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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Anaesthesiology and Surgical
Intensive Care Medicine, Division of Experimental and Clinical
Hemostasis, Institute of Medical Microbiology, University of
Münster, Münster, Germany; the Department of Microbial
Pathogenesis, Gesellschaft für Biotechnologische Forschung,
Braunschweig, Germany; the Department of Chemical Engineering,
University of Florida, Gainesville, FL; Moyne Institute of Preventive
Medicine, Trinity College, Dublin, Ireland; and the Department of
Hematology, University of Utrecht, Utrecht, The Netherlands.
Endovascular infection is a highly critical complication of
invasive Staphylococcus aureus disease. For colonization,
staphylococci must first adhere to adhesive endovascular foci. Von
Willebrand factor (vWF) is a large, multimeric glycoprotein mediating
platelet adhesion at sites of endothelial damage. Earlier it was
demonstrated that vWF binds to and promotes the surface adhesion of
S. aureus, prompting this effort to identify the vWF
adhesin. In Western ligand assays of S. aureus lysates,
staphylococcal protein A (SPA) was recognized by purified vWF. Surface
plasmon resonance demonstrated the binding of soluble vWF to
immobilized recombinant protein A with a Kd of
1.49 × 10 The induction of endovascular infections involves
complex interactions between surface components on the invading
organism and various host determinants. Staphylococcus
aureus is a major pathogen in endovascular infections, such as
infective endocarditis, suppurative thrombophlebitis, or vascular or
heart valve prosthetic infection.1 Among the host factors
potentially contributing to endovascular infections, various plasma and
extracellular matrix proteins Among the factors released by endothelial cells and by platelets,
constitutively or on stimulation, is von Willebrand factor (vWF), a
large multifunctional glycoprotein characterized by high molecular
weight multimers of up to 15 million daltons, consisting of subunits of
250 to 270 kd.12 After release, multimers are subject to
plasma protease cleavage resulting in multimer heterogeneity, and they
are subsequently bound to various subendothelial components, such as
collagens, proteoglycans, and glucosaminoglycans.13 Immobilized vWF is pivotal for platelet adhesion at sites of vascular injury in the presence of high shear stress, an adhesion reaction mediated through the GPIb-IX-V complex on resting and the GPIIb-IIIa complex on stimulated platelets.14 Previously, we
described the interaction of S. aureus with vWF both in
suspension and immobilized on the surface. Binding isotherms revealed a
dose-dependent binding reaction to S. aureus Cowan 1 and
other S. aureus isolates, adsorption kinetics showed
saturable immobilization of vWF on solid substrates, and, consequently,
solid-phase vWF could be demonstrated to promote adhesion of various
S. aureus isolates by 2 to 3 orders of
magnitude.15
In this study, the promotion of adhesion was found to be sensitive to
trypsin pretreatment of the bacteria. Furthermore, though the adhesion
of all other clinical and laboratory isolates was promoted by
surface-adsorbed vWF, the adhesion of S. aureus Wood 46, a
protein A-negative strain, was not promoted on vWF-polymethyl methacrylate (PMMA). Therefore, we hypothesized that an adhesin, possibly of the MSCRAMM (microbial surface components recognizing adhesive matrix molecules) family of staphylococcal surface
proteins,16 might be involved in the interaction of
S. aureus with vWF and therefore investigated the nature of
the putative vWF adhesin by use of various biochemical and molecular
experimental approaches.
Bacteria
Preparation of bacterial lysates
Purification and labeling of vWF vWF was purified from Haemate-HS500 (provided by Centeon Pharma, Marburg, Germany) as previously described.15,20 In brief, cryoprecipitates were subjected to size chromatography (using a column containing BioGel A-15m [Bio-Rad]) in a Tris-HCl buffer (50 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L Na+ citrate, pH 7.35) (TBS) containing a proteinase inhibitor cocktail consisting of phenylmethylsulfonyl fluoride (1 mmol/L; Sigma), leupeptin hemisulfate (5 µmol/L; Sigma), and aprotinin (10 µmol/L; Trasylol; Bayer, Leverkusen, Germany). Eluted fractions were analyzed for vWF content as previously described15 by use of the BCA protein assay, a vWF ELISA, and an assay for ristocetin cofactor activity. Only the first 3 fractions containing high-molecular-weight vWF and shown to be more than 99% pure were used.15For labeling of vWF with fluorescein-isothiocyanate (FITC), a
previously described method21 was modified as follows. TBS containing vWF was supplemented with 2 mmol/L Ca++, and the
pH of the solution was adjusted to 9.5 by the addition of
Na+ carbonate. FITC (10 mg/mL), isomer I, on Celite 10%
(Calbiochem, La Jolla, CA) was solubilized in dimethyl sulfoxide, added
to vWF-TBS in a 1:10 ratio, and incubated for 30 minutes at 21°C. Unbound label was separated using a Sephadex G25 PD10 column
(Pharmacia, Freiburg, Germany) equilibrated with HEPES buffer (10 mmol/L HEPES, 140 mmol/L NaCl). The concentration of FITC-labeled vWF
was calculated as follows: vWF
(mg/mL) = [A280 Western ligand assays and amino terminal amino acid sequencing of eluted proteins Either purified vWF or whole bacterial lysate (40 µg/mL) was subjected to SDS-PAGE using 7.5% acrylamide slab gels. After migration, proteins were transferred to nitrocellulose membranes (membrane BA85; Schleicher & Schuell, Dassel, Germany) using a semidry trans-blot apparatus (Bio-Rad, Munich, Germany), and then the membranes were blocked using bovine serum albumin (BSA; 3% wt/vol) for 6 hours in PBS. Membranes were washed with PBS, incubated at gentle agitation with PBS containing DIG-labeled ligand (40 µg in 30 mL, 4 hours, room temperature), then washed 3 times in PBS/0.05% Tween 20 (10 minutes). For the detection of bound ligand, the digoxigenin detection kit (Boehringer) was used according to the manufacturer's instructions with alkaline phosphatase-coupled anti-DIG Fab fragments and nitroblue-tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as color reagents. For N-terminal sequencing of blotted proteins, lysates were transferred after SDS-PAGE separation to polyvinylidene difluoride membranes. The vWF binding band was localized after detection by Western ligand analysis, eluted, and analyzed with an ABI 494-A automated sequencer (Applied Biosystems, Weiterstadt, Germany).Detection of vWF binding by surface plasmon resonance Surface plasmon resonance (SPR) was determined using a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). The detection principle of SPR is based on an optical phenomenon allowing the detection of changes in mass concentration on the surface of a sensor chip. For the determination of vWF binding to immobilized recombinant protein A (rSPA) (Sigma), 6150 relative response units (RU) rSPA (corresponding to 1.6 ng) was immobilized on sensor chip CM5 by amine coupling using 10 mmol/L NaAc buffer (pH 4.0). The design of the Biacore instrument allows the simultaneous detection of vWF binding to the immobilized rSPA and an uncoated control surface under continuous flow conditions in the detection chamber. HEPES buffer (25 mmol/L HEPES, 100 mmol/L NaCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 0.005% surfactant P20 [pH 7.4]) was used for instrument equilibration and protein injection, and regeneration after ligand binding was achieved using glycine buffer (100 mmol/L glycine, 500 mmol/L NaCl, pH 4.0). The flow rate used was 30 µL/minute. If not indicated otherwise, vWF was used at a concentration of 250 µg/mL. In some experiments, vWF was digested using S. aureus V8 (Sigma). For IgG binding studies, pooled human immunoglobulin (Venimmun; Sandoz, Nuremberg, Germany) was used.Flow cytometric analysis of FITC-vWF binding to S. aureus Bacteria from a fresh overnight culture were diluted at a concentration of 120 000 cells/µL in TBS buffer containing 2 mmol/L Ca++ (pH 7.4). FITC-labeled vWF (final concentration, 0-150 µg/mL) was incubated with the bacterial suspension (10 minutes at room temperature). After washing and sonication, bacteria (5000 cells/determination) were analyzed in a flow cytometer (FACScan; Becton Dickinson, Heidelberg, Germany) using an excitation wavelength of 488 nm at the FACScan standard configuration with a 530-nm bandpass filter. Data were obtained from fluorescence channels in a logarithmic mode.Solid-phase adhesion assays For radiometric analysis of S. aureus adhesion to solid surfaces, a previously described assay was used.22 Briefly, solutions containing indicated concentrations of purified vWF were allowed to adsorb to PMMA coverslips (60 minutes at 37°C). Thereafter, coverslips were washed with PBS and incubated in a shaking water bath with [3H]-thymidine-labeled S. aureus cells (4 × 106 CFU in 1 mL PBS containing 0.5% human serum albumin [HSA]) (60 minutes at 37°C). After adhesion, unbound microorganisms were removed from coverslips by washing twice, and PMMA-adherent radioactivity was determined. For the determination of S. aureus binding to preadsorbed PMMA under flow conditions, slides (60 × 24 mm) were preincubated with 5 mL vWF (50 µg/mL) (60 minutes at 37°C), then placed in a parallel-plate flow chamber (CytoDyne, San Diego, CA). To maintain a well-defined laminar flow field between the parallel plates of the flow chamber, a syringe pump containing PBS/Ca++Mg2+/HSA buffer and either S. aureus NCTC 8325-4 or its spa
mutant, DU 5875, introduced the cell suspension into the chamber at a constant flow rate. The shear rate was calculated to be 110 s 1 according to the formula,
S = 6Q/wh2, where S is the shear rate (s1), Q is the volumetric flow rate (0.008 cm3/s), w is chamber width (0.7 cm), and
h is the chamber gap thickness (0.025 cm).23
Analysis of the attached bacteria over time was performed using an
inverted Axiovert 135 TV (Zeiss, Oberkochen, Germany) microscope, a
motorized stage table (Märzhäuser, Wetzlar, Germany), and
algorithms for an image analysis software package (Optimas 5.1; Media
Cybernetics, Silver Spring, MD). A macro was written in the Optimas
Analytical Language for Images (Media Cybernetics) to recursively
capture and store digital images of 10 different microscopic fields at
each time point. Counts of attached bacteria were subsequently
performed by digital image analysis of the captured images (gray level
thresholding and size discrimination) to distinguish bacteria from the
background in the bright-field images.
Detection of vWF binding to staphylococcal proteins by Western ligand analysis Whole-cell lysates of S. aureus strains Cowan 1, NCTC 8325-4, and Newman were prepared using lysostaphin, resolved on 7.5% SDS-polyacrylamide gels, and transferred to nitrocellulose. Blot membranes were blocked and incubated with purified DIG-labeled vWF (37°C at 240 minutes). After washing, vWF bound to blot membranes was detected in a color reaction. As shown in Figure 1A (left), a single band of 50 to 55 kd was identified in all 3 test strains. Parallel blots performed with DIG-labeled anti-protein A antibodies revealed a strong reaction of bands of identical size compared with those detected with DIG-vWF (Figure 1A, right). Control reactions prepared with nitrocellulose-transferred whole- cell staphylococcal lysates were developed using all reagents, with the exception of DIG-labeled vWF. These controls revealed no color reaction of the blot membrane (Figure 1A, center). For further characterization, the blotted proteins of S. aureus Cowan 1 and 8325-4 recognized by vWF were analyzed using automated N-terminal sequencing, and the following amino acid sequence was obtained: N-Ala-Gln-His-Asp-Glu-Ala-Gln-Gln-Asn-Ala-Phe-Tyr-Gln-Val-Leu-Asn-Met-Pro-Asn-Leu-Asn-Ala-Asp-Gln-Arg-Asn-Gly-Phe-Ile-Gln-(Ser)-Leu-Lys-Asp-Asp. This sequence is 100% identical to the published sequence of SPA.24,25 To confirm that SPA is the molecule recognized by vWF in the Western ligand assays, lysates of thespa
deletion mutants DU 5889, DU 5873, and DU 5875 were separated by
SDS-PAGE, blotted, and incubated with DIG-labeled vWF. These
ligand-binding assays revealed no interaction of molecules contained in
nitrocellulose-blotted S. aureus lysate with soluble vWF
(Figure 1B, lane 1'-3').
Protein A interacts with vWF To confirm further the interaction of vWF with SPA, in a reciprocal binding reaction recombinant human (rh) vWF was subjected to SDS-PAGE, blotted onto nitrocellulose, and incubated with DIG-labeled rSPA. After development of the color reaction, a single band recognized by SPA and corresponding to a protein with an apparent molecular weight of 250 kd was detected (Figure 1C, right lane). This protein was the same size as rhvWF in Coomassie-stained SDS-PAGE (Figure 1C, left lane). In addition to Western ligand analysis, the interaction of vWF with SPA was evaluated using SPR. For this purpose, rSPA was immobilized on a CM5 sensor chip and exposed to purified vWF, and binding was analyzed using a Biacore 2000 instrument (Biacore AB) (Figure 2A). After the injection of vWF, a linear increase in RU was measured on the rSPA-coupled surface, whereas the control surface showed no response increase. Assuming a mean of 10 vWF monomers per multimeric molecule,15 the kon was calculated to be 3.07 × 103 mol/L-1 s-1. At the end of the injection, the response signal on the rSPA surface remained elevated, and it decreased at a koff rate of 4.58 × 10-5 s-1. The resultant Kd was calculated as 1.49 × 108 mol/L. After surface regeneration, the
RU value on the rSPA surface was identical to that on the control
surface. The binding of vWF was found to be dose dependent when vWF
concentrations ranging from 200 to 500 µg/mL were tested (Figure 2A,
insert). HSA or BSA (3 µmol/L) did not bind to rSPA surfaces (Figure
2B). As expected, injections of IgG (1060 nmol/L) resulted in a
response increase on rSPA surfaces as a result of a saturable binding
reaction (Figure 2C). After the saturation of binding sites, no further
increase in response could be observed with the injection of vWF.
However, regeneration of the surface restored the ability of rSPA to
recognize the vWF ligand (Figure 2C). Taken together, these findings
clearly demonstrate the dose-dependent, reversible, and specific
character of the interaction of vWF with SPA. Because S. aureus
expresses a serine protease (V8 protease), the effect of vWF
pretreatment with V8 protease on the vWF-SPA interaction was further
determined. Digestion of vWF with V8 protease (4 U/mL) was found to be
complete after 10 minutes, as demonstrated by SDS-PAGE (not shown). As shown in Figure 2D, binding of vWF fragments to SPA yielded an increase
in RU similar to that observed using undigested vWF.
Binding of soluble vWF to S. aureus wild-type strains,
to spa mutants were further explored. Both mutants of
S. aureus Newman and NCTC 8325-4 showed greatly reduced
binding compared with the wild-type strain (Figure 4). Although relative fluorescence values
of vWF bound to DU 5873 were approximately one tenth of those observed
with wild-type S. aureus Newman, the
spa-deficient strain DU 5875 exhibited approximately one
tenth of the binding values observed with wild-type strain NCTC 8325-4. Complementation of the deletion mutants fully restored binding.
Although binding isotherms of the wild-type and complemented mutants of
S. aureus Newman were almost superimposable, vWF bound to
the complemented strain DU83/256 to a larger extent than to the
wild-type strain S. aureus NCTC 8325-4 (Figure 4).
Adhesion of S. aureus wild-type strains,
spa mutant DU5875 was significantly less (adhesion,
1.2 ± 0.5 × 104; 24% of wild type;
P = .003 [unpaired t test]). With the
complementation of DU5875, adhesion could not only be restored but was
found to be higher when compared with the wild-type strain (adhesion,
6.1 ± 1.3 × 104; 124% with wild type) (Figure
5A). Similar results were observed when
Cowan 1 and its SPA-deficient mutant were tested (Figure 5B). Although
human IgG interacts with SPA, S. aureus adheres to
immobilized vWF even at physiological concentrations of IgG. Adhesion
to vWF-adsorbed surfaces was inhibitable only to 48% in the presence
of excess amounts (10 000 µg/mL) of IgG (Figure 6).
To determine staphylococcal adhesion to vWF-adsorbed surfaces
under well-defined flow conditions, a suspension containing S. aureus NCTC 8325-4 cells (5 × 107 CFU/mL in
PBS-HSA) was introduced into the parallel-plate flow chamber at a shear
rate of 110/s. Attaching cells were detected by video-enhanced light
microscopy and analyzed using image analysis. Adhesion of the wild-type
strain increased as a function of time and exhibited an almost linear
increase in the number of adherent cells during the first 10 minutes of
perfusion and saturation of adhesion after prolonged times of
perfusion. Cell density values of 8.78 ± 2.09 × 105 cells/cm2 were achieved after 20 minutes of perfusion (Figure 7). In
contrast to the wild-type strain, the
In this report, we describe the identification of a novel function
of SPA as an adhesin for a platelet and extracellular matrix protein,
vWF. SPA recognition in Western ligand assays of staphylococcal whole-cell lysates as the putative vWF-binding adhesin prompted the
detailed analysis of the role of SPA in the interaction of S. aureus with vWF by evaluating spa-deficient allelic
replacement mutants and spa-complemented Seven surface proteins from S. aureus have been
characterized so far at the molecular level. Five proteins SPA is an exoprotein that binds to the Fc region of immunoglobulins of
most mammalian species.31 It consists of 5 extracellular domains (designated E, D, A, B, and C), cell wall-spanning regions (Xr and Xc), and an 18- to 20-residue
hydrophobic membrane spanning domain distal to LPXTG.24,25
Each extracellular domain can bind 1 IgG molecule through its Fc For identification of the vWF adhesin, we selected a strategy that has
already been successfully used for other staphylococcal adhesins.45,46 In Western ligand assays, we could identify a single protein band recognized by soluble vWF that weighed 50 to 55 kd. Because the reported molecular weights of SPA ranged from 42 kd47 to 57 kd,25,48 it was suggested that the
vWF-recognized protein could be SPA. This could be confirmed by Western
immunoblot experiments and N-terminal amino-acid sequencing and by the
observation that Western ligand experiments using soluble vWF exposed
to immobilized lysates of To establish its role clearly as staphylococcal adhesin, a putative
MSCRAMM molecule must be demonstrated to mediate the interaction of
whole bacterial cells with extracellular matrix molecules. In previous
reports, flow cytometry has been successfully used to monitor the
interaction of staphylococci with whole cells.8 Thus, we
modified and adapted this technique, which we have established for the
determination of vWF binding to platelets52 for
staphylococci. Our findings confirm and extend the previously published
data on vWF-S. aureus interaction by demonstrating both the
saturation ability of binding and binding inhibition with the addition
of unlabeled ligand. Half-maximal binding concentrations were found to
approximate 50 µg/mL. Given this value to correspond to an estimate
of Kd, the apparent dissociation constant would
be in the order of nanomolar considering the multimeric size of the molecule, suggesting binding affinities similar to those of
fibronectin53 or thrombospondin.54 To evaluate
further the role of SPA, we tested SPA not only confers binding of soluble ligand, it promotes the
adhesion of staphylococcal cells to vWF-adsorbed surfaces. A first
indication for the role of SPA in the interaction with surface-bound
vWF was given by our previous observation, demonstrating the lack of
adhesion promotion of S. aureus Wood 46.15 In
this study, the role of SPA in surface adhesion assays was evidenced by
the demonstration of significantly decreased adhesion of the Interaction of S. aureus with vWF occurs in vivo in an IgG-containing milieu. Our observations demonstrating the IgG-binding SPA as the adhesin for vWF and the inhibition of vWF interaction with immobilized SPA on pretreatment with IgG may suggest that this interaction does not take place in vivo. On the other hand, when investigating other S. aureus-binding plasma proteins, such as fibronectin or fibrinogen, it has been clearly demonstrated that attachment to these adhesive molecules occurs even in the presence of plasma concentrations of the ligand,55 that adhesion to ex vivo catheter material is promoted by these ligands,56,57 and, most important, that staphylococcal deletion mutants deficient in adhesins for fibronectin- or fibrinogen-binding proteins are less virulent in endovascular infection models irrespective of large plasma concentrations of the ligand.3,7 In line with these observations, our experiments revealed the inhibition of adherence to immobilized vWF by maximally 50% in the presence of large (10 000 µg/mL) concentrations of IgG. For fibrinogen, it has been suggested that high local concentration58 and conformational changes59 confer the effect of the surface-immobilized ligand on platelet binding and activation. Although we have previously shown that vWF is significantly adsorbed on PMMA,15 it remains an open issue whether similar mechanisms contribute to the retained adhesion of S. aureus to immobilized vWF, even in the presence of large concentrations of IgG. In conclusion, in experiments using site-directed S. aureus mutants for the evaluation of binding with and adhesion to vWF, we provide compelling evidence that SPA confers interaction with this adhesive extracellular matrix protein. The molecular architecture of SPA fulfills all characteristics of most staphylococcal adhesins of the MSCRAMM family (all except EbpS, which is not LPXTG anchored). SPA expression has been demonstrated to be coregulated by 2 repressor systems, agr and sar,19,60 resulting in decreased expression in stationary growth phases, a characteristic that SPA shares with other MSCRAMMs. Most recently, S. aureus isolates from patients with Kawasaki disease, an acute vasculitis of young children complicated by coronary artery abnormalities, have been shown to express high levels of SPA.61 Together with our findings, this observation attributes to SPA a hitherto unknown function in staphylococcal pathogenesis. Further investigations will focus on the binding domains on both molecules involved in the SPA-vWF interaction and on the application of this in vitro evidence to adequate experimental models, such as endovascular infection. These issues are the subjects of current investigations in our laboratories.
We thank Susanne Weber and Marion Schiphorst for excellent technical help, Dorothea Voss and Centeon Pharma for providing Haemate HS, and M. A. Schmidt for helpful discussions.
Submitted December 16, 1999; accepted May 18, 2000.
Supported by Deutsche Forschungsgemeinschaft, Collaborative Research Center 293, Project A6, and the German Minister for Education, Science, Research, and Technology (grant 01 KI 9750/9). Supported in part by The Wellcome Trust (T. J. F.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mathias Herrmann, Department of Medical Microbiology, University of Münster, Domagkstrasse 10, 48129 Münster, Germany; e-mail: mathias.herrmann{at}uni-muenster.de.
1. Ing M, Baddour LM, Bayer AS. Bacteremia and infective endocarditis: pathogenesis, diagnosis, and complications. In: Crossley K,Archer GL, eds. The Staphylococci in Human Disease. New York: Churchill Livingstone; 1997:331-354. 2. Foster TJ, Höök M. Surface protein adhesins of Staphylococcus aureus. Trends Microbiol. 1998;6:484-488[Medline] [Order article via Infotrieve]. 3. Moreillon P, Entenza JM, Francioli P, et al. Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis. Infect Immun. 1995;63:4738-4743[Abstract]. 4. Cheung AL, Eberhardt KJ, Chung E, et al. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis. J Clin Invest. 1994;94:1815-1822.
5.
Cheung AL, Yeaman MR, Sullam PM, Witt MD, Bayer AS.
Role of the sar locus of Staphylococcus aureus in induction of endocarditis in rabbits.
Infect Immun.
1994;62:1719-1725 6. Flock JI, Hienz SA, Heimdahl A, Schennings T. Reconsideration of the role of fibronectin binding in endocarditis caused by Staphylococcus aureus. Infect Immun. 1996;64:1876-1878[Abstract].
7.
Kuypers JM, Proctor RA.
Reduced adherence to traumatized rat heart valves by a low-fibronectin-binding mutant of Staphylococcus aureus.
Infect Immun.
1989;57:2306-2312 8. Yeaman MR, Sullam PM, Dazin PF, Norman DC, Bayer AS. Characterization of Staphylococcus aureus-platelet binding by quantitative flow cytometric analysis. J Infect Dis. 1992;166:65-73[Medline] [Order article via Infotrieve]. 9. Sullam PM, Bayer AS, Foss WM, Cheung AL. Diminished platelet binding in vitro by Staphylococcus aureus is associated with reduced virulence in a rabbit model of infective endocarditis. Infect Immun. 1996;64:4915-4921[Abstract]. 10. Herrmann M, Lai QJ, Albrecht RM, Mosher DF, Proctor RA. Adhesion of Staphylococcus aureus to surface-bound platelets: role of fibrinogen/fibrin and platelet integrins. J Infect Dis. 1993;167:312-322[Medline] [Order article via Infotrieve]. 11. Yeaman MR. The role of platelets in antimicrobial host defense. Clin Infect Dis. 1997;25:951-968[Medline] [Order article via Infotrieve]. 12. Titani K, Kumar S, Takio K, et al. Amino acid sequence of human von Willebrand factor. Biochemistry. 1986;25:3171-3184[Medline] [Order article via Infotrieve]. 13. Ruggeri ZM, Ware JA. von Willebrand factor. FASEB J. 1993;7:308-316[Abstract]. 14. Ruggeri ZM, De Marco L, Gatti L, Bader R, Montgomery RR. Platelets have more than one binding site for von Willebrand factor. J Clin Invest. 1983;72:1-12. 15. Herrmann M, Hartleib J, Kehrel B, Montgomery RR, Sixma JJ, Peters G. Interaction of von Willebrand factor with Staphylococcus aureus. J Infect Dis. 1997;176:984-991[Medline] [Order article via Infotrieve]. 16. Patti JM, Allen BL, Hook M. MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol. 1994;48:585-617[Medline] [Order article via Infotrieve].
17.
Patel AH, Nowlan P, Weavers ED, Foster TJ.
Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.
Infect Immun.
1987;55:3103-3110 18. McDevitt CA, Francois P, Vaudaux PE, Foster TJ. Identification of the ligand binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus. Mol Microbiol. 1995;16:895-907[Medline] [Order article via Infotrieve]. 19. Patel AH, Kornblum J, Kreiswirth BN, Novick RP, Foster TJ. Regulation of the protein A-encoding gene in Staphylocccus aureus. Gene. 1992;114:25-34[Medline] [Order article via Infotrieve].
20.
Sodetz JM, Pizzo SV, McKee PA.
Relationship of sialic acid to function and in vivo survival of human factor VIII/von Willebrand factor protein.
J Biol Chem.
1977;252:5538-5546
21.
Goto S, Salomon DR, Ruggeri ZM.
Characterization of the unique mechanism mediating the shear-dependent binding of soluble von Willebrand factor to platelets.
J Biol Chem.
1995;270:23352-23361
22.
Vaudaux PE, Waldvogel FA, Morgenthaler JJ, Nydegger UE.
Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence.
Infect Immun.
1984;45:768-774 23. Dickinson RB, Nagel JA, Foster TJ, Proctor RA, Cooper SL. Quantitative comparison of two distinct fibrinogen receptor populations in mediating adhesion of Staphylococcus aureus to surfaces under flow. Infect Immun. 1995;63:3143-3150[Abstract].
24.
Loefdahl S, Guss B, Uhlen M, Philipson L, Lindberg M.
Gene for staphylococcal protein A.
Proc Natl Acad Sci U S A.
1983;80:697-701
25.
Uhlen M, Guss B, Nilsson B, Gatenbeck S, Philipson L, Lindberg M.
Complete sequence of the staphylococcal gene encoding protein A: a gene evolved through multiple duplications.
J Biol Chem.
1984;259:1695-1702 26. Ruta A. Quantitative study of mechanisms of bacterial adhesion to surfaces. Gainesville, FL: University of Florida; 1998. Thesis. 27. Dickinson RB, Cooper SL. Analysis of shear-dependent bacterial adhesion kinetics to biomaterial surfaces. AICHE J. 1995;41:2160.
28.
Schneewind O, Fowler A, Faull KF.
Structure of the cell wall anchor of surface proteins in Staphylococcus aureus.
Science.
1995;268:103-106
29.
Mazmanian SK, Liu G, Ton-That H, Schneewind O.
Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall.
Science.
1999;285:760-763
30.
Park PW, Rosenbloom J, Abrams WR, Mecham RP.
Molecular cloning and expression of the gene for elastin-binding protein (ebpS) in Staphylococcus aureus.
J Biol Chem.
1996;271:15803-15809 31. Forsgren A, Ghetie V, Lindmark R, Sjöquist J. Protein A and its exploitation. In: Easmon CSF,Adlam C, eds. Staphylococci and Staphylococcal Infections. London: Academic Press; 1983:429-480. 32. Kozlowski LM, Soulika AM, Silverman GJ, Lambris JD, Levinson AI. Complement activation by a B cell superantigen. J Immunol. 1996;157:1200-1206[Abstract]. 33. Torigoe H, Shimada I, Waelchli M, Saito A, Sato M, Arata Y. 15N nuclear magnetic resonance studies of the B domain of staphylococcal protein A: sequence specific assignments of the imide 15N resonances of the proline residues and the interaction with human immunoglobulin G. FEBS Lett. 1990;269:174-176[Medline] [Order article via Infotrieve].
34.
Peterson PK, Verhoef J, Sabath LD, Quie PG.
Effect of protein A on staphylococcal opsonization.
Infect Immun.
1977;15:760-764 35. Inganas MS, Johansson SGO, Benninch HH. Interaction of human polyclonal IgE and IgG from different species with protein A from Staphylococcus aureus: demonstration of protein A-reactive sites located on the F(ab')2 fragments of human IgG. Scand J Immunol. 1980;12:23-31[Medline] [Order article via Infotrieve]. 36. Roben PW, Salem AN, Silverman GJ. VH3 family antibodies bind domain D of staphylococcal protein A. J Immunol. 1995;154:6437-6445[Abstract]. 37. Starovasnik MA, Skelton NJ, O'Connell MP, Keley RF, Reilly D, Fairbrother WJ. Solution structure of the E-domain of staphylococcal protein A. Biochemistry. 1996;35:15558-15569[Medline] [Order article via Infotrieve]. 38. Forsgren A, Svedjelund A, Wigzell H. Lymphocyte stimulation by protein A of Staphylococcus aureus. Eur J Immunol. 1976;6:207-213[Medline] [Order article via Infotrieve]. 39. Sasso EH, Silverman GJ, Mannik M. Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup. J Immunol. 1991;147:1877-1883[Abstract]. 40. Espersen F. Complement activation by clumping factor and protein A from Stahylococcus aureus strain E 2371. Acta Pathol Microbiol Immunol Scand. 1985;93:59-64.
41.
Callegan MC, Engel LS, Hill JM, O'Callaghan RJ.
Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and protein A in phagocytosis.
Infect Immun.
1994;62:2478-2482 42. Forsgren A. Pathogenicity of Staphylococcus aureus mutants in general and local infections. Acta Pathol Microbiol Immunol Scand Sect B. 1972;80:564-570.
43.
Jonsson P, Lindberg M, Haraldsson I, Wadström T.
Virulence of Staphylococcus aureus in a mouse mastitis model: studies of alpha hemolysin, coagulase, and protein A as possible virulence determinants with protoplast fusion and gene cloning.
Infect Immun.
1985;49:765-769
44.
Foster TJ, O'Reilly M, Phonimdaeng P, Cooney J, Patel AH, Bramley AJ.
Genetic studies of virulence factors of Staphylococcus aureus: properties of coagulase and 45. Liang OD, Flock J-I, Wadström T. Isolation and characterisation of a vitronectin-binding surface protein from Staphylococcus aureus. Biochim Biophys Acta. 1995;1250:110-116[Medline] [Order article via Infotrieve].
46.
McGavin MH, Krajewska Pietrasik D, Rydén C, Höök M.
Identification of a Staphylococcus aureus extracellular matrix-binding protein with broad specificity.
Infect Immun.
1993;61:2479-2485 47. Bjork I, Petersson BA, Sjöquist J. Some physicochemical properties of protein A from Staphylococcus aureus . Eur J Biochem. 1972;29:579-584[Medline] [Order article via Infotrieve]. 48. Frenay HM, Bunschoten AE, Schouls LM, et al. Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. Eur J Clin Microbiol Infect Dis. 1996;15:60-64[Medline] [Order article via Infotrieve]. 49. Szabo A, Stolz L, Granzow R. Surface plasmon resonance and its use in biomolecular interaction analysis (BIA). Curr Opin Struct Biol. 1995;5:699-705[Medline] [Order article via Infotrieve]. 50. Kelly CG, Younson JS, Hikmat B, et al. A synthetic peptide adhesion epitope as a novel antimicrobial agent. Nat Biotechnol. 1999;17:42-47[Medline] [Order article via Infotrieve]. 51. Arvidson S. Extracellular enzymes from Staphylococcus aureus. In: Easmon CSF,Adlam C, eds. Staphylococci and Staphylococcal Infections. London: Academic Press; 1983:745-808.
52.
Kehrel B, Wierwille S, Clemetson KJ, et al.
Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, GPIIb/IIIa and vWf do not.
Blood.
1998;91:491-499
53.
Proctor RA, Mosher DF, Olbrantz PJ.
Fibronectin binding to Staphylococcus aureus.
J Biol Chem.
1982;257:14788-14794
54.
Herrmann M, Suchard SJ, Boxer LA, Waldvogel FA, Lew PD.
Thrombospondin binds to Staphylococcus aureus and promotes staphylococcal adherence to surfaces.
Infect Immun.
1991;59:279-288 55. Vaudaux PE, Yasuda H, Velazco MI, et al. Role of host and bacterial factors in modulating staphylococcal adhesion to implanted polymer surfaces. J Biomater Appl. 1990;5:134-153. 56. Vaudaux PE, Pittet D, Haeberli A, et al. Fibronectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters. J Infect Dis. 1993;167:633-641[Medline] [Order article via Infotrieve]. 57. Vaudaux PE, Francois P, Proctor RA, et al. Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts. Infect Immun. 1995;63:585-590[Abstract]. 58. Salzman EW, Lindon J, Mcmanama G, Ware JA. Role of fibrinogen in activation of platelets by artificial surfaces. Ann N Y Acad Sci. 1987;516:184-193[Medline] [Order article via Infotrieve].
59.
Lindon JN, McNamana G, Kushner L, Merrill EW, Salzman EW.
Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces?
Blood.
1986;68:355-362 60. Cheung AL, Eberhardt K, Heinrichs JH. Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus. Infect Immun. 1997;65:2243-2249[Abstract].
61.
Wann ER, Fehringer AP, Ezepchuk YV, et al.
Staphylococcus aureus isolates from patients with Kawasaki disease express high levels of protein A.
Infect Immun.
1999;67:4737-4743
© 2000 by The American Society of Hematology.
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  B. V. Lowder, C. M. Guinane, N. L. Ben Zakour, L. A. Weinert, A. Conway-Morris, R. A. Cartwright, A. J. Simpson, A. Rambaut, U. Nubel, and J. R. Fitzgerald Recent human-to-poultry host jump, adaptation, and pandemic spread of Staphylococcus aureus PNAS, November 17, 2009; 106(46): 19545 - 19550. [Abstract] [Full Text] [PDF]
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|
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 N. Merino, A. Toledo-Arana, M. Vergara-Irigaray, J. Valle, C. Solano, E. Calvo, J. A. Lopez, T. J. Foster, J. R. Penades, and I. Lasa Protein A-Mediated Multicellular Behavior in Staphylococcus aureus J. Bacteriol., February 1, 2009; 191(3): 832 - 843. [Abstract] [Full Text] [PDF]
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|
 C. L. Ventura, R. Higdon, E. Kolker, S. J. Skerrett, and C. E. Rubens Host Airway Proteins Interact with Staphylococcus aureus during Early Pneumonia Infect. Immun., March 1, 2008; 76(3): 888 - 898. [Abstract] [Full Text] [PDF]
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 N. N. Nickerson, L. Prasad, L. Jacob, L. T. Delbaere, and M. J. McGavin Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing J. Biol. Chem., November 23, 2007; 282(47): 34129 - 34138. [Abstract] [Full Text] [PDF]
|
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A. C. DeDent, M. McAdow, and O. Schneewind Distribution of Protein A on the Surface of Staphylococcus aureus J. Bacteriol., June 15, 2007; 189(12): 4473 - 4484. [Abstract] [Full Text] [PDF]
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 N. Procopio Evagrio George, Q. Wei, P. Kyun Shin, K. Konstantopoulos, and J. M. Ross Staphylococcus aureus Adhesion via Spa, ClfA, and SdrCDE to Immobilized Platelets Demonstrates Shear-Dependent Behavior Arterioscler Thromb Vasc Biol, October 1, 2006; 26(10): 2394 - 2400. [Abstract] [Full Text] [PDF]
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|
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R. A. Brady, J. G. Leid, A. K. Camper, J. W. Costerton, and M. E. Shirtliff Identification of Staphylococcus aureus Proteins Recognized by the Antibody-Mediated Immune Response to a Biofilm Infection. Infect. Immun., June 1, 2006; 74(6): 3415 - 3426. [Abstract] [Full Text] [PDF]
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 L. A. Marraffini, A. C. DeDent, and O. Schneewind Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria Microbiol. Mol. Biol. Rev., March 1, 2006; 70(1): 192 - 221. [Abstract] [Full Text] [PDF]
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 N. Harraghy, J. Kormanec, C. Wolz, D. Homerova, C. Goerke, K. Ohlsen, S. Qazi, P. Hill, and M. Herrmann sae is essential for expression of the staphylococcal adhesins Eap and Emp Microbiology, June 1, 2005; 151(6): 1789 - 1800. [Abstract] [Full Text] [PDF]
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E. J. Walsh, L. M. O'Brien, X. Liang, M. Hook, and T. J. Foster Clumping Factor B, a Fibrinogen-binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) Adhesin of Staphylococcus aureus, Also Binds to the Tail Region of Type I Cytokeratin 10 J. Biol. Chem., December 3, 2004; 279(49): 50691 - 50699. [Abstract] [Full Text] [PDF]
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B. Fournier and A. Klier Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS-ArlR two-component system of Staphylococcus aureus Microbiology, November 1, 2004; 150(11): 3807 - 3819. [Abstract] [Full Text] [PDF]
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 J. Bjerketorp, A. Rosander, M. Nilsson, K. Jacobsson, and L. Frykberg Sorting a Staphylococcus aureus phage display library against ex vivo biomaterial J. Med. Microbiol., October 1, 2004; 53(10): 945 - 951. [Abstract] [Full Text] [PDF]
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F. M. Roche, R. Downer, F. Keane, P. Speziale, P. W. Park, and T. J. Foster The N-terminal A Domain of Fibronectin-binding Proteins A and B Promotes Adhesion of Staphylococcus aureus to Elastin J. Biol. Chem., September 10, 2004; 279(37): 38433 - 38440. [Abstract] [Full Text] [PDF]
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 P. Pawar, P. K. Shin, S. A. Mousa, J. M. Ross, and K. Konstantopoulos Fluid Shear Regulates the Kinetics and Receptor Specificity of Staphylococcus aureus Binding to Activated Platelets J. Immunol., July 15, 2004; 173(2): 1258 - 1265. [Abstract] [Full Text] [PDF]
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 S. Niemann, N. Spehr, H. Van Aken, E. Morgenstern, G. Peters, M. Herrmann, and B. E. Kehrel Soluble Fibrin Is the Main Mediator of Staphylococcus aureus Adhesion to Platelets Circulation, July 13, 2004; 110(2): 193 - 200. [Abstract] [Full Text] [PDF]
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C. Heilmann, G. Thumm, G. S. Chhatwal, J. Hartleib, A. Uekotter, and G. Peters Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis Microbiology, October 1, 2003; 149(10): 2769 - 2778. [Abstract] [Full Text] [PDF]
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S. R. Clarke, L. G. Harris, R. G. Richards, and S. J. Foster Analysis of Ebh, a 1.1-Megadalton Cell Wall-Associated Fibronectin-Binding Protein of Staphylococcus aureus Infect. Immun., December 1, 2002; 70(12): 6680 - 6687. [Abstract] [Full Text] [PDF]
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R. C. Massey, S. R. Dissanayeke, B. Cameron, D. Ferguson, T. J. Foster, and S. J. Peacock Functional Blocking of Staphylococcus aureus Adhesins following Growth in Ex Vivo Media Infect. Immun., October 1, 2002; 70(10): 5339 - 5345. [Abstract] [Full Text] [PDF]
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P. Vaudaux, P. Francois, C. Bisognano, W. L. Kelley, D. P. Lew, J. Schrenzel, R. A. Proctor, P. J. McNamara, G. Peters, and C. Von Eiff Increased Expression of Clumping Factor and Fibronectin-Binding Proteins by hemB Mutants of Staphylococcus aureus Expressing Small Colony Variant Phenotypes Infect. Immun., October 1, 2002; 70(10): 5428 - 5437. [Abstract] [Full Text] [PDF]
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S. J. Peacock, C. E. Moore, A. Justice, M. Kantzanou, L. Story, K. Mackie, G. O'Neill, and N. P. J. Day Virulent Combinations of Adhesin and Toxin Genes in Natural Populations of Staphylococcus aureus Infect. Immun., September 1, 2002; 70(9): 4987 - 4996. [Abstract] [Full Text] [PDF]
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B. SHENKMAN, D. VARON, I. TAMARIN, R. DARDIK, M. PEISACHOV, N. SAVION, and E. RUBINSTEIN Role of agr (RNAIII) in Staphylococcus aureus adherence to fibrinogen, fibronectin, platelets and endothelial cells under static and flow conditions J. Med. Microbiol., September 1, 2002; 51(9): 747 - 754. [Abstract] [Full Text] [PDF]
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J. Bjerketorp, M. Nilsson, A. Ljungh, J.-I. Flock, K. Jacobsson, and L. Frykberg A novel von Willebrand factor binding protein expressed by Staphylococcus aureus Microbiology, July 1, 2002; 148(7): 2037 - 2044. [Abstract] [Full Text] [PDF]
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 S. W. Kerrigan, I. Douglas, A. Wray, J. Heath, M. F. Byrne, D. Fitzgerald, and D. Cox A role for glycoprotein Ib in Streptococcus sanguis-induced platelet aggregation Blood, June 28, 2002; 100(2): 509 - 516. [Abstract] [Full Text] [PDF]
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R. Downer, F. Roche, P. W. Park, R. P. Mecham, and T. J. Foster The Elastin-binding Protein of Staphylococcus aureus (EbpS) Is Expressed at the Cell Surface as an Integral Membrane Protein and Not as a Cell Wall-associated Protein J. Biol. Chem., January 4, 2002; 277(1): 243 - 250. [Abstract] [Full Text]
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M. Hussain, K. Becker, C. von Eiff, J. Schrenzel, G. Peters, and M. Herrmann Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of Staphylococcus aureus with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins J. Bacteriol., December 1, 2001; 183(23): 6778 - 6786. [Abstract] [Full Text] [PDF]
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 P. Becker, W. Hufnagle, G. Peters, and M. Herrmann Detection of Differential Gene Expression in Biofilm-Forming versus Planktonic Populations of Staphylococcus aureus Using Micro-Representational-Difference Analysis Appl. Envir. Microbiol., July 1, 2001; 67(7): 2958 - 2965. [Abstract] [Full Text] [PDF]
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B. Shenkman, E. Rubinstein, A. L. Cheung, G. E. Brill, R. Dardik, I. Tamarin, N. Savion, and D. Varon Adherence Properties of Staphylococcus aureus under Static and Flow Conditions: Roles of agr and sar Loci, Platelets, and Plasma Ligands Infect. Immun., July 1, 2001; 69(7): 4473 - 4478. [Abstract] [Full Text] [PDF]
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K. Savolainen, L. Paulin, B. Westerlund-Wikstrom, T. J. Foster, T. K. Korhonen, and P. Kuusela Expression of pls, a Gene Closely Associated with the mecA Gene of Methicillin-Resistant Staphylococcus aureus, Prevents Bacterial Adhesion In Vitro Infect. Immun., May 1, 2001; 69(5): 3013 - 3020. [Abstract] [Full Text] [PDF]
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I. R. Siboo, A. L. Cheung, A. S. Bayer, and P. M. Sullam Clumping Factor A Mediates Binding of Staphylococcus aureus to Human Platelets Infect. Immun., May 1, 2001; 69(5): 3120 - 3127. [Abstract] [Full Text] [PDF]
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F. M. McAleese, E. J. Walsh, M. Sieprawska, J. Potempa, and T. J. Foster Loss of Clumping Factor B Fibrinogen Binding Activity by Staphylococcus aureus Involves Cessation of Transcription, Shedding and Cleavage by Metalloprotease J. Biol. Chem., August 3, 2001; 276(32): 29969 - 29978. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
such as fibronectin, fibrinogen,
thrombospondin, collagen, elastin, and vitronectin
(0.350 × A495)/E]
using an extinction coefficient E of 0.7 for vWF. For labeling of vWF
with digoxigenin (DIG), a commercial kit (Digoxigenin Labeling Kit;
Boehringer, Mannheim, Germany) was used according to the
manufacturer's instructions. After labeling, all protein preparations
were checked for degradation using SDS-PAGE.
) followed by HEPES buffer (
) and
surface regeneration with glycine buffer (
) as described in
"Materials and methods," and RU was determined. (inset) Response
measured as a function of time using different vWF concentrations
(250/300/350/400/450/500 µg vWF/mL, lower through upper line)
calculated by subtracting RUvWF-CM5-RUCM5. (B)
Response curves after the injection of HSA (upper 2 lines) and BSA
(lower 2 lines) followed by the injection of HEPES buffer. (C) Response
after 4 injections of pooled human IgG (1060 nmol/L) (
, Cowan 1; 
, Newman; 
,
8325-4). (B) Inhibition of vWF binding to S. aureus strains
by the addition of excess unlabeled vWF over FITC-vWF (200 µg/mL)
(symbol denotation as in A). (C, D) Inhibition of vWF binding to
S. aureus Cowan 1 (C) and Newman (D) by the addition of
polyclonal anti-SPA (
, complemented mutant. Shown are mean values ± SD of 3 determinations performed in quintuplicate.
-amino group of lysyl.
binding sites (the classical site).
-toxin but not to
SPA.
-toxin and protein A in the pathogenesis of S. aureus infections. In:
Novick RP, ed.
Molecular Biology of the Staphylococci. New York: VCH; 1990:403-417.