered saline
containing
only plasminogen and tPA (Fig.
3). The enhancement
was dependent
on the concentration of the type 1 fimbriae (data
not shown). BSA
at 100 µg/ml caused only a marginal enhancement
of the
tPA-catalyzed activation. The formation of plasmin activity
in the
presence of the fimbriae was greatly decreased when 0.8
mM EACA was
added to the protein mixture, again indicating that
the
immobilization of plasminogen on the purified fimbriae was
necessary
for the observed enhancement. No difference was observed
between the
type 1 fimbriae purified from the wild-type strain
S. enterica SH401 and those purified from the recombinant
E. coli strains (data not shown). The formation of
plasmin on the bacterial
cells or on the purified fimbriae was not
affected by the addition
of 2.5 mM
-methyl-
D-mannoside into the suspension (data not
shown).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Binding of plasminogen (A) and enhancement of
tPA-catalyzed plasmin formation (B) on purified type 1 fimbriae of
S. enterica. (A) Plasminogen was added at two
concentrations, 10 µg/ml (bars a to d) and 50 µg/ml (bars e
to h), to plastic coated with fimbriae, and the binding was
measured by time-resolved fluorometry. Binding was assessed in the
absence (bars a, c, e, and g) or presence (bars b, d, f, and h) of 10 mM EACA. Bars a, b, e, and f show the binding to type 1 fimbriae,
and bars c, d, g, and h show the binding to plastic coated with
BSA. (B) Enhancement of tPA-catalyzed plasmin formation was
measured at a fimbria concentration of 10 µg/ml in the absence
() or presence () of 0.8 mM EACA. Levels of plasmin formation in
the presence of BSA (100 µg/ml) () or in buffer alone ( )
are also shown. , plasmin activity when tPA or plasminogen was
omitted in the tests performed with type 1 fimbriae.
|
|
Function of the G fimbria of E. coli as a
PlgR.
Because the G-fimbriated, uropathogenic
E. coli strain IHE11165 expresses PlgR activity
(data not shown), we tested the binding of plasminogen to the
G-fimbriated recombinant strain E. coli IHE11088(pRR-5). To evaluate the role of the fimbrial lectin
activity, the test also included the strain IHE11088(pHUB110),
expressing G-fimbrial filaments deleted in frame for the amino acids
Gly-94 and Thr-95 of the GafD lectin subunit and lacking the
GlcNAc-binding property (34). Both G-fimbriated strains
bound plasminogen and the plasminogen-aprotinin complex more
efficiently than did the nonfimbriated strains IHE11088 (Fig.
4) and IHE11088(pACYC184) (data not
shown). Again, the binding of plasminogen was inhibited by a small
amount of EACA.
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 4.
Binding of 125I-plasminogen
(125I-plg) onto G-fimbriated bacterial cells. Bars a to d
show E. coli IHE11088(pRR-5) with the G fimbria gene
cluster, bars e to h show E. coli IHE11088(pHUB110)
expressing G fimbriae that lack the GlcNAc-binding property,
and bars i to l show the nonfimbriated E. coli strain
IHE11088. Binding in the absence of EACA and aprotinin is shown in bars
a, e, and i, and binding in the presence of 4 mM EACA is shown in
bars b, f, and j. Bars c, g, and k show the binding in the presence
of 150 kIU of aprotinin, and bars d, h, and l show the binding in
the presence of both EACA and aprotinin.
|
|
The
E. coli strain IHE11088(pRR-5), expressing G
fimbriae, and the strain IHE11088(pHUB110), expressing
nonadhesive G fimbriae,
enhanced plasmin formation compared to the
activation seen in
the presence of IHE11088 (Fig.
5) or IHE11088(pACYC184) (data
not
shown) cells lacking fimbriae. In the presence of EACA, the
formation of plasmin was inhibited and no endogenous plasminogen
activation was detected. The wild-type G fimbriae from
E. coli IHE11088(pRR-5) and the mutated G
fimbriae from IHE11088(pHUB110)
both bound plasminogen and enhanced
the tPA-catalyzed plasmin
formation in an EACA-inhibitable
manner (Fig.
6). Tested at 10
µg/ml, the ability of the G fimbriae to enhance the
tPA-catalyzed
plasminogen activation was similar to that
shown by laminin, a
characterized ECM target for plasminogen
binding (Fig.
6).
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 5.
Enhancement of tPA-catalyzed plasmin formation in
the presence of E. coli IHE11088(pRR-5) expressing
G fimbriae (A), of E. coli IHE11088(pHUB110)
expressing G fimbriae devoid of GlcNAc-binding activity (B), and of
the nonfimbriated strain IHE11088 (C). Also shown are plasmin formation
in the presence of bacteria, plasminogen, and tPA (); plasmin
formation when 0.4 mM EACA was added to the suspension (); plasmin
formation when tPA was omitted (); plasmin formation when
plasminogen was omitted (); and tPA-catalyzed plasmin formation
without added bacteria ().
|
|
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 6.
Binding of plasminogen and enhancement of
tPA-catalyzed plasmin formation on purified G fimbriae of
E. coli. (A) The binding of plasminogen to purified
fimbriae was measured by time-resolved fluorometry. Bars a and b show
binding to the wild-type G fimbriae, bars c and d show binding
to the mutated G fimbriae, and bars e and f show binding to BSA.
Binding in the absence of EACA is shown in bars a, c, and e;
binding in the presence of 10 mM EACA is shown in bars b, d, and f.
Levels of plasmin formation in the presence of the G fimbriae from
E. coli IHE11088(pRR-5) (B) and of the mutated G
fimbriae from E. coli IHE11088(pHUB110) (C) are
shown. Plasmin formation in the presence of fimbriae (), in the
presence of fimbriae and 0.4 mM EACA (), without added tPA (),
and without added plasminogen () is shown. (D) For comparison,
plasmin formation in the presence of laminin (), laminin and 0.4 mM
EACA (), and BSA () is shown.
|
|
| |
DISCUSSION |
Our results demonstrate that the type 1 fimbriae of S. enterica serovar Typhimurium as well as the G fimbriae of
E. coli function as PlgRs. This was inferred from
two lines of evidence. First, the purified fimbriae efficiently bound
plasminogen and enhanced tPA-mediated activation of plasminogen.
Second, expression of both fimbria gene clusters in E. coli resulted in enhanced plasmin formation and plasminogen
binding. Our results also give evidence that the
carbohydrate-binding activity of the fimbriae is not involved in
the interaction with plasminogen.
A similar enhancement of plasminogen binding and of
tPA-mediated activation was observed with the complete G fimbriae
and with the mutated fimbriae lacking GlcNAc binding. This
indicates that the plasminogen binding and acquisition of a correct
formation for tPA-mediated activation are not dependent on the
lectin activity of the G fimbria. The mutated GafD lectin subunit
encoded by pHUB110 is expressed in a few copies in the G-fimbria
filament, similarly to the wild-type GafD lectin (35).
Furthermore, we found that the receptor analog for the type 1 fimbriae, -methyl-D-mannoside, had no effect on plasmin
formation. In contrast, the binding and activation processes
observed with both fimbrial types were efficiently inhibited by a low
concentration of the lysine analog EACA. This indicates that the
fimbria-plasminogen interactions follow the general mechanism of
plasminogen-receptor interactions that involve the lysine-binding
kringle domains of plasminogen (27, 37). The results thus
indicate that the fimbrial filaments are functioning as targets in
their interaction with plasminogen.
The expression of S. enterica fim genes on the plasmid
pMK25 as well as of the gaf gene cluster on the
plasmids pRR-5 and pHUB110 enhanced binding and activation of
plasminogen on recombinant E. coli cells. These results
indicate that the fim and the gaf gene clusters
are sufficient to confer plasminogen-binding capacity on bacterial
cells. The variable and seemingly lower PlgR activity exhibited by
the recombinant strains, compared to that shown by the purified
fimbriae, most likely resulted from fimbrial phase variation, which
rendered the bacterial cell population heterogeneous in regard to
fimbriation. The type 1 and the G fimbriae are not the only surface
appendages of Salmonella or E. coli that
have the capacity to bind plasminogen and enhance its activation by tPA. Curli, or thin aggregative fimbriae, have also been identified as
PlgRs (39). We have not detected any expression of curli by Typhimurium strain SH401 under the growth conditions we used. Additionally, other research (23) indicates that the
flagella of S. enterica also function as PlgRs. Finally,
PlgR activity is associated with the S fimbriae of
meningitis-associated E. coli (32). It is
notable that many of the identified bacterial PlgRs are filamentous
structures, such as fimbriae, flagella, or M proteins (1),
which bear morphological similarity to fibrin, a major eukaryotic
target for plasminogen binding and plasmin activity
(37). On the other hand, it is apparent that not all fimbrial filaments express PlgR activity (e.g., PlgR activity in P-fimbrial variants occurring on human uropathogenic E. coli has not been detected [13]). A detailed
comparison of the various enterobacterial fimbrial types in
regard to PlgR function, however, has not been performed.
Furthermore, our knowledge of the fimbrial subunits and of amino acid
sequences recognized by plasminogen is very limited (32).
Overall, the binding of plasminogen to lysine and lysine
analogs is well characterized (27), but other structural
requirements for PlgR activity have remained poorly defined. A
plasminogen-binding 13- to 16-mer repeat domain has been
identified in the PAM surface protein of group A streptococci (46); this sequence, however, exhibits no significant
identity to the sequences of the major and minor fimbrial subunits of
this study. Our present results indicate that the lectin activity
of fimbriae is not involved in the PlgR function; however, this
does not rule out the possibility that certain regions of the FimH or the GafD adhesins are physically involved in plasminogen
binding.
It has been shown that PlgRs on the surfaces of Typhimurium
SH401 cells function under physiological conditions to create bacterium-bound plasmin activity that can be targeted at laminin and BM
(22), which are adhesion targets for the fimbriae used in
this study (18, 36). Adhesion to laminin and activation of
plasminogen and other latent proteases are important in tumor cell
invasion through tissue barriers (4, 30). In vitro evidence has been presented in favor of the hypothesis that these
characteristics also contribute to bacterial penetration through BM;
this process has been termed bacterial metastasis in an analogy to the
behavior of metastatic tumor cells (21, 44). The
contribution to virulence of our present findings still needs to be
assessed in vivo. To date, the role of plasminogen activation in vivo
has been tested in two animal models. The plasminogen
activator surface protein was found to enhance the spread of
Yersinia pestis into circulation (40), and
plasminogen activation was required for dissemination of B. burgdorferi in the tick vector and for enhancement of
spirochetemia in mice (3). Interestingly, both of
these bacterial species also express ECM- and BM-binding
properties (10, 12, 21). In particular, research that
associates the expression of the plasminogen activator of Y. pestis with an increased adhesiveness to laminin (22)
stresses the common occurrence of laminin adherence and PlgR
function in invasive enterobacterial pathogens. BMs are considered to
function as reservoirs for plasminogen, metalloproteinases (collagenases), and plasminogen activators (7, 26, 29). Hence, a close association of bacteria with BM in tissues might bring
them to a microenvironment where formation and targeting of plasmin
activity can effectively promote bacterial metastasis. The major
finding of the present study is a physical link between adhesiveness to
tissues and immobilization of plasminogen on a surface filament of two
invasive bacterial pathogens.
| |
ACKNOWLEDGMENTS |
This work has been supported by grants from the University of
Helsinki, the Sigrid Jusélius Foundation, and the Academy of Finland (grant numbers 29346, 42103, and 42107).
We thank Raili Lameranta for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
General Microbiology, Department of Biosciences, P.O. Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland. Phone:
358-9-70859260. Fax: 358-9-70859262. E-mail:
timo.korhonen{at}helsinki.fi.
Editor:
P. E. Orndorff
| |
REFERENCES |
| 1.
|
Berge, A., and U. Sjöbring.
1993.
PAM, a novel plasminogen-binding protein from Streptococcus pyogenes.
J. Biol. Chem.
268:25417-25424[Abstract/Free Full Text].
|
| 2.
|
Boyle, M. D. P., and R. Lottenberg.
1997.
Plasminogen activation by invasive human pathogens.
Thromb. Haemostasis
77:1-10[Medline].
|
| 3.
|
Coleman, J. L.,
J. A. Gebbia,
J. Piesman,
J. L. Degen,
T. H. Buggs, and J. L. Benach.
1997.
Plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochetemia in mice.
Cell
89:1111-1119[Medline].
|
| 4.
|
Danø, K.,
P. A. Andreasen,
J. Grøndahl-Hansen,
P. Kristensen,
L. S. Nielsen, and L. Skriver.
1985.
Plasminogen activators, tissue degradation and cancer.
Adv. Cancer Res.
44:139-266[Medline].
|
| 5.
|
Elliot, S. J.,
N. Nandapalan, and B. J. Chang.
1991.
Production of type 1 fimbriae by Escherichia coli HB101.
Microb. Pathog.
10:481-486[Medline].
|
| 6.
|
El Mazouari, K.,
E. Oswald,
J.-P. Hernalsteens,
P. Lintermans, and H. De Greve.
1994.
F17-like fimbriae from an invasive Escherichia coli strain producing cytotoxic necrotizing factor type 2 toxin.
Infect. Immun.
62:2633-2638[Abstract/Free Full Text].
|
| 7.
|
Farina, A. R.,
A. Tiberio,
A. Tacconelli,
L. Cappabianca,
A. Guline, and A. R. Mackay.
1997.
Identification of plasminogen in Matrigel and its activation by reconstitution of this basement membrane extract.
BioTechniques
21:904-909.
|
| 8.
|
Fuchs, H.,
R. Wallich,
M. M. Simon, and M. D. Kramer.
1994.
The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor.
Proc. Natl. Acad. Sci. USA
91:12594-12598[Abstract/Free Full Text].
|
| 9.
|
Goguen, J. D.,
N. P. Hoe, and Y. V. B. K. Subrahmanyam.
1995.
Proteases and bacterial virulence: a view from the trenches.
Infect. Agents Dis.
4:47-54[Medline].
|
| 10.
|
Guo, B. P.,
S. J. Norris,
L. C. Rosenberg, and M. Höök.
1995.
Adherence of Borrelia burgdorferi to the proteoglycan decorin.
Infect. Immun.
63:3467-3472[Abstract].
|
| 11.
|
Harrington, D. J.
1996.
Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease.
Infect. Immun.
64:1885-1891[Medline].
|
| 12.
|
Kienle, Z.,
L. Emödy,
C. Svanborg, and P. W. O'Toole.
1992.
Adhesive properties conferred by the plasminogen activator of Yersinia pestis.
J. Gen. Microbiol.
138:1679-1687.
|
| 13.
| Korhonen, T. K. Unpublished data.
|
| 14.
|
Korhonen, T. K.
1979.
Yeast cell agglutination by purified enterobacterial pili.
FEMS Microbiol. Lett.
6:421-425.
|
| 15.
|
Korhonen, T. K.,
K. Lounatmaa,
H. Ranta, and N. Kuusi.
1980.
Characterization of type 1 pili of Salmonella typhimurium LT2.
J. Bacteriol.
144:800-805[Abstract/Free Full Text].
|
| 16.
|
Korhonen, T. K.,
E.-L. Nurmiaho,
H. Ranta, and C. Svanborg Edén.
1980.
New method for isolation of immunologically pure pili from Escherichia coli.
Infect. Immun.
27:569-575[Abstract/Free Full Text].
|
| 17.
|
Korhonen, T. K.,
R. Virkola,
K. Lähteenmäki,
Y. Björkman,
M. Kukkonen,
T. Raunio,
A.-M. Tarkkanen, and B. Westerlund.
1992.
Penetration of fimbriate enteric bacteria through basement membranes: a hypothesis.
FEMS Microbiol. Lett.
100:307-312.
|
| 18.
|
Kukkonen, M.,
T. Raunio,
R. Virkola,
K. Lähteenmäki,
P. H. Mäkelä,
P. Klemm,
S. Clegg, and T. K. Korhonen.
1993.
Basement membrane carbohydrate as a target for bacterial adhesion: binding of type 1 fimbriae of Salmonella enterica and Escherichia coli to laminin.
Mol. Microbiol.
7:229-237[Medline].
|
| 19.
|
Kuusela, P., and O. Saksela.
1990.
Binding and activation of plasminogen at the surface of Staphylococcus aureus.
Eur. J. Biochem.
193:759-765[Medline].
|
| 20.
|
Kuusela, P.,
M. Ullberg,
O. Saksela, and G. Kronvall.
1992.
Tissue-type plasminogen activator-mediated activation of plasminogen on the surface of group A, C, and G streptococci.
Infect. Immun.
60:196-201[Abstract/Free Full Text].
|
| 21.
|
Lähteenmäki, K.,
R. Virkola,
R. Pouttu,
P. Kuusela,
M. Kukkonen, and T. K. Korhonen.
1995.
Bacterial plasminogen receptors: in vitro evidence for a role in degradation of the mammalian extracellular matrix.
Infect. Immun.
63:3659-3664[Abstract].
|
| 22.
| Lähteenmäki, K., R. Virkola, A. Sarén,
L. Emödy, and T. K. Korhonen. Expression of the
plasminogen activator Pla of Yersinia pestis enhances
bacterial attachment to the mammalian extracellular matrix. Submitted
for publication.
|
| 23.
|
Lähteenmäki, K.,
B. Westerlund,
P. Kuusela, and T. K. Korhonen.
1993.
Immobilization of plasminogen on Escherichia coli flagella.
FEMS Microbiol. Lett.
106:309-314[Medline].
|
| 24.
|
Lottenberg, R.,
C. C. Broder,
M. D. P. Boyle,
S. J. Kain,
B. L. Schroeder, and R. Curtiss, III.
1992.
Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor.
J. Bacteriol.
174:5204-5210[Abstract/Free Full Text].
|
| 25.
|
Lottenberg, R.,
D. Minning-Wenz, and M. D. P. Boyle.
1994.
Capturing host plasmin(ogen): a common mechanism for invasive pathogens?
Trends Microbiol.
2:20-24[Medline].
|
| 26.
|
Mackay, A. R.,
D. E. Gomez,
D. W. Cottam,
R. C. Rees,
A. M. Nason, and U. P. Thorgeirsson.
1993.
Identification of the 72-kDa (MMP-2) and 92-kDa (MMP-9) gelatinase/type IV collagenase in preparations of laminin and Matrigel.
BioTechniques
15:1048-1051[Medline].
|
| 27.
|
Mangel, W. F.,
B. Lin, and V. Ramakrishnan.
1990.
Characterization of an extremely large, ligand-induced conformational change in plasminogen.
Science
248:69-73[Abstract/Free Full Text].
|
| 28.
|
Markwell, M. A. K., and C. F. Fox.
1978.
Surface-specific iodination of membrane proteins of viruses and eukaryotic cells using 1,3,4,6-tetrachloro-3,6-diphenylglycouril.
Biochemistry
17:4807-4817[Medline].
|
| 29.
|
McGuire, A. R., and N. W. Seed.
1989.
The interaction of plasminogen activator with reconstituted basement matrix and extracellular macromolecules produced by cultured epithelial cells.
J. Biol. Chem.
40:215-227.
|
| 30.
|
Mignatti, P., and D. B. Rifkin.
1993.
Biology and biochemistry of proteinases in tumor invasion.
Physiol. Rev.
73:161-195[Free Full Text].
|
| 31.
|
Pancholi, V., and V. A. Fischetti.
1992.
A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity.
J. Exp. Med.
176:415-426[Abstract/Free Full Text].
|
| 32.
|
Parkkinen, J.,
J. Hacker, and T. K. Korhonen.
1991.
Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis.
Thromb. Haemostasis
65:483-486[Medline].
|
| 33.
|
Purcell, B. K.,
J. Pruckler, and S. Clegg.
1987.
Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.
J. Bacteriol.
169:5831-5834[Abstract/Free Full Text].
|
| 34.
|
Saarela, S.,
S. Taira,
E.-L. Nurmiaho-Lassila,
A. Makkonen, and M. Rhen.
1995.
The Escherichia coli G-fimbrial lectin protein participates both in fimbrial biogenesis and in recognition of the receptor N-acetyl-D-glucosamine.
J. Bacteriol.
177:1477-1484[Abstract/Free Full Text].
|
| 35.
| Saarela, S., J. Tanskanen, B. Westerlund-Wikström, and T. K. Korhonen. Unpublished
data.
|
| 36.
|
Saarela, S.,
B. Westerlund-Wikström,
M. Rhen, and T. K. Korhonen.
1996.
The GafD protein of the G (F17) fimbrial complex confers adhesiveness of Escherichia coli to laminin.
Infect. Immun.
64:2857-2860[Abstract].
|
| 37.
|
Saksela, O., and D. B. Rifkin.
1988.
Cell-associated plasminogen activation: regulation and physiological functions.
Annu. Rev. Cell Biol.
4:93-126.
|
| 38.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 39.
|
Sjöbring, U.,
G. Pohl, and A. Olsén.
1994.
Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA).
Mol. Microbiol.
14:443-452[Medline].
|
| 40.
|
Sodeinde, O. A.,
Y. V. B. K. Subrahmanyam,
K. Stark,
T. Quan,
Y. Bao, and J. D. Goguen.
1992.
A surface protease and the invasive character of plague.
Science
258:1004-1007[Abstract/Free Full Text].
|
| 41.
|
Ullberg, M.,
G. Kronvall,
I. Karlsson, and B. Wiman.
1990.
Receptors for human plasminogen on gram-negative bacteria.
Infect. Immun.
58:21-25[Abstract/Free Full Text].
|
| 42.
|
Ullberg, M.,
G. Kronvall, and B. Wiman.
1989.
New receptor for human plasminogen on gram positive cocci.
APMIS
97:996-1002[Medline].
|
| 43.
|
Ullberg, M.,
P. Kuusela,
B.-E. Kristiansen, and G. Kronvall.
1992.
Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoeae and formation of surface-associated plasmin.
J. Infect. Dis.
166:1329-1334[Medline].
|
| 44.
|
Virkola, R.,
K. Lähteenmäki,
T. Eberhard,
P. Kuusela,
L. van Alphen,
M. Ullberg, and T. K. Korhonen.
1996.
Interaction of Haemophilus influenzae with the mammalian extracellular matrix.
J. Infect. Dis.
173:1137-1147[Medline].
|
| 45.
|
Westerlund, B., and T. K. Korhonen.
1993.
Bacterial proteins binding to the mammalian extracellular matrix.
Mol. Microbiol.
9:687-694[Medline].
|
| 46.
|
Wistedt, A. C.,
U. Ringdahl,
W. Müller-Esterl, and U. Sjöbring.
1995.
Identification of a plasminogen-binding motif in PAM, a bacterial surface protein.
Mol. Microbiol.
18:569-578[Medline].
|
| 47.
|
Zavizion, B.,
J. H. White, and A. J. Bramley.
1997.
Staphylococcus aureus stimulates urokinase-type plasminogen activator expression by bovine mammary cells.
J. Infect. Dis.
176:1637-1640[Medline].
|
Infection and Immunity, October 1998, p. 4965-4970, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hurmalainen, V., Edelman, S., Antikainen, J., Baumann, M., Lahteenmaki, K., Korhonen, T. K.
(2007). Extracellular proteins of Lactobacillus crispatus enhance activation of human plasminogen. Microbiology
153: 1112-1122
[Abstract]
[Full Text]
-
Sun, H.
(2006). The interaction between pathogens and the host coagulation system.. Physiology
21: 281-288
[Abstract]
[Full Text]
-
Tapper, H., Herwald, H.
(2000). Modulation of hemostatic mechanisms in bacterial infectious diseases. Blood
96: 2329-2337
[Full Text]
-
Monroy, V., Amador, A., Ruiz, B., Espinoza-Cueto, P., Xolalpa, W., Mancilla, R., Espitia, C.
(2000). Binding and Activation of Human Plasminogen by Mycobacterium tuberculosis. Infect. Immun.
68: 4327-4330
[Abstract]
[Full Text]
Top
Abstract
Introduction
