1Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore; and 2Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland
Submitted 26 January 2005 ; accepted in final form 3 May 2005
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ABSTRACT |
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adhesion; red blood cell
S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins such as fibrinogen and IgG (10, 50). Fibrinogen binds to RBC membrane both nonspecifically and specifically through its A chain (28) and can be blocked by the peptide Arg-Gly-Asp-Ser (26). Fibrinogen binding to RBC causes the aggregation of adjacent RBCs (16, 28). In contrast, IgG binds to RBC through at least two types of nonspecific (nonimmunological) mechanisms: loosely fixed and firmly fixed binding (40). IgG bound to RBC also causes cell aggregation (28). Because S. aureus produces surface proteins specific to fibrinogen [clumping factors A (ClfA) and B (ClfB)] and IgG (protein A), S. aureus also can bind to RBC in the presence of fibrinogen or IgG. These binding characteristics have been used to identify S. aureus (10).
RBCs are the most abundant cells in human blood and have been shown to interact with numerous pathogens (2, 6, 17, 21, 35, 38, 43, 47, 51). These pathogens bind to sugar groups of RBC surface glycoproteins, especially sialic acid residues. Because a RBC expresses a wide variety of sialyoglycoproteins on its surface and because sialoglycoprotein functions are not well known, it has been postulated that these sialoglycoproteins function as decoy receptors to attract pathogens away from target tissues (14). However, some pathogens, such as Malaria parasites, use RBC sialoglycoproteins as receptors to invade RBC through specific molecular interactions (7, 11, 18, 30, 37, 42). RBC sialoglycoproteins are also known to be the binding sites for bacterial toxins such as Escherichia coli and Vibrio cholerae hemolysins (9, 52).
In this study, we report a new shear-dependent mechanism of S. aureus binding to RBC that is independent of fibrinogen and IgG. The binding of bacteria to RBC is maximal at the shear rate of 100 s1, and the adhesion is stable under shear rates as high as 2,000 s1. Sialoglycoprotein(s) on the RBC surface are likely the major receptor(s) for the adhesion and high molecular weight plasma constituents are required. The new binding mechanism demonstrates high sensitivity to the anticoagulant heparin. The responsible plasma molecule and adhesin on S. aureus remain to be identified.
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MATERIALS AND METHODS |
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Bacteria culture and staining. The S. aureus (Newman) strain was chosen because it is well characterized and produces various receptor proteins capable of mediating adhesion to extracellular matrix proteins and blood cells such as platelets (36). To study the involvement of two major bacterial receptors to the adhesion of S. aureus to RBC, the following mutant strains of Newman strain were also used: ClfA negative mutant (ClfA; DU5876) and protein A negative mutant on S. aureus (SPA; DU5971) (36). Cultures were grown in tryptic soy broth (Difco, Detroit, MI), and glycerol stocks were prepared and stored at 80°C (33). Bacteria were inoculated into tryptic soy broth medium from glycerol stock and cultivated at 37°C to optical density 67 (at 600 nm) with constant rotation (33). Cells were harvested and washed three times with Dulbecco's phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ containing 0.2% bovine serum albumin. Bacterial cells were stained with 60 µg/ml of hexidium iodide (HI; Molecular Probes, Eugene, OR) for 1 h at room temperature with constant rotation (29). Cells were then washed three times in DPBS/0.2% bovine serum albumin to remove excess dye and resuspended in the same buffer.
Cell preparation. Human blood was drawn from healthy volunteers by venipuncture into sodium citrate (0.38% wt/vol) anticoagulant. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared by centrifugation of whole blood at 160 g for 15 min (platelet-rich plasma), followed by further centrifugation of the blood at 1,900 g for 15 min (PPP) (31). Serum and buffy coat were removed as previously described (44). RBCs were washed twice with DPBS and stained with 20 µM 5-(and -6)-carboxyfluorescein diacetate, succinimidyl ester [5(6)-CFDA SE; Molecular Probes] for 20 min at room temperature in the dark (15). The final RBC concentration was adjusted to the desired levels of 1 to 40% hematocrit with platelet-poor plasma. To study the effect of plasma on bacteria-RBC binding, RBCs were also diluted by DPBS with plasma content from 0 to 100%. In some experiments, RBCs diluted with DPBS were preincubated with fibrinogen (1,000 µg/ml), fibronectin (100 µg/ml), thrombospondin (5 µg/ml), vitronectin (20 µg/ml), laminin (5 µg/ml), von Willebrand factor (7.5 µg/ml), IgG (1,000 µg/ml), or a cocktail of all these plasma proteins for 1 h at room temperature before the rheometry assay. In other experiments, RBCs diluted with PPP were preincubated with heparin (0 to 1,000 U/ml), or 5 mM EDTA for 30 min at room temperature before being mixed with bacteria for the adhesion experiment.
Cone-and-plate rheometry assay.
5(6)-CFDA SE-stained RBC (180 µl; 1 to 40% hematocrit) and 20 µl of HI-stained bacteria (0.088 3.52 x 108 cells/ml) were mixed briefly in a microcentrifuge tube at room temperature and then placed onto the stationary plate of a cone-and-plate rheometer (RS150; cone angle 1°, plate diameter 35 mm; Haake, Paramus, NJ), which was maintained at 37°C during the entire experiment (20, 31). Shear rates varied from 25 to 2,000 s1 for prescribed periods of time ranging from 30 to 120 s. Static conditions were achieved by setting the shear rate to 0 s1. On termination of shear exposure, 5 µl of samples were obtained and instantly fixed with 1 ml of 1% formaldehyde before flow cytometric analysis (31). To prevent nonspecific cell binding to the cone-and-plate of the rheometer, these parts were precoated with 4% bovine serum albumin for 1 h.
RBC treatment with enzyme. To identify the receptor(s) responsible for the binding of bacteria to RBC, unstained RBC was pretreated with trypsin (1 mg/ml), chymotrypsin (1 mg/ml), or neuraminidase (0.5 U/ml) for 1 h at 37°C (9). Immediately thereafter, RBC was washed twice with DPBS containing Ca2+/Mg2+ and stained with 5(6)-CFDA SE as described above. RBC concentration was diluted to 10% hematocrit during enzyme treatment and adjusted to 20% hematocrit after staining. Matched control specimens were exposed to the identical sequence of steps (i.e., incubation time at 37°C, staining procedures) without enzyme addition.
Analysis of heterotrophic aggregation by flow cytometric analysis. The cellular composition of stable aggregates generated in the rheometer was determined using a FACSCalibur flow cytometer (Becton Dickinson) according to a dual-color flow cytometric methodology (31). 5(6)-CFDA SE-stained RBC and HI-stained bacteria were identified on the basis of their characteristic forward-scatter, side-scatter, and fluorescence profiles. 5(6)-CFDA SE and HI are excited efficiently at 490 nm by the argon laser of a flow cytometer, and their emission spectra are well separated [525 nm for 5(6)-CFDA SE and 605 nm for HI], thereby allowing simultaneous two-color fluorescence measurements. At least 50,000 5(6)-CFDA SE-stained RBC events from the RBC gate in FSC-SSC diagram were collected to determine the extent of S. aureus binding to RBC. Unmixed 5(6)-CFDA SE-stained RBC and HI-stained bacteria were used to set thresholds (Fig. 1, A and B, horizontal and vertical lines) that separate nonadherent single RBCs and single bacteria (SB) from bacteria-RBC heteroaggregates (HA), respectively. The percentage of bacterial cells bound to RBCs (%HA) was calculated as the ratio of the number of HAs to the sum of the number of SBs and the number of HAs. That is, %HA = 100 x HA/(HA + SB). Although some HAs seem to have more than one bacterial cell, because they are negligible with <5% of total HAs as shown in the flow cytometeric diagram, we considered all HAs containing SB cells only.
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Statistics. Data are expressed as means ± SE. Statistical significance of differences between means was determined by ANOVA at a 95% confidence interval (equivalent to a P value <0.05). If means were shown to be significantly different, multiple comparisons by pairs were performed using Tukeys test.
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RESULTS |
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To elucidate whether the observed adhesion is related to the known mechanism that involves ClfA or protein A on S. aureus, bacterial mutants that do not express either ClfA (ClfA) or protein A (SPA) were used to quantitatively compare the adhesion levels among the wild-type and mutant strains. For these experiments, 180 µl of RBCs (20% hematocrit) and 20 µl of bacteria (1.76 x 108 cells/ml) were mixed and subjected to shear (100 s1) in a cone-and-plate rheometer for 60 s. The wild-type Newman strain produced an adhesion level of 41.8 ± 5.6%, whereas the ClfA and SPA yielded adhesion levels of 45.0 ± 13.4% and 71.8 ± 4.5%, respectively (Fig. 2). This study shows that mutant strains bind to RBC with similar (ClfA) or even higher (SPA) levels compared with the wild-type Newman strain, suggesting that neither ClfA nor protein A of the S. aureus is the bacterial binding protein for the observed adhesion to RBC.
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A baseline level (10.1 ± 2.4%) of bacterial adhesion to RBCs was observed for 30 s of static (no flow) incubation (Fig. 3A). This level did not change significantly as the incubation time increased up to 120 s under static conditions. As hydrodynamic shear rate increased from 25 to 2,000 s1, bacterial adhesion to RBC showed a maximum level of 28.5 ± 4.4% at shear 100 s1, and the adhesion level changed slightly in the shear range from 25 to 400 s1. At the shear 2,000 s1, adhesion level (11.2 ± 2.5%) decreased to nearly the level of the static baseline. The increased levels of adhesion on shear application are all statistically significant, except at shear 2,000 s1 with shear time of 30 and 120 s. The adhesion level increased progressively with shear exposure time (60, 120 s) and peaked at shear rate 100 s1. The extent of adhesion showed a maximum value of 72.7 ± 5.7% at shear rate of 100 s1 for 120 s and decreased with shear rate >100 s1. The increase of adhesion on shear time is statistically significant at shear rate of 100 s1 (60 s, 120 s) and 400 s1 (120 s).
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Effect of RBC concentration on adhesion. We next investigated the effects of RBC concentration on the adhesion of bacteria to RBCs. For this purpose, we adopted two different methods. First, we fixed the S. aureus final concentration (1.76 x 107 cells/ml) and three different concentrations (36, 18, and 3.6% of final hematocrit) of RBCs were mixed with bacteria and subjected to shear rate levels ranging from 0 to 2,000 s1 for 60 s in a cone-and-plate rheometer. With 36% hematocrit, >50% of bacteria bound to RBC even under static conditions (Fig. 4). As shear rate increased, the adhesion levels revealed a pattern similar to that of 18% hematocrit. The adhesion levels were >80% under all the shear rates up to 2,000 s1. In distinct contrast, when RBC concentration decreased to 3.6% hematocrit, the adhesion levels were <11% with little change on shear. Although the adhesion level with 3.6% hematocrit showed maximum value under the shear rate of 400 s1, this value was not statistically different from those of other shear rates. As RBC concentration decreases, the collision frequency as well as the number of RBC binding sites for bacteria also decrease, which ultimately results in the decreased adhesion of bacteria to RBC with lower RBC concentrations.
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To characterize the molecular mechanism of this adhesion process on the RBC side, we turned our focus to the identification of the responsible molecule(s) on the RBC surface. RBCs were treated with trypsin, chymotrypsin, or neuraminidase to remove the surface proteins or the sialic acid residues of the glycoproteins on the RBC, respectively. Trypsin and chymotrypsin are proteolytic enzymes, whereas neuraminidase is the sialidase enzyme, which catalyzes the hydrolysis of 23-,
26-, and
28-linked N-acetyl-neuraminic acid (sialic acid) residues from glycoproteins and oligosaccharides. After enzyme treatment, bacteria were mixed with the RBCs and exposed to a shear rate of 100 s1 for 60 s. The adhesion level for the control without enzyme treatment was 38.8 ± 9.8% (Fig. 6). Incubation of RBCs for 1 h at 37°C decreased the extent of S. aureus binding to RBCs. Trypsin treatment abolished receptor function on RBCs completely, as the adhesion level after trypsin treatment was only 2.9 ± 0.3%. Chymotrypsin was slightly less effective than trypsin (7.8 ± 2.0%). Neuraminidase, which cleaves the sialic acids from the glycoprotein, significantly decreased heteroaggregation, but was the least effective among three enzymes (19.4 ± 7.6%). Because trypsin, chymotrypsin, and neuraminidase inhibited the adhesion of bacteria to RBC with higher inhibition by trypsin, we hypothesize that the receptor on RBC for S. aureus is a sialylated, protease-sensitive epitope.
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DISCUSSION |
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The binding of S. aureus to RBC is maximal at a shear rate of 100 s1. As the shear rate increases over 100 s1, the adhesion level decreases, and at shear rate 2,000 s1, the adhesion level is only slightly higher than the static condition at 120 s (Fig. 3A). The HAs, once formed, are resistant to breakage at high shear rate 2,000 s1, suggesting that the low adhesion of bacteria at high shear rate might be attributed to the inability of participating receptor-ligand pair(s) to form bonds at short contact times (Fig. 3B). However, the lack of adhesion at high shear rate may also be due to disaggregation of bonds that have just formed, because these will not necessarily be as strong as those allowed to mature before application of the high shear. These data suggest that S. aureus-RBC aggregates may form preferentially in microcirculation and postcapillary venules, which have relatively low wall shear rates. However, once formed, the aggregates may survive much higher shear rates, such as those found in the arterial circulation.
Bacterial mutants which do not express either ClfA (ClfA) or protein A (SPA) bind to RBC with similar (ClfA) or even higher (SPA) levels than the parental wild-type strain under the same adhesion conditions, suggesting that neither ClfA nor protein A is the bacterial binding protein for the adhesion (Fig. 2). That is, the adhesion reported in this study is different from the widely known mechanism in the hemagglutination test.
The presence of plasma is required for adhesion of S. aureus to RBC. As we increased the plasma content of the RBC before mixing with bacteria, the adhesion level increased dramatically from a baseline level (5.7 ± 0.8%) without plasma to a maximum level (85.8 ± 7.7%) with 20% plasma in DPBS buffer (Fig. 5A). The baseline adhesion may represent nonspecific binding between bacteria and RBC. Addition of 10% plasma is enough to elicit maximal adhesion, whereas 100% plasma, surprisingly, decreases the adhesion slightly. It is noteworthy that the filtrate of plasma produced using an ultrafiltration membrane with 100,000 nominal molecular weight limit failed to support the adhesion of bacteria to RBC (data not shown). This observation suggests that small molecules such as ions are not the key factor supplied by plasma addition; rather, high molecular weight molecules such as proteins, which cannot pass through this membrane, might be involved.
The decrease in adhesion with 100% plasma may be explained by the presence of an inhibition protein(s) in plasma. The dilution of plasma could reduce the inhibition resulting in increased adhesion when only 1050% of plasma is used. Because the protein A deletion mutant strain showed a much higher adhesion level than the wild-type Newman strain (Fig. 2), IgG might be the inhibitor. Exogenous IgG addition to the RBC diluted with 20% plasma showed decreased adhesion compared with the control, consistent with this explanation (data not shown).
To investigate the involvement of plasma proteins to the adhesion, we added various plasma proteins to the RBC-S. aureus mixture before the application of shear. Although the surface proteins of S. aureus for fibrinogen (ClfA) and IgG (protein A) are not involved in the adhesion, we further investigated the possible involvement of fibrinogen and IgG because they are known to bind to RBC and cause RBC aggregation (28, 41). Fibrinogen (1,000 µg/ml), von Willebrand factor (7.5 µg/ml), and IgG (1,000 µg/ml) were added separately to the RBC solution before being mixed with bacteria. The adhesion did not increase compared with the control sample without plasma (Fig. 5B). That is, different plasma protein(s) seem to be involved in the adhesion.
We also evaluated plasma proteins involved in sickle RBC binding to endothelial cells. Thrombospondin (TSP) released from activated platelets binds to endothelial cells and is thought to promote sickle RBC adhesion to the endothelium (5, 45). Binding of fibronectin to sickle RBCs makes them more adherent (23). Laminin, a major component of the subendothelium, also supports significant adhesion of sickle RBC (25). The addition of these plasma proteins also failed to support the adhesion of bacteria to RBC. A "cocktail" of all of the above- mentioned proteins also could not support the adhesion.
Although the addition of TSP failed to support the binding of S. aureus to RBC, it is interesting to note that heparin blocked the adhesion because heparin is known to inhibit the adhesion of sickle RBC to endothelial cells via TSP (19) (Fig. 7). In addition, it can function against P-selectin-mediated tumor cell adhesion (24) and inflammation (34). Heparin also has been used as an anticoagulant. At this moment, the mechanism of inhibition by heparin is not clear. EDTA, a divalent cation chelating agent, also blocked the adhesion of bacteria to RBC in the presence of plasma, suggesting that divalent ions are involved in the adhesion.
The flow cytometry data demonstrating adhesion of bacteria to RBC in the absence of any platelet contamination suggests that platelets are not directly involved in the observed adhesion (Fig. 1). Furthermore, microparticle-free PPP, which was prepared by ultracentrifugation of PPP at 77,000 g for 1 h (48), showed similar adhesion to the control PPP (data not shown), although microparticles produced from platelets have been shown to be involved in the adhesive interaction between various cells (1, 32).We also added platelet factor-4 to the RBC suspended in the buffer. This addition did not increase the adhesion compared with buffer (data not shown). These results demonstrate that platelet-derived microparticles and platelet itself are not involved in the observed adhesion.
Attachment of pathogens to host cell surface receptors is essential to invade target cells. Many pathogens use sugar groups of surface glycoproteins, in particular, sialic acid residues, as receptors for invasion (22). RBC glycoproteins, such as glycophorin A and C (GYPA and GYPC), are known to be used as receptors for viruses, bacteria, parasites, and toxins (3). They are also known to be sensitive to the enzymatic cleavage, such as trypsin and neuraminidase (49). Of these, neuraminidase cleaves the sialic acid residue of the GYPA and GYPC. Because trypsin, chymotrypsin, and neuraminidase inhibited the adhesion of bacteria to RBC with higher inhibition by trypsin (Fig. 6), we hypothesize that the receptor on RBC for S. aureus is a sialylated, protease-sensitive epitope. To verify the possible involvement of either GYPA or GYPC on bacterial adhesion, we performed inhibition tests for bacterial adhesion to RBC using monoclonal antibodies against GYPA and GYPC. When the monoclonal antibodies for either GYPA or GYPC proteins were incubated with RBC before being mixed with bacteria, the adhesion level was reduced in a dose-dependent manner with either antibody (data not shown). Isotype control antibody did not reduce the adhesion level. However, the addition of either antibody also caused homotypic aggregation of RBCs that may reduce the adhesion level of bacteria to RBC. Furthermore, the homotypic aggregation of RBC and the reduction of bacterial adhesion to RBC were also observed with a monoclonal antibody against glycophorin B, which is not a sialylated glycoprotein. The trends were the same when RBCs were perfused over a bacterial layer in a parallel plate flow chamber. With these results, it is not clear whether GYPA or GYPC is involved in the bacterial adhesion. The addition of Fab fractions of these antibodies may eliminate the homotypic aggregation of RBCs and reveal whether those glycophorins are the receptor(s) for the bacterial adhesion to RBC. However, antibodies available commercially are at low concentration, making fractionation and recovery of significant quantities of Fab fragments impossible at this time.
In conclusion, this work demonstrates a new mechanism of S. aureus-RBC binding under hydrodynamic shear conditions via unknown RBC sialoglycoprotein(s). The binding requires plasma protein(s) other than fibrinogen and IgG and does not involve the S. aureus adhesions ClfA or protein A. At this time, the clinical significance of this binding mechanism is not clear. However, the binding of S. aureus to RBCs might limit the bacterial adherence to host components such as platelets, which initiate colonization. Studies are currently under way to probe this possibility and to identify the molecular constituents responsible for the reported adhesion.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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