Next Article 
Infection and Immunity, April 2001, p. 1977-1982, Vol. 69, No. 4
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.1977-1982.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Plasminogen Binding and Activation by
Mycoplasma fermentans
Amichai
Yavlovich,1
Abd A.-R.
Higazi,2 and
Shlomo
Rottem1,*
Department of Membrane and Ultrastructure
Research, The Hebrew University-Hadassah Medical
School,1 and Department of Clinical
Biochemistry, Hadassah Hospital, Mount Scopus,2
Jerusalem, Israel
Received 23 June 2000/Returned for modification 14 September
2000/Accepted 15 December 2000
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ABSTRACT |
The binding of plasminogen to Mycoplasma fermentans was
studied by an immunoblot analysis and by a binding assay using
iodine-labeled plasminogen. The binding of 125I-labeled
plasminogen was inhibited by unlabeled plasminogen, lysine, and lysine
analog 
-aminocaproic acid. Partial inhibition was obtained by a
plasminogen fragment containing kringles 1 to 3 whereas almost no
inhibition was observed with a fragment containing kringle 4. Scatchard
analysis revealed a dual-phase interaction, one with a dissociation
constant (kd) of 0.5 µM and the second with a
kd of 7.5 µM. The estimated numbers of
plasminogen molecules bound were calculated to be 110 and 790 per
cell, respectively. Autoradiograms of ligand blots containing M. fermentans membrane proteins incubated with
125I-labeled plasminogen identified two plasminogen-binding
proteins of about 32 and 55 kDa. The binding of plasminogen to M. fermentans enhances the activation of plasminogen to plasmin by
the urokinase-type plasminogen activator (uPA), as monitored by
measuring the breakdown of chromogenic substrate S-2251. Enhancement
was more pronounced with the low-molecular-weight and the single-chain
uPA variants, known to have low plasminogen activator activities. The
binding of plasminogen also promotes the invasion of HeLa cells by
M. fermentans. Invasion was more pronounced in the presence
of uPA, suggesting that the ability of the organism to invade host
cells stems not only from its potential to bind plasminogen but also from the activation of plasminogen to plasmin.
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INTRODUCTION |
Mycoplasmas (class
Mollicutes) are wall-less prokaryotes widely distributed in
nature. Most mycoplasmas are parasites, exhibiting strict host and
tissue specificities, and almost all of them are bound to the surface
of the host cell (22, 24). Human pathogen Mycoplasma
fermentans was isolated from the urogenital tract several decades
ago (25). The interest in this organism has recently increased because of its possible role in the pathogenesis of rheumatoid arthritis and reports indicating that this organism may
function as a cofactor accelerating the progression of human immunodeficiency virus disease (17, 23).
Plasminogen (Pg) is a 92-kDa plasma glycoprotein. This protein is
activated in vivo to the serine protease plasmin by urokinase-type and
tissue-type Pg activators (uPA and tPA, respectively) by cleavage of a
single peptide bond (R561-V562) yielding two
chains that remain connected by two disulfide bridges (26). Plasmin participates in several physiological and
pathophysiological processes such as fibrinolysis, pericellular
proteolysis, tissue penetration of cancer cells, and neuronal cell
death (19, 21, 26). The active domain of Pg is located in
the COOH terminal of the molecule, whereas the NH2 terminal
contains five characteristic triple-loop structures (kringles) that
mediate the interaction of Pg with a variety of ligands such as fibrin,
the 
2-plasmin inhibitor, etc. (21). This
interaction is between lysine-binding sites in the kringles and
exposed COOH-terminal lysines in the ligands (26).
Therefore, lysine or lysine analogs such as -aminocaproic acid
(ACA) mimic COOH-terminal lysine by inhibiting the interaction.
Many eucaryotic cells express surface structures that interact with Pg,
and specific receptors have been described (21). Recently
it became evident that Pg is also capable of interacting with
receptors on several prokaryotic organisms, including both gram-positive (4, 15, 18, 34) and gram-negative bacteria (12, 13, 20, 32, 33). In the present study we extend the
group of bacteria capable of binding Pg to include the wall-less M. fermentans and show that Pg interacts with two surface
proteins of this organism. The association of Pg with M. fermentans greatly enhances its uPA-associated activation. The
possible role of Pg binding in M. fermentans-host
interactions is discussed.
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MATERIALS AND METHODS |
Organisms and growth conditions.
M. fermentans
strain PG-18 was used throughout the study. In some experiments strains
M-52, M-32, AOU, and Z-62 (kindly provided by P. Hannan, Surrey, United
Kingdom) and the incognitus strain (obtained from the American Type
Culture Collection, Manassas, Va.) were also utilized. The organisms
were grown in Channock medium supplemented with 5 to 10% horse serum
(7). The cultures were grown for 24 to 72 h at
37°C. Growth was monitored by measuring the absorbance of the culture
at 640 nm and by recording pH changes in the growth medium. The
organisms were collected by centrifugation at 12,000 × g for 20 min, washed twice, and resuspended in a cold solution of 10 mM Tris-HCl in 250 mM NaCl (pH 7.5; TN buffer) to a
protein concentration of 0.5 to 1 mg/ml. The number of viable cells was
determined by the plating method and is presented as CFU. Membrane and
soluble-fraction preparations were obtained from intact cells by
ultrasonic treatment as described before (27). The
membranes were washed twice and resuspended in 10 mM Tris-HCl buffer
(pH 7.5).
Pg binding assay.
A qualitative determination of Pg binding
to M. fermentans strains was performed by immunodot blot
analysis. M. fermentans cells (1 mg of protein) were
incubated with 25 µg of Pg in TN buffer. After 0.5 to 2 h of
incubation at 37°C, the cells were pelleted, washed three times, and
resuspended in the TN buffer. The washed-cell suspension was than
immobilized on a nitrocellulose membrane with a Bio-Dot apparatus
(Bio-Rad Laboratories). Alternatively, the bound Pg was released from
the M. fermentans cells by incubating the cells in 10 mM
ACA for 20 min at 37°C. The intact cells were removed by
centrifugation at 8,000 × g, and the supernatant fluid containing the Pg released was immobilized. The nitrocellulose membranes were processed by (i) blocking for 1 h at room
temperature with skim milk, (ii) incubation for 16 h at 4°C with
goat anti-human Pg antiserum, and (iii) incubation at room temperature
for 1 h with horseradish peroxidase-conjugated mouse anti-goat
immunoglobulin G. Blots were developed by using the
o-dianisidine substrate (Sigma) according to the
manufacturer's recommendations.
For quantitative binding experiments, Pg preparations were labeled with
125I (Amersham, Little Chalfont, United Kingdom) by the
chloramine-T method. Pg (25 µg in 100 µl of 10 mM
Na2HPO4, pH 7.5) was mixed with 0.5 mCi of
125I. Chloramine-T was then added to a final
concentration of 1 mg/ml, and the samples were incubated at room
temperature for 1 min. The reaction was stopped by the addition of 100 µl of sodium metabisulfite (1 mg/ml) and 50 µl of KI (0.3 mg/ml).
The labeled protein was separated from free 125I by gel
filtration on a Sephadex G-25 column. Labeled Pg was diluted to
appropriate concentrations and kept at 
20°C. The specific activity
of the labeled Pg was 10,000 cpm/ng. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis before and after reduction confirmed that no degradation of Pg had occurred.
Pg binding was assayed at 37°C in a reaction mixture (total volume of
0.5 ml) containing
M. fermentans cells (0.5 mg of cell
protein, 5 × 10
11 CFU/ml) with 15 ng of radiolabeled
Pg in TN buffer. After 5 to
45 min of incubation, the reaction mixtures
were then transferred
onto the surface of 0.5 ml of silicone oil
(XF-1792-B; Dexter
Hysol, Olean, N.Y.) in 1.5-ml plastic centrifuge
tubes and centrifuged
at 12,800 ×
g for 2 min. Under these
conditions, the cells pass
through the silicone oil, forming a pellet
at the bottom of the
tube, while the aqueous phase remains on top of
the silicone oil
(
28). The aqueous and silicone phases
were removed by suction,
the tips of the centrifuge tubes containing
the cell pellets were
cut off, and radioactivity was counted. To
measure the extracellular
space, [
14C]inulin (Amersham)
was used instead of Pg. Since inulin cannot
bind or enter the cells,
[
14C]inulin counts in the cell pellet were taken as a
measure of
extracellular space. This space was found to be 5 ± 0.5 µl per
mg of cell protein. For Scatchard analysis, serial
dilutions of
labeled Pg (1 ng/ml to 20 µg/ml) were added to
M. fermentans using
the procedure described above. Experiments were
done in triplicate.
Specific binding was calculated by subtracting
nonspecific binding
from total binding. The ratio of bound/free Pg was
plotted as
a function of bound Pg using Scatchard analysis
(
14). Linear
regression analysis was used for curve
fitting; the slope represented
1/
kd, where
kd is the dissociation constant, and the
x intercept
represented the total number of
receptors.
Inhibition of Pg binding.
Inhibition assays were performed
by preincubating 250 µl of M. fermentans PG-18 cells (0.5 mg of protein) with 100 µl of each inhibitor for 1 h at 4°C.
The inhibitors used were lysine, ACA, glycine, glucose, bovine serum
albumin (BSA), unlabeled Pg, Pg-lysine-binding site I (kringles 1 to
3), and Pg-lysine-binding site II (kringle 4). All inhibitors were the
products of Sigma. The inhibitors were tested at concentrations of 10 to 100 µg/ml. Subsequently, 10 µl of radiolabeled Pg (containing
100,000 cpm) was added and the binding assay was performed as described above.
Pg activation assay.
Pg was purified from human plasma as
described by Deutsch and Mertz (6). Pg activation was
assayed as described by Parkkinen et al. (20) with minor
modifications; the reaction mixture contained cells (5 µg of protein;
5 × 109 CFU), 150 ng of Pg, 10 ng of two-chain uPA
(tcuPA; American Diagnostic Inc.), 2 µl of a 2-mg/ml concentration of
chromogenic substrate S-2251
(H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline
dihydrochloride; Bio-Fine, Stockholm, Sweden), and
phosphate-buffered saline (PBS) to 150 µl. In some experiments, tcuPA
was replaced by low-molecular-weight tcuPA (LMW-tcuPA) or by
single-chain uPA (scuPA) (American Diagnostic Inc.). After various
periods of incubation (up to 2 h) at room temperature, hydrolysis
of S-2251 that resulted in the formation of p-nitroanilide
was measured spectroscopically at 405 nm. Controls were run without Pg
to assess a direct amidolytic activity on S-2251 or without exogenous
tcuPA to assess uPA-like activity of the cells and to assure that the
Pg was not contaminated with plasmin.
Internalization of M. fermentans.
The ability of
M. fermentans to penetrate eucaryotic cells was measured
with a HeLa cell culture as previously described (1). In
brief, HeLa cells were seeded in 24-well plates at a density of
105 cells per well and after 24 h of incubation the
cultures were inoculated with 10 µl of untreated M. fermentans cells or cells treated at 37°C for 30 min with Pg (25 µg) with or without tcuPA (50 U). The treated cells were harvested,
washed twice, and resuspended in TN buffer. The M. fermentans preparations were utilized at a multiplicity of
infection of 100. The infected HeLa cell cultures were incubated for
3 h, washed twice with PBS, and incubated for an additional 2 h in Dulbecco modified Eagle medium (DMEM) (1 ml/well) containing 400 µg of gentamicin ml
1 and 0.01% Triton X-100. The
medium was then removed and the HeLa cells were trypsinized,
resuspended, diluted in DMEM, and plated. The invasion of HeLa cells by
M. fermentans was also studied by immunofluorescence
staining of HeLa cells permeabilized by Triton X-100 using a polyclonal
rabbit anti-M. fermentans antiserum followed by analysis
with a Sarastro Phoibos 1000 laser scanning confocal microscope
(Molecular Dynamics, Sunnyvale, Calif.) as previously described
(2).
Analytical methods.
Protein was determined by the method of
Bradford (3) using BSA as the standard. NADH dehydrogenase
activity was determined spectrophotometrically (27) in the
presence of sodium deoxycholate (1 mg/ml). For proteolytic digestion of
surface proteins, 1 mg of M. fermentans cell protein was
incubated with proteinase K (25 µg) for 30 min at 37°C in TN buffer
containing 10 mM CaCl2. Identification of membrane proteins
that bind Pg was performed by binding I125-labeled Pg or by
an immunoblot assay (3). In brief, membrane proteins were
separated by SDS-polyacrylamide gel electrophoresis (16)
and the protein bands were electroblotted onto nitrocellulose paper in
a Tris-glycine-SDS solution at 250 mA for 2 h at 10°C. The
nitrocellulose paper was then incubated with I125-labeled
Pg (10 ng/ml) for 1 h at room temperature, washed three times with
0.1% Tween 20 in PBS, dried, and autoradiographed (Fuji X-ray film).
For immunoblotting, the nitrocellulose paper was incubated with Pg (50 µg/ml) for 1 h at room temperature and then (i) washed several
times in 0.1% Tween 20 in PBS solution (ii) incubated for 16 h at
4°C with monospecific goat anti-human Pg polyclonal antibodies
(Sigma), and (iii) incubated for 1 h at room temperature with
mouse anti-goat immunoglobulin G peroxidase-conjugated antibodies
(Sigma). Pg-bound bands were detected with ECL Western blotting
detection reagents (Amersham) according to the manufacturer's instructions.
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RESULTS AND DISCUSSION |
Pg binding to M. fermentans.
Pg binding,
determined by the immunoblot assay, revealed that M. fermentans cells are capable of binding Pg. The levels of Pg bound
to the urogenital isolates (PG-18 and incognitus), respiratory isolates
(M-39 and M-52), and a strain isolated from leukemic bone marrow (Z-62)
were almost the same (data not shown). The binding of Pg to M. fermentans was further studied by monitoring the association of
125I-labeled Pg to M. fermentans cells (Fig.
1). Figure 1 shows that urogenital strain
PG-18 and pulmonary isolate M-52 reacted with Pg to about the same
extent. With both strains, binding was increased with the increase in
the amount of mycoplasmas present in the reaction mixture, indicating
that Pg binding was directly correlated with the presence of
mycoplasmas and not with nonspecific factors.
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FIG. 1.
Binding of125I-Pg to M. fermentans. 125I-Pg (10 ng, 100,000 cpm) was incubated
with M. fermentans strain PG-18 ( ) or M-52 ( ) at
37°C for 45 min. Binding was assayed as described in Materials and
Methods. The results are the means ± standard deviations of three
independent experiments with duplicate samples.
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There were no significant differences in
125I-labeled Pg
binding among
M. fermentans PG-18 cells grown in a medium
containing
various concentrations of horse serum (4 to 20%) or between
cells
washed with TN buffer containing
ACA (1 mg/ml), known to elute
bound Pg from various bacteria (
19), and cells washed with
TN
buffer alone (data not shown). These results suggest that Pg-related
molecules that might be present in the horse serum component of
the
growth medium do not occupy the Pg binding sites on
M. fermentans cells. Levels of Pg binding for cells harvested at the
early exponential
(
A640 = 0.08; pH = 7.2), late exponential (
A640 = 0.23; pH
6.4),
or stationary (
A640 = 0.25; pH 6.0)
phase of growth were almost
the same and were not affected by
pretreating the cells with a
10 mM concentration of either NaF, sodium
arsenate, or sodium
iodoacetate (data not shown). Isolated membrane
preparations obtained
from
M. fermentans strain PG-18 cells
exhibited a Pg binding activity
similar to that of intact cells.
However, treatment of the membrane
preparations or of intact
M. fermentans cells with proteinase
K reduced the Pg binding activity
by over 85%. The proteolytic
treatment of the intact cells did not
affect the intracellular
NADH dehydrogenase activity of
M. fermentans; thus, proteolysis
apparently did not affect cell
intactness. These results suggest
that the Pg binding component is a
protein present on the
M. fermentans cell
surface.
The results of experiments in which unlabeled Pg competed with
125I-labeled Pg are shown in Fig.
2. Inhibition of
125I-labeled
Pg association with
M. fermentans increased with increasing
amounts of unlabeled Pg. Inhibition reached almost 50% at a 100-fold
excess of unlabeled Pg and almost 100% at a 1,000-fold excess.
These
results demonstrate that a major portion of Pg binding to
M. fermentans is specific. To estimate
kds for
receptor interaction,
Scatchard plots were made (inset to Fig.
2);
these plots showed
a two-phase interaction, indicating the possibility
of two different
receptor structures, one with a
kd value of 0.5 µM and the second
with a
kd value of 7.5 µM. The calculated numbers of
Pg molecules
bound per bacteria were 110 for the first phase and 790 for the
second. Multiple Pg-binding proteins, often with both high and
low affinities, have also been found in other bacteria which bind
Pg
(
12,
19,
33). Pg binding to
M. fermentans was
very little
affected by EDTA (10 mM) and was independent of
Ca
2+ or Mg
2+ (1 to 10 mM) added to the reaction
mixture. However, a twofold
increase in binding was observed with
Zn
2+. The highest Zn
2+ activity was obtained at
a concentration of
1 mM (data not shown).
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FIG. 2.
Pg binding to M. fermentans cells. M. fermentans cells were incubated with increasing concentrations of
Pg and binding was determined as described in Materials and Methods.
(Inset) Scatchard plot analysis of the binding process. The results are
means ± standard deviations from four independent experiments
with duplicate samples.
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The binding of
125I-Pg to
M. fermentans was
specific, as nonlabeled Pg, lysine, and
ACA markedly inhibited the
binding, whereas
another amino acid (Gly), glucose, or albumin had no
effect (Fig.
3). The figure also shows
that the Pg fragment containing the
first three kringles (Pg-1) had a
pronounced inhibitory effect
on
125I binding, whereas the
fragment containing kringle 4 (Pg-2) had
very little effect,
suggesting a role for the first three kringles
(k
1 to
k
3) of Pg in the interaction(s) with the cell surface
receptor(s)
of
M. fermentans. These three kringles (mainly
kringle 2) were
also shown to interact with a Pg-binding surface
protein of group
A streptococci (
34). The inhibition
obtained with lysine or
with lysine analog
ACA reveals the
significance in the binding
process of the lysine-binding sites on the
heavy chain of Pg (
19,
21).
ACA, which has been shown
to inhibit Pg binding to various
bacteria (
19), was found
to be a very efficient inhibitor of
Pg binding to
M. fermentans (Fig.
4). As expected,
ACA was capable
of eluting bound Pg from the cell surface of
M. fermentans. The
elution was more efficient in the
presence of EDTA. Thus, whereas
in the absence of EDTA only about 55%
of the bound Pg was eluted
by 10 µg of
ACA/ml (Fig.
4), in the
presence of 10 mM EDTA, over
90% of the bound Pg was eluted (data not
shown).
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FIG. 3.
Inhibition of125I-Pg binding to M. fermentans. M. fermentans cells (0.5 mg of protein)
were incubated with the various inhibitors for 1 h at 4°C.
Lysine, glycine, ACA (ACA), glucose, and BSA were added to a final
concentration of 100 µg/ml. Pg, Pg-1 (Pg-lysine-binding site I), and
Pg-2 (Pg-lysine-binding site II) were added to 10 µg/ml.
125I-Pg (10 ng) was then added, and the binding experiment
was performed as described in Materials and Methods. The results are
the means ± standard deviations of four experiments with
duplicate samples.
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FIG. 4.
Effect of ACA on the binding of125I-Pg to
M. fermentans. Cells (0.5 mg of protein) were incubated with
various concentrations of ACA for 1 h at 4°C.
125I-Pg (10 ng) was then added, and binding was determined
as described in Materials and Methods. The results are the means ± standard deviations from four independent experiments with duplicate
samples.
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Autoradiograms of ligand blots containing protein of
M. fermentans PG-18 membranes incubated with
125I-labeled
Pg are shown in Fig.
5. The solubilized
membranes showed
binding of
125I-labeled Pg to ~32-and
~55-kDa protein bands. Similar results
were obtained when blots of
M. fermentans membrane protein were
reacted with
monospecific polyclonal antibodies raised against
human Pg (data not
shown). No
125I-labeled protein bands were detected with
the soluble-protein
fraction of
M. fermentans cells or with
membrane preparations
obtained from proteinase K-treated
M. fermentans cells (data not
shown). Radioactivity analysis of the
125I-labeled protein bands revealed that the levels of
labeling of
the 55- and 32-kDa protein bands were 32% ± 4% and 52% ± 6%, respectively,
of the total radioactivity bound to the
nitrocellulose blots.
The presence of two binding sites is consistent
with the Scatchard
plot analysis of the kinetics of Pg binding to
M. fermentans (Fig.
2), which showed high- and low-affinity
sites. Multiple Pg binding
sites, often with both high and low
affinities, in other bacteria
have also been described (
12,
19,
33).
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FIG. 5.
Analysis of Pg-binding proteins in M. fermentans membranes. Membrane proteins (20 µg) were resolved by
electrophoresis on an SDS-11% polyacrylamide gel and electroblotted
to nitrocellulose paper as described in Materials and Methods. Lane A,
Coomassie blue-stained gel; lane B, blot incubated with
125I-Pg followed by autoradiography to detect bound Pg;
lane C, protein standards.
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Activation of Pg-bound M. fermentans.
Figure
6A shows that M. fermentans
PG-18 cells enhance the tcuPA-mediated activation of Pg. In the absence
of cells, activation of Pg was significantly lower, reaching, after 45 min of incubation, about 60% of the activation levels observed in the
presence of mycoplasmas, suggesting that the interaction of Pg or tcuPA
with the cells facilitates the formation of active plasmin. The effect of M. fermentans cells on tcuPA activation was less
pronounced than the effect of these cells on tPA (tissue Pg activator)
enhanced activity of Pg (30). The figure also shows that
no hydrolysis of S-2251 was observed in a medium containing M. fermentans cells and Pg, but without tcuPA, suggesting that
M. fermentans does not produce plasmin activators such as
streptokinase and staphylokinase (19).
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FIG. 6.
Effect of M. fermentans on the activation of
Pg by uPA. Pg was incubated with uPA preparations in the presence ( )
or absence () of M. fermentans cells (5 × 109) at room temperature for up to 2 h. Hydrolysis of
S-2251 as a result of plasmin formation was measured
spectrophotometrically as described in Materials and Methods. The uPA
preparations utilized were tcuPA (A), LMW-tcuPA (B), and scuPA (C).
 , control with M. fermentans cells but without uPA. The
results are the means ± standard deviations of three independent
experiments with duplicate samples.
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uPA is synthesized as a single-chain molecule (scuPA) that is activated
by the hydrolysis of a peptide bond yielding two polypeptide
chains
that are held together by a disulfide bridge (
11). The
conversion of scuPA to tcuPA exposes the catalytic site to Pg.
Thus, in
the absence of a uPA receptor, tcuPA initiates the process
of plasmin
formation, whereas plasmin formation by scuPA is very
low
(
11). Indeed, Fig.
6C shows that in the absence of
M. fermentans no plasmin formation was observed with scuPA.
Low plasmin levels
were detected with a low-molecular-weight
variant of tcuPA (LMW-tcuPA)
(Fig.
6B) obtained by proteolysis.
This variant contains the protease
domain but lacks the growth factor
domain at the amino terminal
(
11). Although
M. fermentans cells did not bind any of the three
uPA preparations
utilized (data not shown), with all the preparations,
plasmin formation
in the presence of
M. fermentans cells was significantly
higher than that in the absence of the mycoplasma (Fig.
6). The
effect
of
M. fermentans was most pronounced with scuPA (Fig.
6C).
These results indicate that Pg bound to
M. fermentans
undergoes
conformational changes that modulate its susceptibility to
activation
by scuPA, LMW-tcuPA, and
tcuPA.
Despite the reports of the capability of bacteria to bind Pg and/or
plasmin, the importance of such binding for virulence
has not yet been
established. The interaction of
M. fermentans with the host
Pg system allows the bacteria to acquire a surface-associated
host
protease that cannot be regulated (
18). Unlike other
bacteria
M. fermentans does not appear to secrete endogenous
serine proteases.
Thus, the acquisition of host plasmin, a serine
protease with
broad substrate specificity, is an important strategy for
M. fermentans.
This may give the organisms, after they
penetrate the epithelial
layer of the urogenital system, the ability to
escape easily from
a fibrin network deposited by the host and hydrolyze
matrix proteins
(
19), permitting their spread through
connective tissues and
evasion of the inflammatory response.
Nonetheless,
M. fermentans does not produce plasmin
activators such as streptokinase and
staphylokinase (
19).
Thus, the generation of plasmin relies
on host Pg activators. These
activators are released at the site
of infection by macrophages and
monocytes (
19,
21).
Invasion of HeLa cells by Pg-bound M. fermentans.
Although M. fermentans is considered to be a surface
parasite associated with the surfaces of host cells (22,
24), this organism has been detected also within cells
(31). The ability of M. fermentans to invade
eucaryotic cells was studied using a HeLa cell line. The bactericidal
antibiotic gentamicin in combination with a low concentration of Triton
X-100 was utilized to kill mycoplasmas that had not entered the cells,
allowing the quantitation of the internalized organism
(1). Table 1 shows that no internalization of untreated
M. fermentans cells by HeLa cells was observed. Under the
same conditions, Mycoplasma penetrans was shown to be
invasive and over 48,000 CFU were internalized by 106 HeLa
cells (data not shown). Nevertheless, when M. fermentans cells were incubated with Pg, internalization was apparent (Table 1). Internalization of Pg-treated
M. fermentans was more pronounced with the tcuPA-treated
Pg-bound M. fermentans cells. Internalization was also
observed by immunofluorescence. When HeLa cells were infected with
tcuPA-treated Pg-bound M. fermentans and incubated with
anti-M. fermentans antibodies followed by a second
fluorescein isothiocyanate conjugated-antibody, numerous foci of
fluorescence were observed on the HeLa cell surface as well as within
the cells (data not shown). Internalization was not detected in control HeLa cells infected with untreated M. fermentans. Bacterial
invasion of eucaryotic cells is a complex process that involves a
variety of bacterial and host cell factors. It has been recently shown that bacterial invasion is based on the ability of several bacteria to
bind sulfated polysaccharides (9) or fibronectin
(10). It was suggested that these compounds form a
molecular bridge between the bacteria and different types of eukaryotic
surface proteins (4, 8) that enables invasion. The finding
that in the presence of tcuPA invasion was more pronounced suggests also that the ability of M. fermentans to invade host cells
stems not only from its potential to bind Pg but also from its
activation to plasmin. Plasmin, a protease with broad substrate
specificity, may alter M. fermentans cell surface proteins
and thereby enable its internalization. The enhanced invasiveness of
plasmin-coated Borrelia burgdorferi was described recently
(5). Proteolytic modification of bacterial and/or host
cell surface proteins is an emerging theme among bacterial pathogens.
Thus, the Pg activator of Yersinia pestis degrades the
bacterial outer membrane protein and is associated with virulence
(29) and a secreted protease was shown to stimulate the
fibronectin-dependent uptake of Streptococcus pyogenes into
eucaryotic cells (4).
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ACKNOWLEDGMENTS |
The technical assistance of Edna Hiss and Avigail Katzenel was
greatly appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Membrane and Ultrastructure Research, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. Phone: 972-2-6578148. Fax:
972-2-678 4010. E-mail: Rottem{at}cc.huji.ac.il.
Editor:
V. J. DiRita
| |
REFERENCES |
| 1.
|
Andreev, J.,
Z. Borovsky,
I. Rosenshine, and S. Rottem.
1995.
Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein.
FEMS Microbiol. Lett.
132:189-194[CrossRef][Medline].
|
| 2.
|
Borovsky, Z.,
M. Tarshis,
P. Zhang, and S. Rottem.
1998.
Protein kinase C activation and vacuolation in HeLa cells invaded by Mycoplasma penetrans.
J. Med. Microbiol.
47:915-922[Abstract/Free Full Text].
|
| 3.
|
Bradford, M. M.
1976.
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[CrossRef][Medline].
|
| 4.
|
Chaussee, M. S.,
R. L. Cole, and J. P. M. van Putten.
2000.
Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells.
Infect. Immun.
68:3226-3232[Abstract/Free Full Text].
|
| 5.
|
Coleman, J. L.,
E. J. Roemer, and J. L. Benach.
1999.
Plasmin-coated Borrelia burgdorferi degrades soluble and insoluble components of the mammalian extracellular matrix.
Infect. Immun.
67:3929-3936[Abstract/Free Full Text].
|
| 6.
|
Deutsch, D. G., and E. T. Mertz.
1970.
Plasminogen purification from human plasma by affinity chromatography.
Science
170:1095-1096[Abstract/Free Full Text].
|
| 7.
|
Deutsch, J.,
M. Salman, and S. Rottem.
1995.
An unusual polar lipid from the cell membrane of Mycoplasma fermentans.
Eur. J. Biochem.
227:897-902[Medline].
|
| 8.
|
Duensing, T. D., and J. P. M. van Putten.
1998.
Vitronectin binds to gonococcal adhesin OpaA through a glycosaminoglycan molecular bridge.
Biochemistry
334:133-139.
|
| 9.
|
Duensing, T. D.,
J. S. Wing, and J. P. M. van Putten.
1999.
Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenesis.
Infect. Immun.
67:4463-4468[Abstract/Free Full Text].
|
| 10.
|
Dziewanowska, K.,
J. M. Platt,
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].
|
| 11.
|
Higazi, A. A.-R., and D. Cines.
1996.
Regulation of single chain urokinase by small peptides.
Thromb. Res.
84:243-225[CrossRef][Medline].
|
| 12.
|
Hu, L. T.,
G. Perides,
R. Noring, and M. S. Klempner.
1995.
Binding of human plasminogen to Borrelia burgdorferi.
Infect. Immun.
63:3491-3496[Abstract].
|
| 13.
|
Khin, M. M.,
M. Ringer,
P. Aleljung,
T. Wadstrom, and B. Ho.
1996.
Binding of human plasminogen and lactoferrin by Helicobacter pylori coccoid forms.
J. Med. Microbriol.
45:433-439.
|
| 14.
|
Klotz, I. M.
1982.
Number of reactor sites from Scatchard graphs: facts and fantasies.
Science
214:1247-1249.
|
| 15.
|
Kuusela, P., and O. Saksela.
1990.
Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand.
Eur. J. Biochem.
193:759-765[Medline].
|
| 16.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[CrossRef][Medline].
|
| 17.
|
Lo, S. C.
1992.
Mycoplasma and AIDS, p. 525-548.
In
J. Maniloff, R. N. Finch, and J. B. Baseman (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C.
|
| 18.
|
Lottenberg, R.,
C. C. Broder, and M. D. Boyle.
1987.
Identification of a specific receptor for plasmin on a group A streptococcus.
Infect. Immun.
55:1914-1918[Abstract/Free Full Text].
|
| 19.
|
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[CrossRef][Medline].
|
| 20.
|
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].
|
| 21.
|
Plow, E. F.,
T. Herren,
A. Redlitz,
L. A. Miles, and J. L. Hoover-Plow.
1995.
The cell biology of the plasminogen system.
FASEB J.
9:939-945[Abstract].
|
| 22.
|
Razin, S., and E. Jacobs.
1992.
Mycoplasma adhesion.
J. Gen. Microbiol.
138:407-422[Free Full Text].
|
| 23.
|
Razin, S.,
D. Yogev, and Y. Naot.
1998.
Molecular biology and pathogenicity of mycoplasmas.
Microbiol. Mol. Biol. Rev.
62:1094-1156[Abstract/Free Full Text].
|
| 24.
|
Rottem, S., and Y. Naot.
1998.
Subversion and exploitation of host cells by mycoplasmas.
Trends Microbiol.
6:436-440[CrossRef][Medline].
|
| 25.
|
Ruiter, M., and H. M. M. Wentholt.
1952.
The occurrence of a pleuropneumonia-like organism in the fuso-spirillary infections of the human genital mucosa.
J. Investig. Dermatol.
18:313-323[Medline].
|
| 26.
|
Saksela, O., and D. P. Rifkin.
1988.
Cell-associated plasminogen activation: regulation and physiological functions.
Annu. Rev. Cell Biol.
4:93-126[CrossRef].
|
| 27.
|
Salman, M., and S. Rottem.
1995.
The cell membrane of Mycoplasma penetrans: lipid composition and phospholipase A1 activity.
Biochim. Biophys. Acta
1235:369-377[Medline].
|
| 28.
|
Shirvan, M. W.,
S. Shuldiner, and S. Rottem.
1989.
Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.
J. Bacteriol.
171:4417-4424[Abstract/Free Full Text].
|
| 29.
|
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].
|
| 30.
|
Tarshis, M.,
B. Morag, and M. Mayer.
1993.
Mycoplasma cells stimulate in vitro activation of plasminogen by purified tissue-type plasminogen activator.
FEMS Microbiol. Lett.
106:201-204[CrossRef][Medline].
|
| 31.
|
Taylor-Robinson, D.,
H. A. Davies,
P. Saratheandra, and P. M. Furr.
1991.
Intracellular location of mycoplasmas in cultured cells demonstrated by immunocytochemistry and electron microscopy.
Int. J. Exp. Pathol.
73:705-714.
|
| 32.
|
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].
|
| 33.
|
Ullberg, M.,
P. Kuusela,
B. E. Kristiansen, and G. Kronvall.
1992.
Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoae and formation of surface-associated plasmin.
J. Infect. Dis.
166:1329-1334[Medline].
|
| 34.
|
Wistedt, A. C.,
H. Kotarsky,
D. Marti,
W. Ringdahl,
F. J. Castellino,
J. Schaller, and U. Sjobring.
1998.
Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM.
J. Biol. Chem.
273:24420-24424[Abstract/Free Full Text].
|
Infection and Immunity, April 2001, p. 1977-1982, Vol. 69, No. 4
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.4.1977-1982.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
