Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under hydrodynamic shear conditions

Pyong Kyun Shin,1 Parag Pawar,2 Konstantinos Konstantopoulos,2 and Julia M. Ross1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Staphylococcus aureus infection begins when bacterial cells circulating in blood adhere to components of the extracellular matrix or endothelial cells of the host and initiate colonization. S. aureus is known to exhibit extensive interactions with platelets. S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins, such as fibrinogen and IgG. Herein we report a new binding mechanism of S. aureus to RBC independent of those plasma proteins. To characterize the new adhesion mechanism, we experimentally examine the binding kinetics and molecular constituents mediating the new adhesive interactions between S. aureus and RBCs under defined shear conditions. The results demonstrate that the receptors for fibrinogen (clumping factor A) and IgG (protein A) of S. aureus are not involved in the adhesion. S. aureus binds to RBCs with maximal adhesion at the shear rate 100 s–1 and decreasing adhesion with increasing shear. The heteroaggregates formed after shear are stable when subjected to the shear rate 2,000 s–1, indicating that intercellular contact time rather than shear forces controls the adhesion at high shear. S. aureus binding to RBC requires plasma, and 10% plasma is sufficient for maximal adhesion. Plasma proteins involved in the cell-cell adhesion, such as fibrinogen, fibronectin, von Willebrand factor, IgG, thrombospondin, laminin, and vitronectin are not involved in the observed adhesion. The extent of heteroaggregation is dramatically reduced on RBC treatment with trypsin, chymotrypsin, or neuraminidase, suggesting that the receptor(s) mediating the heteroaggregation process is a sialylated glycoprotein on RBC surface. Adhesion is divalent cation dependent and also blocked by heparin. 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 or IgG and does not involve the S. aureus adhesins clumping factor A or protein A.

adhesion; red blood cell


STAPHYLOCOCCUS AUREUS is a well-known pathogenic organism that causes a variety of blood-borne infectious diseases, including infective endocarditis (8). Previous studies have shown that S. aureus can interact with vascular endothelial cells (39), extracellular matrix (12), and platelets (36, 46). Infection begins when S. aureus cells circulating in blood adhere to components of the extracellular matrix, a forming thrombus, or endothelial cells of the host and initiate colonization. Several surface proteins of S. aureus have high specificity to matrix and plasma proteins and have been shown to mediate these adhesion processes (4, 12).

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{alpha} 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 s–1, and the adhesion is stable under shear rates as high as 2,000 s–1. 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Trypsin, chymotrypsin, neuraminidase, fibrinogen, fibronectin, thrombospondin, vitronectin, laminin, and IgG were purchased from Sigma (St. Louis, MO). Soluble von Willebrand factor (factor VIII free) was purchased from Haematologic Technologies (Essex Junction, VT).

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 6–7 (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 s–1 for prescribed periods of time ranging from 30 to 120 s. Static conditions were achieved by setting the shear rate to 0 s–1. 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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Fig. 1. Detection of Staphylococcus aureus-red blood cell (RBC) heteroaggregates by flow cytometry and electron microscopy. A: bacteria-RBC specimen after 60 s without shear. B: bacteria-RBC specimen subjected to shear for 60 s at 100 s–1. C: electron micrograph showing heterotypic aggregates composed of a single S. aureus cell and one RBC cell (magnification, x10,500; scale bar = 2 µm). 5-(And -6)-carboxyfluorescein diacetate, succinimidyl ester [5(6)-CFDA SE]-stained RBC (20% hematocrit) and hexidium iodide-stained S. aureus (1.76 x 107 cells/ml) were subjected to either 100 s–1 or 0 s–1 for 60 s at 37°C in the absence of platelet. On termination of shear, aliquots (5 µl) were immediately fixed with 1% formaldehyde and subsequently analyzed by a dual-color flow cytometric technique.

 
Electron microscopy. Samples containing RBC-S. aureus cell aggregates were prepared for electron microscopy by standard procedures described previously (31). Briefly, sheared specimens were fixed in 1.5% glutaraldehyde for 1 h at RT and postfixed for 1 h in Palade's 1% osmium tetroxide at 4°C, and subsequently incubated in Kellenberger's uranyl acetate solution overnight. After dehydration with a graded series of ethanol, cells were embedded in Epon. After polymerization, ultrathin sections were obtained on a Leica Ultracut UCT microtome equipped with a diamond knife. Sections were then stained with uranyl acetate and lead citrate before being viewed with a transmission electron microscope (model 420, Phillips).

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 Tukey’s test.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adhesion of S. aureus to RBC. When RBCs (18% final hematocrit) and S. aureus were allowed to interact under static (no flow) conditions, a baseline level of S. aureus binding to RBC was observed (Fig. 1A). However, application of fluid shear significantly potentiated the binding of bacteria to RBC. For instance, >50% of the total bacterial population fell into the RBC-bacteria HA region after shear exposure to 100 s–1 for 60 s (Fig. 1B). Most RBC-S. aureus cell aggregates (>95%) were present as SB cells with one or occasionally two RBCs bound to their surface, as confirmed by transmission electron microscopy (Fig. 1C).

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 s–1) 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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Fig. 2. Effects of bacterial receptors on the adhesion of S. aureus to RBC. Wild-type S. aureus (1.76 x 107 cells/ml), clumping factor A negative mutant bacteria (ClfA), or protein A negative mutant bacteria (SPA) were mixed with platelet-poor plasma (PPP)-diluted RBC (20% hematocrit) in the cone-and-plate rheometer and subjected to shear for 60 s at 100 s–1. Data represent means ± SE of 3–5 donors.

 
The kinetics of the S. aureus to RBC binding events were studied under various hydrodynamic shear conditions for defined periods of time. More specifically, 200 µl of RBC and bacteria mixture was subjected to shear (0 to 2,000 s–1) in a cone-and-plate rheometer for 30, 60, and 120 s. The ratio of RBCs to bacteria was ~100:1.

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 s–1, bacterial adhesion to RBC showed a maximum level of 28.5 ± 4.4% at shear 100 s–1, and the adhesion level changed slightly in the shear range from 25 to 400 s–1. At the shear 2,000 s–1, 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 s–1 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 s–1. The extent of adhesion showed a maximum value of 72.7 ± 5.7% at shear rate of 100 s–1 for 120 s and decreased with shear rate >100 s–1. The increase of adhesion on shear time is statistically significant at shear rate of 100 s–1 (60 s, 120 s) and 400 s–1 (120 s).



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Fig. 3. Kinetics of the adhesion of S. aureus to the RBC. RBC concentration was adjusted to 20% hematocrit by dilution with platelet-poor plasma. RBCs were mixed with hexidium iodide-stained S. aureus cells (1.76 x 107 cells/ml) in the cone-and-plate rheometer. A: bacteria-RBC specimens were subjected to well-defined levels of shear for 30 s ({blacksquare}), 60 s ({bullet}), or 120 s ({blacktriangleup}). *P < 0.05 with respect to 30 s. {dagger}P < 0.05 with respect to static (0 s–1) condition. B: bacteria-RBC specimen was subjected to well-defined levels of shear for 60 s or 120 s. For shear rate change, first shear rate was maintained for 60 s and followed by second shear rate for 60 s. *P < 0.05 with respect to control shear and time (100 s–1 for 120 s). Data represent means ± SE of 3–5 experiments.

 
To examine the effects of hydrodynamic shear rate not only on the formation of heterotypic aggregates but also on the strength of adhesion, we subjected bacteria-RBC cell suspensions to well-defined levels of shear. Application of low shear rate (100 s–1) for both 60 s (Fig. 3B, bar c) and 120 s (Fig. 3B, bar d) led to a statistically significant increase in bacteria-RBC cell binding compared with either static (Fig. 3B, bars a and b) or high shear (2,000 s–1) conditions (Fig. 3B, bars e and f). The integrity of the bacteria-RBC aggregate formed under low shear rate was preserved when exposed to subsequent high shear for 60 s (Fig. 3B, bar g), thus suggesting that HAs, once formed, are resistant to disaggregation with increasing shear. In contrast, bacteria and RBCs subjected to high shear for 60 or 120 s did not aggregate (Fig. 3B, bars e and f). However, they retain the ability to form heterotypic aggregates on subsequent exposure to low shear for 60 s (Fig. 3B, bar h). Consequently, the increased heteroaggregation observed at low shear rates might be attributed to the longer intercellular contact time.

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 s–1 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 s–1. 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 s–1, 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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Fig. 4. Effects of RBC concentration on the adhesion of S. aureus to the RBC with a fixed bacteria number. RBC concentration was adjusted by dilution with platelet-poor plasma. Various concentrations of RBC were mixed with hexidium iodide-stained S. aureus cells (1.76 x 107 cells/ml) in the cone-and-plate rheometer and subjected to well-defined levels of shear for 60 s. *P < 0.05 with respect to 36% RBC concentration. Data represent means ± SE of 3–5 experiments.

 
Second, the RBC-to-bacteria ratio was fixed to 100:1 and the adhesion of bacteria to RBC was measured with various RBC concentrations (36, 18, 8, 3.6, and 0.8% hematocrit) under static and shear rate of 100 s–1 for 60 s. That is, the relative receptor density on RBC for bacteria was maintained constant. This RBC-to-bacteria ratio corresponded to the bacteria concentration of 1.76 x 107 cells/ml with 18% hematocrit of RBC. So, for example, for 36% RBC, two times higher bacteria concentration was used in this experiment than in the previous experiment. Although the relative receptor density for bacteria remained constant, the adhesion levels increased along with RBC concentration (Table 1). For instance, under shear rate 100 s–1, the adhesion levels increased linearly up to 18% hematocrit of RBC. With 36% hematocrit, the adhesion level did not increase proportionally with RBC concentration, suggesting that the adhesion efficiency decreased as RBC concentration reached 36% hematocrit. Under static condition, the adhesion level increased linearly from 3.6 to 36% hematocrit, but to a significantly lesser extent than in the presence of shear.


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Table 1. Effects of RBC concentration on the adhesion of S. aureus to RBC with fixed RBC-to-bacteria ratio

 
Elucidation of receptor responsible for S. aureus binding to RBC. Because adhesion of S. aureus to RBC was independent of ClfA and protein A of S. aureus and was concentration and shear dependent, we investigated the potential contributions of plasma proteins to adhesion. For this purpose, buffer was added to the PPP to get the desired plasma concentration ranging from 0 to 100%. The RBC concentration (18% of final hematocrit) was then adjusted with the use of these mixtures of PPP and buffer. Figure 5A shows the extent of bacterial adhesion to RBC measured under shear rate 100 s–1 for 60 s as a function of relative plasma concentration. Plasma content from 10 to 50% revealed the similar adhesion levels with a maximum adhesion (85.8 ± 7.7%) at 20% plasma while the adhesion level dropped with 100% plasma (65.2 ± 14.3%). Without plasma, the adhesion level was very low (5.7 ± 0.8%). This result suggests that the adhesion of S. aureus to RBCs is specific and that a plasma protein(s) is required.



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Fig. 5. Effects of plasma concentration and various plasma proteins on the adhesion of S. aureus to the RBC. A: RBC concentration (20% hematocrit) was adjusted by dilution with the mixture of platelet-poor plasma (PPP) and Dulbecco's PBS (DPBS) buffer. PPP and DPBS buffer were mixed to get the desired plasma concentration. RBC were mixed with hexidium iodide-stained S. aureus cells (1.76 x 107 cells/ml) in the cone-and-plate rheometer and subjected to shear for 60 s at 100 s–1. *P < 0.05 with respect to 20% plasma concentration. B: effects of various plasma protein additions on the adhesion of S. aureus to the RBC. RBCs were diluted with buffer to 20% hematocrit. Fibrinogen (Fg, 1,000 µg/ml), fibronectin (Fn, 100 µg/ml), thrombospondin (TSP, 5 µg/ml), vitronectin (Vn, 20 µg/ml), laminin (Ln, 5 µg/ml), von Willebrand factor (vWF, 7.5 µg/ml), or IgG (1,000 µg/ml) were added separately or all together (Mix) to the RBC solution before mixing with bacteria (1.76 x 107 cells/ml) in the cone-and-plate rheometer and subjected to shear for 60 s at 100 s–1. As positive and negative controls, plasma-diluted RBC (Plasma) or buffer-diluted RBC without plasma protein addition (Buffer) were mixed with bacteria before shear. *P < 0.05 with respect to positive control (Plasma). Data represent means ± SE of 3–5 donors.

 
To investigate the involvement of plasma proteins to the adhesion, we added various plasma proteins, whose receptor proteins are produced in S. aureus, to the RBCs diluted with PBS buffer. 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), or IgG (1,000 µg/ml) were added separately or all together to the RBC solution before being mixed with bacteria. The adhesion did not increase compared with the control in any case (Fig. 5B). That is, different plasma protein(s) and bacterial receptor(s) seem to be involved in the adhesion. The results of this study showing that fibrinogen, IgG, or von Willebrand factor did not support the adhesion separately or together are consistent with the observations with mutant strains herein.

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 {alpha}2–3-, {alpha}2–6-, and {alpha}2–8-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 s–1 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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Fig. 6. Involvement of glycoproteins in the adhesion of S. aureus to the RBC. A: effects of enzyme treatment of RBC on the adhesion. RBC concentration (10% hematocrit) was adjusted by dilution with DPBS buffer. Samples were treated with trypsin (1 mg/ml), chymotrypsin (1 mg/ml), and neuraminidase (0.5 U/ml) for 1 h at 37°C. After being washed, RBCs were resuspended into PPP. Enzyme-treated RBCs were mixed with hexidium iodide-stained S. aureus cells (1.76 x 107 cells/ml) in the cone-and-plate rheometer and subjected to shear for 60 s at 100 s–1. *P < 0.05 with respect to control. Data represent means ± SE of 3 donors.

 
To further characterize the adhesion, we added EDTA (5 mM) or heparin to the RBC solution (18% final hematocrit) diluted with PPP. The extent of S. aureus-RBC HAs is abrogated on addition of EDTA, suggesting that their molecular interaction is dependent on divalent cations (data not shown). Heparin was added from 0 to 1,000 U/ml (final concentration) to the RBC and incubated for 30 min at room temperature before mixing with bacteria. The adhesion was measured under shear 100 s–1 for 60 s. With 100 U/ml or higher heparin concentration, the adhesion of bacteria to RBC was dropped to the level without plasma (6.8 ± 2.9%) (Fig. 7). That is, the contribution of plasma to the adhesion was blocked completely with heparin concentration >100 U/ml. With 10 U/ml heparin, the blocking was highly donor dependent. The adhesion level varied from 91.6 to 9.7% depending on the donor.



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Fig. 7. Effects of heparin on the adhesion of S. aureus to the RBC. RBC concentration (20% hematocrit) was adjusted by dilution with PPP. Heparin was added at the desired concentration to RBC and incubated at room temperature for 30 min. After incubation, RBCs were mixed with hexidium iodide-stained S. aureus cells (1.76 x 107 cells/ml) in the cone-and-plate rheometer and subjected to shear for 60 s at 100 s–1. For comparison, RBC diluted with DPBS buffer rather than plasma was used for adhesion experiment without heparin addition (no heparin/no plasma). *P < 0.05 with respect to 0 U/ml heparin concentration. Data represent means ± SE of 3–5 donors.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
S. aureus expresses various surface proteins with specific affinity for plasma proteins and for components of the extracellular matrix (12, 13). These surface proteins enable S. aureus to adhere to the extracellular matrix of the host and to initiate colonization. S. aureus also binds to RBC either specifically or nonspecifically through the mediation of plasma proteins such as fibrinogen and IgG (10, 50). Among the surface proteins expressed on S. aureus, ClfA and protein A are involved in this adhesion. In this study, we describe a new shear-sensitive mechanism of S. aureus adhesion to RBCs. To our knowledge, this is the first study presenting the observation that S. aureus adheres specifically to RBC in the absence of fibrinogen and IgG.

The binding of S. aureus to RBC is maximal at a shear rate of 100 s–1. As the shear rate increases over 100 s–1, the adhesion level decreases, and at shear rate 2,000 s–1, 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 s–1, 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 10–50% 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.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was funded by National Heart, Lung, and Blood Institute Grant HL-66453.


    ACKNOWLEDGMENTS
 
We thank Dr. Monica M. Burdick (Johns Hopkins University, Department of Chemical and Biomolecular Engineering) and J. Michael McCaffery (Johns Hopkins University Integrated Imaging Center) for preparation of sample for electron microscopy analysis and for kind use of the electron microscopy facility, respectively.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Ross, Dept. of Chemical and Biochemical Engineering, Univ. of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250 (e-mail: jross{at}umbc.edu)

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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