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Published as doi: 10.1096/fj.06-7992com.
 (The FASEB Journal. 2007;21:3118-3132.)
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Thrombospondin-1 promotes cellular adherence of Gram-positive pathogens via recognition of peptidoglycan

Claudia Rennemeier*,1, Sven Hammerschmidt*,1,2, Silke Niemann{dagger}, Seiichi Inamura{ddagger}, Ulrich Zähringer{ddagger} and Beate E. Kehrel{dagger}

* University of Wuerzburg, Research Center for Infectious Diseases, Wuerzburg, Germany;

{dagger} Department of Anaesthesiology and Intensive Care, Experimental and Clinical Haemostasis, University Hospital Muenster, Muenster, Germany; and

{ddagger} Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

2Correspondence: Max von Pettenkofer-Institute for Hygiene and Microbiology, Ludwig-Maximilians University Munich, Pettenkoferstrasse 9a, D-80336 Muenchen, Germany. E-mail: hammerschmidt{at}mvp.uni-muenchen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thrombospondin-1 (TSP1) is a matricellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. Here we report a novel role for the lectin TSP1 in pathogen-host interactions. Binding assays and flow cytometric analysis demonstrate that Streptococcus pneumoniae and other Gram-positive pathogens including S. pyogenes, Staphylococcus aureus, and Listeria monocytogenes interact specifically with human TSP1. We also show for the first time that host cell-bound TSP1 promotes adherence of Gram-positive pathogens to human epithelial and endothelial cell lines. Pretreatment of bacteria with sodium periodate but not Pronase E substantially reduced TSP1-mediated bacterial adherence to host cells, suggesting that a glycoconjugate on the bacterial cell surface functions as the receptor for TSP1. Lipoteichoic acids did not affect TSP1-mediated adherence of S. pneumoniae to host cells. In contrast, attachment of S. pneumoniae and other Gram-positive pathogens to host cells via TSP1 was blocked by soluble peptidoglycan, indicating recognition of bacterial peptidoglycan by TSP1. In conclusion, our results demonstrate that recognition of Gram-positive pathogens by TSP1 promotes bacterial colonization of host tissue cells. In this scenario, peptidoglycan functions as adhesin and TSP1 acts as a molecular bridge linking Gram-positive bacteria with receptors on the host cell.—Rennemeier, C., Hammerschmidt, S., Niemann, S., Inamura, S., Zähringer, U., Kehrel, B. E. Thrombospondin-1 promotes cellular adherence of Gram-positive pathogens via recognition of peptidoglycan.


Key Words: colonization • platelet lectin • pneumococci • staphylococci • host tissue cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACTERIAL ADHESION TO HOST CELLS IS THE INITIAL and essential first step in all infectious processes. To combat severe infections, it is important to understand the mechanisms underlying bacterial adhesion to host cells in order to prevent pathogenic bacteria from initiating local inflammations or invasive diseases. Pathogenic, particularly Gram-positive bacteria, recognize various components of the extracellular matrix (ECM) to gain access to host cells of their target tissue. The bacteria can remain extracellular, which allows persistence in the host niche, or they can enter the host cells and spread into subcellular compartments (1) .

Thrombospondin-1 (TSP1) is an extracellular matrix glycoprotein that is made and secreted by cells in response to injury and stress, and displays distinct biological activities on different cell types (2 , 3) . It is rapidly secreted at high levels in inflamed and damaged tissues. As a consequence of its binding to several cell surface receptors, TSP1 exerts multiple effects on cell function, which sometimes appear to be contradictory. TSP1 inhibits angiogenesis and tumor growth; it can regulate T cell function, promote the generation of human peripheral regulatory T cells through the ligation of CD47, activate latent TGF-ß, and is necessary for the maintenance of pulmonary homeostasis (4 5 6 7 8) . Moreover, both adhesive and antiadhesive properties have been attributed to TSP1. TSP1 mediates monocyte binding to endothelium, activation-dependent T cell adhesion through binding to {alpha}4ß1 and {alpha}5ß1 integrins, and it mediates as an alternative to vWF platelet adhesion at high shear rates (9 10 11) . However, TSP1 was also found to regulate focal adhesion disassembly, and TSP1 binding to the coreceptor of a complex of calreticulin and low density lipoprotein receptor-related protein (LRP1) stimulates cell motility (12) .

TSP1 is the main endogenous lectin of human platelets. Formation of the fibrinogen-thrombospondin complex was inhibited by amino sugars mannosamine and glucosamine. These inhibitors further block thrombin-induced platelet lectin activity and thrombospondin lectin activity (13) . Adsorbed thrombospondin also forms a complex with fibronectin via its lectin activity (3) .

Pathogens have developed various strategies to encounter their host niches. In particular, many bacteria such as the Gram-positive streptococci and staphylococci interact specifically with host proteins of the ECM, including fibronectin and vitronectin and cellular receptors. The bacterial surface structures recognizing host proteins have been collectively termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) or adhesins (14) . The largest group of bacterial adhesins or MSCRAMMs is represented by surface-exposed proteins, although other surface components such as polysaccharides and lipids may also function as adhesive molecules (1 , 15) .

The cell envelopes of Gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their host environment. The cell wall peptidoglycan (PG, murein) is a rigid structure, and in Gram-positive bacteria the thick layer of the murein contains, in addition to proteins, complex sugar polymers including lipoteichoic acids (LTA), teichoic acids (TA), and polysaccharides. The PG is one of the conserved components of microorganisms called pathogen-associated molecular patterns (PAMPs). These structures are recognized by molecules of the innate immune system that enable the host to discriminate between self and nonself (16) . The PG has been shown to be a powerful biological effector with different stimulatory activities and can per se be considered a potential virulence factor (17 , 18) .

Here, we indicate that matricellular TSP1 acts as a molecular bridge between Gram-positive pathogens and host tissue cells and that this interaction promotes cellular adherence. Further, we show in blocking experiments that TSP1 recognizes the peptidoglycan of pathogens, indicating a novel and direct role of peptidoglycan for bacterial adherence to and invasion into host tissue cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains, media, and culture conditions
Streptococcus pneumoniae strains NCTC 10319 (Cps+ serotype 35A), ATCC 11733 (Cps+, serotype 2), R800 (nonencapsulated), D39 (Cps+ serotype 2), TIGR4 (Cps+ serotype 4), and its nonencapsulated isogenic mutant TIGR4{Delta}cps (a generous gift of F. Iannelli, Siena, Italy) as well as S. pyogenes M6 strain were cultured in Todd-Hewitt broth (Oxoid, Basingstoke, UK) supplemented with 0.5% yeast extract (THY) and erythromycin (5 µg/ml), kanamycin (200 µg/ml), when appropriate, or grown on blood agar (19) . Neisseria meningitidis MC58{Delta}siaD (nonencapsulated mutant of the serogroup B strain MC58) (20) was grown on GC-agar supplemented with vitamins. The Haemophilus influenzae Rd (a spontaneous capsule-deficient mutant) (21) , Listeria monocytogenes (22) , and Stapylococcus aureus Cowan-1 (23) were cultured in BHI medium or on BHI agar. Legionella pneumophila Corby (24) was grown on active coal agar. Escherichia coli 536 (25) and E. coli BL21 (DE3) (Stratagene, La Jolla, CA, USA) were cultivated on Luria-Bertani (LB) agar or in LB broth. The pathogenic strains used in this study, their genotype, and source are also listed in Table 1 .


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  		Table 1. Bacterial strains and primers used in this study

Treatment of bacteria with Pronase E (Merck, Rahway, NJ, USA) was performed as described previously (26) . Treatment of bacteria ({approx}109 cfu/ml) with 5 µl sodium periodate (NaIO4; 1 mg/ml), which did not affect bacterial viability, was performed for 0.25–0.5 h at 37°C, followed by washing and resuspension in cell culture infection medium.

Construction of pneumococcal mutants
In TIGR4{Delta}cps the capsule locus is replaced by the kanamycin resistance gene aphA3, with the flanking genes dex and aliA still present (27) . To construct D39{Delta}cps, a PCR product amplified with TIGR4{Delta}cps DNA template and primer pair dexB/aliA (primers are listed in Table 1 ) were used. Transformation and molecular analysis of the mutants was performed as described (28 , 29) .

Human thrombospondin-1
Human thrombospondin-1 (TSP1) was isolated and purified from freshly isolated platelets as described (30) , with 2 mM CaCl2 added to all buffers. In brief, human platelets were gently isolated from fresh buffy coats. Quiescent platelets were resuspended in buffer A (150 mM NaCl, 20 mM Tris, 5 mM Glucose, 2 mM CaCl2, 1 mM MgCl2) and activated by thrombin (2 U/ml) (Sigma, Taufkirchen, Germany). Activation was stopped by hirudin (11 U/ml) (Pentapharm, Basel, Switzerland) to carefully prevent thrombin-induced cleavage of TSP1. Fibrin and fibrin-bound proteins were removed from platelet activation supernatant by precipitation. TSP1 was isolated by affinity chromatography using heparin-Sepharose CL-6B (Amersham Biosciences, Freiburg, Germany). The protein was eluted with increasing NaCl concentrations in Tris buffer (50 mM Tris, 2 mM CaCl2, 1 mM MgCl2 with 150 mM NaCl, 250 mM NaCl, 350 mM NaCl, or 500 mM NaCl, respectively, pH 7.4) containing proteinase inhibitors (0.1 mM phenylmethylsulfonylfluoride (Sigma) and 2 µM leupeptin hemisulfate (Sigma). Desalting and buffer exchange was performed by Sephadex G-25 PD-10 column filtration (Amersham Biosciences) equilibrated with TBS buffer (10 mM Tris, 150 mM NaCl, 2 mM CaCl2, pH 7.4). The purified TSP1 gave a single band after SDS-PAGE and silver staining (data not shown). Silver staining did not detect any von Willebrand factor, factor H, or fibronectin contamination and the single protein band was confirmed to be TSP1 by immunoblotting (Fig. 1 C).


Figure 1
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  		Figure 1. Recruitment of platelet-secreted human TSP1 by S. pneumoniae. S. pneumoniae NCTC 10319 were incubated with various amounts (%v/v) of platelet-free and TSP1-containing protein fractions obtained after thrombin activation of freshly isolated human platelets. The protein fraction released from 1 x 105 thrombin-activated platelets (in 200 µl) was defined as 100%. The reaction was carried out in a final volume of 200 µl. Bound TSP1 to pneumococci was measured by flow cytometry (A, B) or illustrated by immunoblot analysis (C). A) Dot plots are shown from flow cytometric analysis representative for binding of platelet-released human TSP1 to pneumococci. Binding was detected using an anti-TSP antibody (Calbiochem; mAb 6.1) and secondary anti-mouse Alexa 488 (MoBiTec). B) Graph demonstrating dose-dependent binding of TSP1 to S. pneumoniae NCTC 10319. Results are expressed as GMFI x percentage of gated bacteria. The graph shows the means and SD of three independent experiments, each done in triplicate. C) Immunoblot analysis of platelet-secreted human TSP1 recruited by S. pneumoniae NCTC 10319. TSP1 was detected with the anti-TSP antibody and secondary anti-mouse peroxidase-conjugated antibody (Jackson ImmunoResearch). Pneumococcal enolase protein was used as a loading control and detected with our polyclonal antienolase and anti-rabbit peroxidase-conjugated antibodies (32) . TSP1: 2 µg of purified human TSP1; secPlatelets: platelet-secreted TSP1 after activation of human platelets with thrombin.

Induction of TSP1 release from human platelets
Human platelet concentrates (kindly provided by the Department for Transfusion Medicine and Immunohaematology, University Hospital Wuerzburg), which contained 1.5 x 104 platelets per milliliter, were centrifuged at 1000 g for 10 min. The sedimented platelets (200 ml platelet concentrate) were washed with 10 ml glucose buffer (30 mM sodium citrate, 100 mM sodium chloride, 3 mM potassium chloride, 9.6 mM D-glucose, pH 6.5) and resuspended in 5 ml glucose buffer. Thrombin (3 U/ml; Sigma) was added under agitation and incubated for 2 min at 37°C. Thereafter, hirudin (6 U/ml) was used to stop the reaction. Samples were frozen overnight at –20°C. Samples were thawed and activated platelets were sedimented by centrifugation (10,000 g for 30 min). The supernatant, which contains the glycoprotein TSP1, was collected, fractionated, and stored at –80°C. Samples of 200 µl contained the released proteins including TSP1 of 1 x 105 platelets.

Flow cytometric analysis of TSP1 binding to S. pneumoniae and other bacteria
A suspension of 1 x 107 bacteria in 100 µl TBS buffer containing 2 mmol Ca2+ was incubated for 15 min at 37°C with FITC-labeled TSP1 or with the secreted proteins of thrombin-activated platelets. Bacteria were washed and analyzed for fluorescence intensity by flow cytometry using a FACSCaliburTM (Becton Dickinson, Bedford, MA, USA) as described (31) . When using the secreted protein fractions of thrombin-activated platelets, pneumococci were washed with TBS Ca2+ and incubated with anti-TSP antibodies (Calbiochem mAb6.1; 1:400; San Diego, CA, USA). Pneumococci-bound TSP1 was detected by incubation with a 1:300 dilution of an anti-mouse Alexa 488 IgG (MoBiTec GmbH, Goettingen, Germany). Finally, the bacteria were washed with TBS Ca2+ and fixed with 1% PFA in TBS Ca2+ in a total volume of 200 µl. The bacteria were detected using log-forward and log-side scatter dot plots, and a gating region was set to exclude debris and larger aggregates of bacteria. Ten thousand bacteria were analyzed for fluorescence using log-scale amplification. The geometric mean fluorescence intensity (GMFI) multiplied by the percentage of labeled bacteria was recorded as a measure for binding activity.

SDS-PAGE and immunoblotting
Whole bacterial cell lysates were subjected to SDS-PAGE with 4% stacking and 8% separating gel after incubation with protein supernatant of platelets (20%). Thereafter, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) using a semidry blotting system (Bio-Rad, Hercules, CA, USA) and probed with antibodies diluted in PBS. TSP1 binding was indicated by immunodetection using the anti-TSP antibody (1:200) together with a horseradish peroxidase-conjugated mouse antibody (Jackson Immunoreseach, West Grove, PA, USA). The enolase of pneumococci was used as a control protein; for immunodetection, we used our antienolase antiserum (32) together with peroxidase-conjugated rabbit antibody (DakoCytomatation, Ft. Collins, CO, USA).

Binding of bacteria to immobilized thrombospondin-1
TSP1 was applied to a 96-well microtiter plate (polystyrene surface) at 4°C overnight. Blocking and labeling of the bacteria with fluorescein-isothiocyanate (FITC) were performed as described previously (29) . Extensively washed FITC-labeled bacteria (2x108) were added to the wells and incubated for 1 h at 37°C for binding. Fluorescence was measured at 485/538 nm (excitation/emission) using a Fluoroscan Ascent reader.

Cell lines and cell culture
Human epithelial cell lines A549 (ATCC CCL-185; type II pneumocytes), Detroit 562 (ATCC CCL 138; human pharynx carcinoma), and HEp-2 (ATCC CCL-23; larynx carcinoma), and human brain-derived endothelial cells (HBMEC) were cultured as described previously (28 , 29) . As indicated by flow cytometric analysis, the host cells did not capture extracellular matrix proteins derived from FBS; Gold, PAA, Cölbe, Germany), including fibronectin (data not shown).

Infection experiments and quantification of pneumococcal adherence to host cells by immunofluorescence staining and microscopy
Pneumococcal adherence to and invasion in host cells were quantified by double immunofluorescence microscopy as described previously (29) . Briefly, host cells were seeded on glass coverslips (diameter, 12 mm) in 24-well plates (Cellstar, Greiner, Germany) and cultivated to confluent monolayers with ~2 x 105 cells (29) . The washed cell monolayers were infected with pneumococci in 500 µl Dulbecco’s minimal essential medium-HEPES (DMEM-HEPES; PAA) and 1% FBS (this incubation does not lead to ECM/serum protein binding of pneumococci) with a multiplicity of infection of 50 bacteria per cell at 37°C under 5% CO2 for 3 h. Nonadherent bacteria were removed by rinsing the cells several times with PBS. After washing, the infections were fixed on glass coverslips with 1.0% paraformaldehyde. For differentiation between extracellular (adherent) and intracellular bacteria, the fixed samples were blocked with PBS/10% FBS and stained with a polyclonal antipneumococcal IgG in combination with a secondary goat-anti-rabbit IgG coupled to Alexa 488 (green). After several washings with PBS, host cells were permeabilized with 0.1% Triton-X-100 for 5 min and intracellular bacteria were incubated with antipneumococcal IgG, followed by incubation with goat anti-rabbit IgG coupled to Alexa 568 (red) (MoBiTec) as described previously (29) . This results in Alexa 568-labeled intracellular bacteria and Alexa488/568-labeled extracellular bacteria (green/yellow). For inhibition experiments, infection assays with TSP1 pretreated host cells were carried out in the presence of soluble peptidoglycan (2 µg to 100 µg), sugars, antibodies, or after pretreatment of the cells with heparitinase.

Quantification of host cell-associated bacteria by plating
HEp-2 cells were infected with S. pneumoniae NCTC 10319, S. aureus Cowan-1, S. pyogenes M6, N. meningitidis MC58{Delta}siaD, or E. coli 536. After 2 h, the cells were washed thoroughly with PBS to remove unbound bacteria. Thereafter, intracellular bacteria were released by incubation of the host cells in 300 µl DMEM-HEPES/1% saponin for 10 min at 37°C. The total number of adherent and intracellular recovered bacteria was monitored after plating the sample aliquots on appropriate solid medium, followed by colony formation and enumeration.

Phagocytosis of bacteria by granulocytes
Human polymorphonuclear leukocytes (PMNs) were isolated from peripheral blood of four healthy volunteers. A total of 10 ml of heparinized blood of a single donor was mixed with 10 ml of PBS (pH 7.4), layered on Ficoll-Histopaque gradient, and centrifuged for 20 min at 800 g. The isolated phase containing PMN cells and partially erythrocytes was depleted of erythrocytes by two consecutive hypotonic shocks (33) . Finally, the PMNs were resuspended in RPMI1640 (PAA) and immediately used for the phagocytosis assay. PMNs (6x104) were incubated for 30 min in 100 µl PBS/1% FBS with 1.5 x 106 FITC-labeled S. pneumoniae (NCTC10319) or S. aureus Cowan1 at 37°C. Attachment to and uptake of pneumococci by phagocytes was investigated in the absence or presence of human TSP1 (0 µg–100 µg/ml) and stopped by centrifugation. The PMNs were fixed with 200 µl PBS/1% FBS/1% paraformaldehyde. Binding and uptake of FITC-labeled bacteria were assessed by flow cytometry using a FACSCaliburTM (Becton Dickinson). The mean fluorescence intensity (MFI) of the entire PMN population was recorded as a measure for bacterial binding and/or uptake.

Purification of peptidoglycan and lipoteichoic acids
Soluble peptidoglycan (sPG) was prepared from S. aureus as described (34) . An aliquot of 30 mg was further purified by gel permeation chromatography on a Toyopearl HW-40S column (1.2 mx2.5 cm) run with 20 mM NH4HCO3. The eluate was monitored at 206 nm (Uvicord S, LKB) and fractions (7.5 ml, Ultrorac, LKB) were collected and lyophilized twice in order to remove residual buffer salt from the samples. Samples representing high molecular fraction 1 (63–74, yield 14.88 mg), medium-sized fraction 2 (75–92, 12.95 mg), and small-sized fraction 3 (93–120, 5.23 mg) were combined and analyzed.

The lipoteichoic acids were prepared according to Draing et al. (35) and were a generous gift from Dr. T. Hartung (University of Konstanz, Germany). The LPS content of sPG fractions was rigorously checked by measuring the endotoxin reactivity of the sPG with Limulus amebocyte lysate (LAL) at 37°C using the Hemachrom Coamatic®-LAL kit (Biochrom, Essen, Germany).

TSP1-sPG binding assay
MaxiSorb ELISA plates (Nunc, Germany) were coated overnight with purified human TSP1 by incubating the plates with 50 µl TSP1 in coating buffer (TBS containing 2 mmol Ca2+) per 96-well at 4°C followed by three washes with TBS. To block nonspecific reactions, the plates were incubated for 5 h with 200 µl of 5% skim milk/1% Tween in coating buffer at 4°C. After repeated washing with TBS, immobilized TSP1 was incubated for 1 h with 3.0 µg sPG in coating buffer. After removing the sPG and washing with TBS, the coated plates were incubated for 1 h with the polyclonal anti-PG antibodies (Serotec; Raleigh, NC, USA: clone 10H6, isotype IgG1) diluted 1:50 in coating buffer at 4°C. Immobilization of TSP1 was verified in a parallel experiment using the anti-TSP1 antibody, and in controls only the antibodies were used to detect unspecific binding. Detection was conducted with a horseradish peroxidase-conjugated mouse antibody (1:5000; Jackson ImmunoReseach) followed by an incubation with the substrate ABTS [2,2-azinobis(3-ethylbenzthiazolinesulfonic acid)] (Sigma) and 2 µl H2O2. Absorbance was read at 405 nm in an ELISA reader (Thermolab, San Fernando, CA, USA).

Statistical analysis
Adherence data were expressed as the mean ± SD. Differences in adherence were analyzed by the 2-tailed unpaired Student’s t test. In all analyzes, a P value of < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recruitment of human TSP1 by Streptococcus pneumoniae
Gram-positive bacteria, including S. pneumoniae (pneumococci), interact with a variety of extracellular matrix proteins (1 , 36) . To investigate whether pneumococci are able to recruit soluble TSP1, freshly isolated platelets were activated with thrombin, and S. pneumoniae NCTC 10319 (serotype 35A) were incubated with serially diluted secreted protein fractions containing the major platelet protein human TSP1. Flow cytometric analysis indicated a dose-dependent binding of human TSP1 secreted by platelets (Fig. 1A, B ). Binding of platelet-released TSP1 to the surface of pneumococci was also confirmed by immunoblot analysis using a TSP1-specific monoclonal antibody for immuno-detection (Fig. 1C ). In summary, these results show that pneumococci are able to recruit TSP1 to the bacterial cell surface.

Interference of the pneumococcal capsular polysaccharide with binding of human TSP1
We recently showed that the bacterial capsular polysaccharide (CPS) interferes with pneumococcal interactions with host cells and proteins (19) . To elucidate whether the CPS affects the interaction of pneumococci with TSP1, we compared the binding of purified and FITC-labeled human TSP1 to S. pneumoniae NCTC 10319, strain D39 (serotype 2), and its isogenic CPS-deficient strain D39{Delta}cps. Flow cytometric analysis indicated a dose-dependent binding of soluble TSP-FITC to the pneumococcal strains used. More important, the results demonstrated a significantly increased TSP1 binding activity of the capsule knockout strain D39{Delta}cps compared with the isogenic wild-type strain D39 (Fig. 2 A, B). Strikingly, binding of TSP1 to D39{Delta}cps was even higher than binding of TSP1 to NCTC 10319 (Fig. 2A, B ), which is a low encapsulated strain (19) .


Figure 2
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  		Figure 2. Binding of soluble human TSP1 to S. pneumoniae strains and effect of the CPS. Pneumococci were incubated with various amounts of purified and FITC-labeled human TSP1 for 15 min at 37°C. Binding of TSP-FITC was analyzed by flow cytometry. A) Binding of FITC-labeled soluble human TSP1 to pneumococcal wild-type NCTC 10319, D39 and the nonencapsulated D39{Delta}cps as determined by flow cytometry. Results are expressed as GMFI multiplied by the percentage of gated bacteria. The graph shows the means and SD of three independent experiments, each done in triplicate. B) Dot plots are shown for representative samples.

Adherence of S. pneumoniae to immobilized TSP1
To investigate whether pneumococci adhere to human TSP1 that was immobilized on polystyrene surfaces, FITC-labeled pneumococci NCTC 10319 (serotype 35A) were used in adherence assays. The results demonstrated adherence of pneumococci to immobilized TSP1. Similar to our results with soluble TSP1, adherence of pneumococci to immobilized TSP1 was dose dependent and increased with higher amounts of immobilized TSP1 (Fig. 3 A, B). To elucidate whether the CPS of pneumococci also interferes with pneumococcal adherence to immobilized TSP1, binding of FITC-labeled capsule knockout strains D39{Delta}cps and TIGR4{Delta}cps to immobilized TSP1 was compared with that of the isogenic wild-type strains TIGR4 (serotype 4) and D39, respectively (Fig. 3C ). Consistent with our results with soluble TSP1, our binding assays indicated a substantially increased TSP1 binding activity of the cps knockout strains compared with the wild-type strains D39 and TIGR4 (Fig. 3C ). Taken together, our binding assays with soluble and immobilized TSP1 indicate that pneumococci interact specifically with human TSP1 and that the cell wall-attached CPS reduces TSP1 binding efficiency.


Figure 3
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  		Figure 3. Pneumococcal adherence to immobilized human TSP1. A) Binding of S. pneumoniae NCTC 10319 (serotype 35A) to different amounts of immobilized TSP1. Different amounts of human TSP1 were coated on the polystyrene surface of a 96-well microtiter plate and binding of FITC-labeled pneumococci was measured at 485/538 (excitation/emission). B) Repesentative immunofluorescence micrographs of pneumococci (NCTC 10319) bound to immobilized TSP1. C) Binding activity of FITC-labeled S. pneumoniae wild-type strains and isogenic capsule knockout strains ({Delta}cps) was determined to 3.0 µg of immobilized TSP1. 100% represents the total amount of FITC-labeled pneumococci used per binding experiment (2x108 bacteria). Values are the means ± SD from at least three independent experiments, each performed in triplicate. *P < 0.05.

TSP1 binding activity of other Gram-positive and Gram-negative pathogens
To test whether besides pneumococci and S. aureus (23) , other bacteria also have the ability to interact with TSP1, we investigated binding of representative Gram-positive and Gram-negative pathogens to immobilized TSP1. We demonstrated binding of the Gram-positive S. aureus, S. pyogenes, and Listeria monocytogenes to TSP1. In contrast, the Gram-negative pathogenic microorganisms Escherichia coli, Legionella pneumophila, Haemophilus influenzae, and Neisseria meningitidis, respectively, showed no significant interaction with TSP1 (Fig. 4 A). To avoid the potential inhibitory effect of the CPS, we have used the capsule-deficient strains of H. influenzae (H.i. Rd) and N. meningitidis (MC58{Delta}siaD) in these studies. Similar to pneumococci, flow cytometric analysis indicated a dose-dependent binding of TSP1 to other Gram-positive bacteria such as S. aureus, but not to Gram-negative bacteria such as E. coli (Fig. 4B, C ). From these data we conclude that TSP1 can interact specifically with several Gram-positive pathogens, but probably not with Gram-negative bacteria.


Figure 4
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  		Figure 4. Interaction of other Gram-positive and Gram-negative pathogens with human TSP1. A) Binding of pathogens to immobilized TSP1. Human TSP1 (3.0 µg) was immobilized and bacteria were labeled with FITC. Binding activity was recorded as the percentage of bound bacteria. Values are the mean ± SD from at least three independent experiments, each performed in triplicate. B, C) Binding of FITC-labeled soluble human TSP1 to E. coli and S. aureus as analyzed by flow cytometry. B) Dots plots are shown for representative samples. C) The graph shows the means and SD of three independent experiments, each done in triplicate. Results are expressed as GMFI x percentage of gated bacteria.

Human TSP1 acts as a molecular bridge between host tissue cells and pneumococci
To elucidate whether pneumococci can exploit the glycoprotein TSP1 as a linking molecule between host cells and bacteria, we investigated the role of TSP1 for adhesion of pneumococci to host cells. To avoid pneumolysin-induced damaging of host cells in extended infections, the pneumolysin knockout pneumococcal strain NCTC 10319{Delta}ply was used in cell culture infections as described previously (29) . Pretreatment of larynx epithelial cells HEp-2 with TSP1 significantly increased adherence of pneumococci to host cells as determined by immunofluorescence microscopy (Fig. 5 A, B). Flow cytometric analysis indicated a dose-dependent binding of TSP1 to host cells. Accordingly, the number of pneumococci attached via TSP1 to host cells depends on the amount of host cell-bound TSP1 (data not shown). Apparently, the mechanism of TSP1-mediated adherence is independent of the host tissue cells, as the positive effect of TSP1 on adherence was observed for several host epithelial cells and human brain-derived endothelial cells HBMEC (Fig. 5A, B ). Host cell-bound TSP1 also significantly increased internalization of pneumococci in host cells as determined by double immunofluorescence microscopy (Fig. 5A ).


Figure 5
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  		Figure 5. Human TSP1 mediates adherence and invasion of pneumococci to human tissue cells. A) Adherence and invasion of S. pneumoniae NCTC 10319{Delta}ply (Cps+, serotype 35A) was determined by double immunofluorescence microscopy after 3 h of infection of epithelial cells Detroit 562, A549, and HEp-2, respectively, or the endothelial cell line HBMEC. The infections were conducted in the absence of TSP1 (none) and in the presence of host cell-bound human TSP1 (3 µg). Results present the means ± SD of at least three independent experiments. *P < 0.05 relative to infections carried out in the absence of TSP1. B) Immunofluorescence microscopy of adherent pneumococci in the absence and presence of TSP1. C) Interference of the pneumococcal capsular polysaccharide with TSP-mediated adherence. Adherence of low encapsulated (ATCC 11733), high encapsulated (D39 and TIGR4), and nonencapsulated (R800, D39{Delta}cps, and TIGR4{Delta}cps) pneumococcal strains (19) to HEp-2 cells was monitored by immunofluorescence microscopy after 3 h of infection. The graph shows the log of host cell attached pneumococci per well (1x105 HEp-2 cells per well) of a 24-well tissue culture plate. Results present the means ± SD of at least three independent experiments. *P < 0.05 compared with infections carried out in the absence of TSP1.

We recently showed that the CPS inhibits pneumococci adherence to host cells (19) . To elucidate the impact of the CPS on TSP1-mediated adherence of pneumococci, we compared the host cell adhesion of encapsulated D39 and TIGR4 with that of their isogenic nonencapsulated mutants. In addition, the low encapsulated pneumococcal strain ATCC 11733 and the nonencapsulated R800 (19) were used in our cell culture infection experiments. As shown recently, the experiments confirmed that the CPS interferes with pneumococcal adherence in the absence of an exogenously added matrix protein, here TSP1 (19) . More important, our data indicated that encapsulated pneumococci are impaired in the exploitation of TSP1 for cellular adherence. In the presence of host cell-bound TSP1, the increase in adherence was relatively low for the encapsulated wild-type strains D39 and TIGR4 compared with the increase in adherence of the capsule-deficient strains D39{Delta}cps and TIGR4{Delta}cps (Fig. 5C ). This suggests that the bacterial adhesin for TSP1 is concealed below the CPS. Taken together, the cell culture infection experiments showed that TSP1 acts as a molecular bridge between host cells and pneumococci and that the CPS interferes with this interaction.

Effect of TSP1 on host cell adherence of other pathogenic bacteria
Our binding studies clearly demonstrated binding of Gram-positive but not Gram-negative bacteria to TSP1. To determine whether the exploitation of TSP1 for adherence is a common mechanism of Gram-positive bacteria, cell culture infection assays were carried out using HEp-2 cells as the host cell line. In the presence of host cell-bound TSP1, the number of host cell-associated bacteria was significantly increased for the Gram-positive bacteria S. aureus, S. pyogenes, and L. monocytogenes, as determined by counting the cfu received after plating the total number of cell-associated and recovered intracellular bacteria (Fig. 6 ). Similar to pneumococci, internalization of Gram-positive S. aureus and S. pyogenes was also increased in the presence of host cell-bound TSP1 (data not shown). By contrast, TSP1 had no effect on the interaction of the Gram-negative E. coli, H. influenzae, and N. meningitidis with HEp-2 cells (Fig. 6) . Apparently, there is a significant difference in binding and exploitation of the lectin TSP1 for bacterial adherence between Gram-positive and Gram-negative bacteria.


Figure 6
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  		Figure 6. Impact of TSP1 on cell adherence and invasion of Gram-positive and Gram-negative bacteria. HEp-2 cells were infected for 2 h with bacteria and the total number of host cell-associated bacteria (i.e., attached and recovered intracellular bacteria) was determined by plating and counting the cfu. The infections were carried out in the absence of human TSP1 (defined as 100%) and in the presence of host cell-bound TSP1. *P < 0.05; n.s., not significant compared with infections carried out in the absence of TSP1.

Characterization of the TSP1 receptor on Gram-positive bacteria
To characterize the bacterial receptor recognized by TSP1, S. pneumoniae were treated with Pronase E or sodium periodate before being used in our infection assays. The impact of this bacterial treatment on TSP1-mediated adherence to host cells was analyzed by immunofluorescence microscopy. Proteolytic pretreatment of pneumococci with Pronase E did not affect TSP1-mediated adherence (Fig. 7 A, B). By contrast, oxidation of pneumococcal glycoconjugates by periodate abolished the TSP1-mediated effect on pneumococcal adherence to host epithelial cells and HBMEC (Fig. 7A, B ). Periodate treatment results in a level of pneumococcal adherence that is similar to infections carried out in the absence of TSP1. These data suggest that surface-exposed glycoconjugate functions as the major bacterial adhesin for TSP1.


Figure 7
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  		Figure 7. Influence of proteolytic and periodate treatment of pneumococci on TSP1-mediated adherence. A) Adherence of S. pneumoniae NCTC 10319{Delta}ply to HEp-2 or HBMEC cells was determined after Pronase E (ProE) or sodium periodate (NaIO4) treatment of the bacteria. Infections were carried out in the absence (none) and in the presence of host cell-bound TSP1 (3.0 µg). Results are presented as the means ± SD for at least three independent experiments. *P < 0.05; n.s., not significant, compared with infections carried out in the absence of TSP1. B) Immunofluorescence micrographs illustrating the effect of Pronase E and periodate treatment of pneumococci for TSP1-mediated bacterial adherence to HEp-2 epithelial cells.

Peptidoglycan blocks TSP1-mediated pneumococcal interaction with host tissue cells
To determine whether bacterial adherence via TSP1 is mediated by recognition of PAMPs of the Gram-positive cell wall such as the LTA or the murein, purified LTA (35) and soluble PG (sPG) (34) were used to block TSP1-mediated pneumococcal adherence to host cells. The results showed a significant inhibition of TSP1-mediated adherence by sPG, whereas the presence of LTA derived from S. pneumoniae or S. aureus had no effect (Fig. 8 ). The effect of sPG was dose dependent (data not shown), indicating the specificity of PG recognition by TSP1. A 50% reduction in TSP1-mediated pneumococcal adherence to HEp-2 cells was observed in the presence of 10 µg/ml of exogenously added sPG (data not shown). PG consists of polysaccharide backbone, which is composed of a polyglycan with ß-(1->4) interlinked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues. In S. aureus MurNAc is substituted by a so-called stem pentapeptide (L-Ala-D-iGln-D-Lys-D-Ala) that, at the {epsilon}-D-Lys amino group, carries the carboxyl terminus of a pentaglycine interlinking peptide (-Gly5-). The amino terminus of this pentaglycine in turn is linked to the D-Ala residue of the next PG strand, thus forming a complex 3-dimensional scaffold (murein). To determine whether one of the PG moieties interferes with TSP1-mediated adherence, GlcNAc and MurNAc monosaccharide part structures of PG (Sigma) were also used in inhibition assays. To determine whether one of the PG moieties interferes with TSP1-mediated adherence, GlcNAc and MurNAc were also used in inhibition assays. The results showed that excessive amounts of GlcNAc can competitively inhibit TSP1-mediated adherence of S. pneumoniae, whereas MurNAc (Fig. 8) and muramyl-dipeptide (data not shown) had no significant inhibitory effect.


Figure 8
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  		Figure 8. Blocking of TSP1-mediated pneumococcal adherence by soluble peptidoglycan. Adherence of S. pneumoniae type NCTC 10319{Delta}ply to human TSP1 pretreated HEp-2 cells (black bars) was investigated in the absence (none) or presence of various glycoconjugates or sugars. LTA Sa, S. aureus LTA (100 µg/ml); LTA Sp, S. pneumoniae LTA (100 µg/ml); sPG, soluble PG (100 µg/ml); GlcNAc (100 mM); MurNAc (100 mM). Results are presented as the means ± SD for at least three independent experiments carried out for 3 h. The gray bar (Co: control infection) shows the basal level of adherence in the absence of host cell-bound TSP1. Results are presented as the means ± SD for at least three independent experiments. *P < 0.05; n.s., not significant, compared to the infection conducted with TSP1 pretreated cells but absence of blocking substance.

Peptidoglycan inhibits TSP1-mediated adherence of Gram-positive bacteria to host cells
Our results showed that sPG purified from Gram-positive bacteria inhibits TSP1-mediated adherence of pneumococci, and therefore we postulated that sPG also affects TSP1-mediated adherence of other Gram-positive pathogens. In cell culture blocking experiments, sPG was evaluated for its ability to inhibit TSP1-mediated adherence and internalization of S. aureus and S. pyogenes to host cells. The amount of attached and recovered internalized bacteria was enumerated by plating the samples on appropriate solid media. Similar to S. pneumoniae, PG significantly blocked the interaction of S. aureus and S. pyogenes with host cells via TSP1 (Fig. 9 ). The results revealed that the inhibitory effect of sPG was not as strong as for pneumococci, suggesting that additional, yet unknown TSP1 receptors may be expressed by S. aureus and/or S. pyogenes. In the absence of TSP1, the presence of sPG did not influence the bacterial interaction with host cells (data not shown). In conclusion, these data demonstrated that the PG of Gram-positive bacteria functions as an adherence molecule for TSP1 and that TSP1 promotes adherence of Gram-positive bacteria to host cells.


Figure 9
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  		Figure 9. Blocking of TSP1-mediated association of Gram-positive bacteria to HEp-2 epithelial cells by sPG. Human TSP1 pretreated epithelial cells HEp-2 were infected with bacteria in the absence (none) or presence of 20 µg sPG. The total number of cell-associated (attached and recovered intracellular) bacteria was determined after 2 h of infection by plating. The number of cell-associated bacteria in the absence of sPG was defined as 100%. *P < 0.05 compared with infections conducted in the absence of sPG.

Binding of soluble peptidoglycan to human TSP1
Binding of sPG to immobilized human TSP1 was measured in an ELISA format. The results demonstrated that sPG bound specifically to human TSP1. The amount of binding varied as a function of the amount of TSP1 immobilized to the ELISA plate (Fig. 10 ). In addition, binding of sPG to a defined amount of immobilized TSP1 (0.5 µg) was dose dependent and saturable (data not shown). These data indicate that sPG is specifically recognized by human TSP1.


Figure 10
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  		Figure 10. Direct binding of sPG to immobilized human TSP1. TSP1 was bound to a microtiter plate and the ability of sPG (3 µg) to bind directly to TSP1 was tested in an ELISA. Binding was detected using an anti-PG antibody (Serotec) and absorbance was measured at 405 nm.

Influence of heparan sulfate proteoglycans on TSP1-mediated bacterial adherence to host cells
TSP1 has been reported to bind to several integrins and several proteoglycans containing heparan sulfate and chondroitin sulfate. Binding of TSP1 to eukaryotic cells can be blocked with heparin and is sensitive to heparitinase treatment of cells (37) . To assess the importance of TSP1 binding to heparan sulfate proteoglycans (HSPGs) on the host cell for TSP1-mediated bacterial adherence, cell culture infections were performed with heparin or heparan sulfate (HS) as blocking reagents. HEp-2 cells were incubated with TSP1 and heparin or HS was added as inhibitor. The infection with pneumococci was carried out after removal of unbound TSP1 and proteoglycans from the host cells. The results revealed that heparin and HS blocked TSP1-mediated pneumococcal adherence to host cells (Fig. 11 ). In addition, treatment of the host cells with heparitinase, followed by treatment of the cells with TSP1, significantly reduced pneumococcal adherence to HEp-2 cells to a level measured when infections were carried out in the absence of TSP1 (Fig. 11) . In contrast, blocking of TSP1 binding by anti-integrin antibodies (anti-ß1 (MAB 1959), anti-ß3 (MAB 2023Z), or anti-{alpha}v (MAB1980); all Chemicon, Temecula, CA, USA) prior to infections with pneumococci showed no notable effects on TSP1-mediated pneumococcal adherence to host cells (data not shown).


Figure 11
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  		Figure 11. Blocking of TSP1-mediated pneumococcal adherence to HEp-2 cells by heparan sulfate proteoglycans. In inhibition assays, HEp-2 cells were incubated with TSP1 (3 µg) in the presence of heparin (20 U) or heparan sulfate (HS; 10 µg/ml). Heparitinase (10 mUnits) was used prior to TSP1 incubation of the host cells. Adherence was determined by immunofluorescence. Results are presented as the means ± SD for at least three independent experiments. *P <0.05, n.s., not significant, relative to infections with host cell-bound TSP1 but absence of inhibitor or enzyme.

The presence of soluble TSP1 prevents uptake of pneumococci and staphylococci by phagocytes
To investigate the influence of human TSP1 on opsonophagocytosis, FITC-labeled S. pneumoniae (NCTC10319) and S. aureus were incubated for 30 min with human PMNs. TSP1 was applied over a range of concentrations, and binding and uptake of pneumococci by PMNs were measured by flow cytometry. Strikingly, a significant reduction of fluorescence was observed in the presence of exogenous soluble TSP1, demonstrating that soluble TSP1 decreases in a dose-dependent manner the number of PMN cell-associated and phagocytosed pneumococci and staphylococci, respectively (Fig. 12 ). These data suggest that opsonization of Gram-positive bacteria with human TSP1 prevents bacterial uptake by primary human phagocytes.


Figure 12
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  		Figure 12. TSP1 affects phagocytosis of S. aureus and S. pneumoniae. FITC-labeled pneumococci (NCTC10319) and staphylococci (S. aureus Cowan1), respectively, were incubated for 30 min with PMNs, to which exogenous soluble TSP1 had been added in increasing concentrations. PMN cell binding and uptake of bacteria by PMNs was quantified by flow cytometry. A) Dot plots are shown for a representative experiment performed with PMNs (PMNs1). B) The values of the graph represent the MFI of the entire PMN population. Phagocytosis of bacteria was quantified in four independent experiments and shown are representative data from two experiments, each done in duplicate. PMNs1 and PMNs2 are phagocytes isolated from the peripheral blood of two independent volunteers. Ctrl: MFI of PMNs in the absence of bacteria.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have shown that the Gram-positive pathogens S. pneumoniae, S. aureus, S. pyogenes, and L. monocytogenes interact with the human lectin TSP1. These pathogens cause a variety of infections of the respiratory tract and severe life-threatening infections including pneumonia, sepsis, septic arthritis, infective endocarditis, toxic shock-like syndrome, meningitis, and listeriosis (1 , 38) . Here we have indicated for the first time a direct and novel role of peptidoglycan in bacterial adherence to host tissue cells. We demonstrated that recognition of pathogens by TSP1 occurs via bacterial PG. Moreover, we have demonstrated that the exploitation of TSP1 as a molecular bridge promotes adherence to and invasion of various Gram-positive pathogens into different host tissue cells.

TSP1 is a large homotrimeric extracellular molecule consisting of multiple domains that bind to a variety of host cell surface receptors and proteins in the extracellular environment (3) . Our binding assays indicated that Gram-positive bacteria, but not the selected Gram-negative bacteria are recognized by TSP1. In a previous study, S. aureus Cowan 1 (the strain that has also been used in our study) was shown to specifically bind TSP1. Binding of TSP1 to S. aureus is blocked by heparin and depends on the presence of Ca2+ ions (23) . The C-terminal domain of TSP1 consists of calcium binding repeats, and the conformation of TSP1 is sensitive to Ca2+ ions (39) . In conclusion, the data suggested a conformation-sensitive interaction of the bacteria with TSP1.

The diverse functions of TSP1 and the cell type-specific effects are mediated via its binding to integrins, including {alpha}vß3, {alpha}IIbß3, and ß1 integrins, the integrin-associated protein (IAP/CD47), and CD36. Moreover, TSP1 binds to extracellular molecules, including heparan sulfate proteoglycans and sulfatides (40 , 41) . Due to its characteristics TSP1 is one of the matricellular proteins that modulate cell behavior, but it does not belong to the structural ECM molecules (4) . Our infection assays indicated that TSP1, which is assembled on the host cell surface of various cell types, recognizes several Gram-positive bacteria and links as a molecular bridge the bacteria with the host cell. This interaction promotes bacterial adherence to and invasion into host cells. In contrast, pretreatment of the bacteria with TSP1 did not promote adherence to host cells. These results suggest that the binding of soluble TSP1 by bacteria induces a conformational change in the TSP1 molecule and that only interaction with the matricellular TSP1 molecule promotes bacterial adherence. Although TSP1 binds to various host cell receptors and structures, blocking experiments suggested that mainly TSP1 bound to heparan sulfate proteoglycans interacts with Gram-positive bacteria and facilitates colonization.

Microorganisms, particularly Gram-positive bacteria, recognize a wide variety of host cell receptors and ECM proteins via surface-exposed MSCRAMMs. These bacterial adhesins interact with ECM proteins such as fibronectin, fibrinogen, and collagen and are usually proteins (1 , 14) . In TSP1 interactions, periodate treatment of pneumococci suggested a surface-exposed glycoconjugate as receptor for TSP1. This is consistent with previous data demonstrating that TSP1 binding to S. aureus was not significantly decreased after proteolytic treatment (23) . In our study, soluble PG but not LTA or wall teichoic acids (WTA) from S. aureus (data not shown) inhibited TSP1-mediated adherence of streptococci and staphylococci to HEp-2 epithelial cells. This suggests that recognition of the pathogens by TSP1 occurs via PG.

The surface-exposed structures, including the LTA, WTA, and PG of Gram-positive bacteria, have potent biological functions and are regarded as potential virulence factors. The PG is one of the conserved components of microorganisms, called PAMPs, that are recognized by molecules of the innate immune system and enable the host to discriminate between the infectious agents from self. Recognition of these PAMPs occurs through pattern recognition receptors (PRR), and the usual consequences are the activation of signal cascades of the innate immune system and stimulation of an immune response. The PG has been shown to be a powerful biological effector with different stimulatory activities (17 , 18) . Several PRR have been identified for the bacterial PG, including Toll-like receptor 2, membrane CD14, cytosolic nucleotide binding oligomerization domain (Nod)-containing proteins, and a family of peptidoglycan recognition proteins (PGRPs) (42 43 44 45 46) . However, there is still a controversial debate as to whether highly purified PGs are indeed sensed by TLR2 (47 , 48) . It has been suggested that TLR2 does not recognize PG, but rather recognizes lipoproteins in PG preparations (18) . The minimum bioactive structure of the PG required for immunostimulation is N-acetylmuramyl-L-alanyl-D-isoglutamate (muramyl dipeptide: MDP); however, the activity is lower than that of soluble PG (49) . In this study the MDP did not inhibit recognition of TSP1 by bacterial PG (data not shown), suggesting that this interaction differs markedly from the immuno-stimulatory role of MDP. Moreover, there seems to be no specificity for the Lys-type PG, which is typical for most Gram-positive bacteria, because the PG of Listeria contains diaminopimelic acid in the stem peptide and recognizes TSP1. We assume that a fine structure may be less critical for recognition of PG by TSP1 than the overall organization and charge of the PG. The minimal component or biologically active structure involved in the TSP1-PG interaction has to be examined using highly purified sPG or synthetic PG part structures.

Pulmonary and meningeal inflammations caused by purified pneumococcal cell wall are indistinguishable from inflammations caused by living pneumococci (38 , 50) . Large-scale genome-wide studies of pneumococcal virulence identified a number of genes that are necessary for virulence and are involved in assembly or turnover of PG (51 52 53 54) . Some of the identified genes encode enzymes such as a carboxypeptidase (SmcB), a ß-N-acetylglucosaminidase (StrH), or a peptidoglycan GlcNAc deacetylase (SpPgdA), which modifies the PG. These modifications function as a defense mechanism against the mammalian immune system (18) . Recently, recognition of the PG of S. pneumoniae by the LPS binding protein (LBP) has been reported (55) . LBP is an acute-phase protein and, like TSP1, recognizes the glycan backbone. It has been suggested that the LBP-PG interaction depends on the basic residues in the N-terminal part of LBP and the iterative polyanionic motif of the PG in the pneumococcal cell wall (55) . However, it is not known whether LBP recognizes exclusively only the pneumococcal PG or also recognizes the PG from other Gram-positive bacteria, as we have shown here for TSP1. Another host protein that recognizes the PG is the mannose binding lectin (MBL), a major innate immune protein present in blood. It has been shown that human MBL binds to PG via its C-type lectin domains. Similar to TSP1, the interaction of MBL and PG can be inhibited more efficiently by GlcNAc than by MurNAc (56) .

Antibacterial antibodies and Gram-positive bacteria, including S. aureus, have been shown to induce aggregation of flowing platelets (57 , 58) . When platelet tethering is initiated the bacteria and platelets may become colocalized on a surface even without interacting directly with each other (57) . It is noteworthy that adherence of streptococci and staphylococci to an immobilized fibrin matrix is largely increased by platelets (59 , 60) . However, soluble fibrin and the release of {alpha}-granule proteins, particularly TSP1, are required for the direct association of staphylococci with platelets (61) . A function for TSP1 as a bridging molecule has been proposed for the phagocytosis of apoptotic neutrophils. TSP1 bound to macrophages or mesangial cells promotes binding and phagocytosis of apoptotic PMNs (62 , 63) . However, this study further reveals that the presence of soluble human TSP1 impairs pneumococcal and staphylococcal PMN cell binding and affords protection from phagocytosis in vitro. This demonstrates that the interaction of TSP1 with bacteria via recognition of the PG is most likely important during colonization and bacterial dissemination, which can result in pneumonia and infective endocarditis.

In summary, the interaction of TSP1 with Gram-positive bacteria via recognition of the PG promotes adherence in the initial step of an infection. Moreover, this interaction may also be important during septicaemia of pathogenic bacteria.


   ACKNOWLEDGMENTS
 
We thank U. Dobrindt, J. Kreft, J. Reidl, M. Steinert, A. Unkmeier, W. Ziebuhr (all University of Würzburg, Germany), C. Heilmann (Institute of Medical Microbiology, University of Muenster), and F. Iannelli (University of Siena, Italy) for providing bacterial strains, and we greatly appreciate the help of T. Hartung (University of Konstanz) for providing purified LTA. The authors also thank J. Morschhaeuser (University of Wuerzburg) for critical reading and helpful discussions. This work was supported by a grant from the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 479, Teilprojekt A7 (to S.H.) and Sonderforschungsbereich 293, Teilprojekt A6 (to B.K.).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication December 20, 2006. Accepted for publication April 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. Bergmann, A. Lang, M. Rohde, V. Agarwal, C. Rennemeier, C. Grashoff, K. T. Preissner, and S. Hammerschmidt
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