INFECTION AND IMMUNITY, Apr. 2001, p. 1977–1982 0019-9567/01/$04.00⫹0 DOI: 10.1128/IAI.69.4.1977–1982.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 4
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
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 M and the second with a kd of 7.5 M. 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-molecularweight 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. 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 ε-aminocaproic acid (εACA) 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). Re-
cently 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. 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 g 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 εACA 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
* 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:
[email protected]. 1977
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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 g in 100 l of 10 mM Na2HPO4, pH 7.5) was mixed with 0.5 mCi of 125 I. 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 l of sodium metabisulfite (1 mg/ml) and 50 l 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 ⫻ 1011 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 (XF1792-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 l per mg of cell protein. For Scatchard analysis, serial dilutions of labeled Pg (1 ng/ml to 20 g/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 l of M. fermentans PG-18 cells (0.5 mg of protein) with 100 l of each inhibitor for 1 h at 4°C. The inhibitors used were lysine, εACA, 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 g/ml. Subsequently, 10 l 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 g of protein; 5 ⫻ 109 CFU), 150 ng of Pg, 10 ng of two-chain uPA (tcuPA; American Diagnostic Inc.), 2 l 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 l. 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 l of untreated M. fermentans cells or cells treated at 37°C for 30 min with Pg (25 g) 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 g 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).
FIG. 1. Binding of125I-Pg to M. fermentans. 125I-Pg (10 ng, 100,000 cpm) was incubated with M. fermentans strain PG-18 (E) or M-52 (䊐) 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.
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 g) 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 g/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 peroxidaseconjugated antibodies (Sigma). Pg-bound bands were detected with ECL Western blotting detection reagents (Amersham) according to the manufacturer’s instructions.
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 (Z62) 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. 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.
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 εACA (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 125Ilabeled 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
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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, εACA (ACA), glucose, and BSA were added to a final concentration of 100 g/ml. Pg, Pg-1 (Pg-lysinebinding site I), and Pg-2 (Pg-lysine-binding site II) were added to 10 g/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.
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 M and the second with a kd value of 7.5 M. 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 Ca2⫹ or Mg2⫹ (1 to 10 mM) added to the reaction mixture. However, a twofold increase in binding was observed with Zn2⫹. The highest Zn2⫹ activity was obtained at a concentration of ⱖ1 mM (data not shown). The binding of 125I-Pg to M. fermentans was specific, as nonlabeled Pg, lysine, and εACA 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 (k1 to k3) 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 εACA reveals the significance in the binding
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FIG. 4. Effect of εACA on the binding of125I-Pg to M. fermentans. Cells (0.5 mg of protein) were incubated with various concentrations of εACA 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.
FIG. 5. Analysis of Pg-binding proteins in M. fermentans membranes. Membrane proteins (20 g) 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.
process of the lysine-binding sites on the heavy chain of Pg (19, 21). εACA, 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, εACA 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 g of εACA/ml (Fig. 4), in the presence of 10 mM EDTA, over 90% of the bound Pg was eluted (data not shown). 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 125Ilabeled 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). 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). 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.
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TABLE 1. Invasion of HeLa cells by M. fermentansa
M. fermentans preparation
Untreated Pg treated Pg ⫹ tcuPA treated
No. of M. fermentans cells invading indicated HeLa cells (CFU/106 HeLa cells) Native
PFA-treated
2,500 ⫾ 500 18,000 ⫾ 2,000 24,000 ⫾ 3,500
⬍1,000 1,500 ⫾ 500 1,500 ⫾ 1,000
a M. fermentans cells were grown to mid-exponential phase and treated with Pg and tcuPA as described in Materials and Methods. Native or paraformaldehyde (PFA; 4%)-treated HeLa cells were infected at a multiplicity of infection of 100 and incubated at 37°C. Internalization was determined after 3 h of incubation as described in Materials and Methods. Values are means ⫾ standard deviations.
FIG. 6. Effect of M. fermentans on the activation of Pg by uPA. Pg was incubated with uPA preparations in the presence (F) or absence (E) 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.
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 tcuPAtreated 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
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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). ACKNOWLEDGMENTS The technical assistance of Edna Hiss and Avigail Katzenel was greatly appreciated. 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. 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. 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. 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. 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. 6. Deutsch, D. G., and E. T. Mertz. 1970. Plasminogen purification from human plasma by affinity chromatography. Science 170:1095–1096. 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. 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. 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. 11. Higazi, A. A.-R., and D. Cines. 1996. Regulation of single chain urokinase by small peptides. Thromb. Res. 84:243–225. 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. 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.
Editor: V. J. DiRita
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