functional blocking of staphylococcus aureus adhesins following … · renal unit. sterile,...

INFECTION AND IMMUNITY, Oct. 2002, p. 5339–5345 Vol. 70, No. 100019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.10.5339–5345.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Functional Blocking of Staphylococcus aureus Adhesins followingGrowth in Ex Vivo Media

Ruth C. Massey,1 Shobana R. Dissanayeke,1 Brian Cameron,1 David Ferguson,1Timothy J. Foster,2 and Sharon J. Peacock1*

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital,Oxford OX3 9DU, United Kingdom,1 and Department of Microbiology, Moyne Institute

of Preventive Medicine, Trinity College, Dublin, Ireland2

Received 4 January 2002/Returned for modification 12 April 2002/Accepted 20 June 2002

Defining the role of Staphylococcus aureus adhesins in disease pathogenesis may depend on the use of bacteriagrown in culture media that more closely reflect the human milieu than conventional broth. This studyexamined the functional effect on S. aureus adhesins following growth in an ex vivo medium containing acomplex mixture of human proteins (used peritoneal dialysate) relative to growth in Todd-Hewitt broth. Theadherence of S. aureus, cultured in dialysate, to fibronectin and fibrinogen was markedly reduced despite theexpresion of full-length ClfA, ClfB, and fibronectin-binding proteins. Growth in dialysate resulted in theacquisition of a surface coat, as visualized by transmission electron microscopy, which was shown to containfibronectin, fibrinogen, and immunoglobulins. Adherence of S. aureus to fibrinogen following growth in dialy-sate was significantly reduced by expression of protein A but was restored following growth in immunoglobulin-depleted dialysate. We conclude that bacterial adherence to solid-phase protein is critically dependent on theculture medium, that S. aureus adhesins may become saturated with target protein prior to contact with solidsurfaces, and that there is an interaction between fibrinogen-binding proteins and immunoglobulin bound toprotein A following contact with host proteins. These findings have important implications for future studiesof S. aureus adhesins.

Bacterial adherence is likely to play a central role in host-to-host transmission and the maintenance of stable carriage ofStaphylococcus aureus. Adhesion is also considered to be im-portant in the disease pathogenesis of this predominantly ex-tracellular pathogen. S. aureus is known to express a range ofcell wall-associated proteins that promote adherence to hostcells, extracellular matrix components, and/or soluble plasmaproteins. These cell wall-anchored proteins include the colla-gen-binding protein Cna (33); the fibrinogen-binding proteinsclumping factor A (ClfA) and B (ClfB) (23, 28); two fibronec-tin-binding proteins, FnBPA and FnBPB (15, 41); and proteinA (20, 45), which can bind Von Willebrand factor and the Fcregion of immunoglobulin G (IgG) (7, 12).

The study of S. aureus adhesin-ligand interactions in vitroand in vivo has relied primarily on comparison of end pointsfor wild-type S. aureus versus isogenic mutants defective in oneor more adhesin or, more recently, on using an expressionsystem in a heterologous host such as Lactococcus lactis. Thesestrategies have made important contributions, providing evi-dence for the involvement of Cna (13), ClfA (27, 37, 40, 44),and FnBPA (37) in the pathogenesis of experimental endocar-ditis and protein A in a subcutaneous-infection model (32). Inaddition, FnBPs have been shown to be important in thepathogenesis of intravenous-device-related infection (46, 47)and in the process of uptake by a range of cell lines (6, 8, 19,21, 34, 42, 43). However, the experimental growth conditions

used to prepare S. aureus normally depend on culture in lab-oratory media, and it seems unlikely that the resulting bacteriaaccurately mirror those in vivo during human infection. Forexample, bacterial adhesins may rapidly interact with solublehost proteins in vivo, and this may inhibit subsequent interac-tions with surface-expressed host protein. This has clear impli-cations for in vitro systems but may also be important in animalmodels where large inocula of broth-grown bacteria injectedinto a blood vessel or the peritoneal cavity may not resemble S.aureus precoated with host proteins during colonization andinvasion.

The purpose of this study was to explore the functions of cellwall-associated adhesins following growth under conditionsmore closely analogous to those in the human host than isachieved by either conventional media or broth supplementedwith one or more host components. The growth medium usedwas peritoneal dialysate from individuals undergoing renal re-placement therapy by continuous ambulatory peritoneal dial-ysis. Fresh dialysate is instilled into the abdominal cavity,where it remains for 6 h while dialysis occurs across the peri-toneal membrane by a process of diffusion. When the fluid isremoved, it contains an array of human proteins at a lowerconcentration than that in the circulation, including fibronectin(approximately 1 to 5% of the level in plasma), fibrinogen(0.5% of the level in plasma), and immunoglobulins (IgG at 1to 2% of the level in serum) (3, 16, 25). This medium is readilyavailable in large quantities and supports the growth of S.aureus (48). We have examined the functional effect on S.aureus adhesins following growth in used peritoneal dialysaterelative to growth in conventional culture media.

* Corresponding author. Mailing address: Department of Microbi-ology, Level 7, The John Radcliffe Hospital, Headington, Oxford, OX39DU, United Kingdom. Phone: 44-1865-220537. Fax: 44-1865-220984.E-mail: [email protected].

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MATERIALS AND METHODS

Chemicals and reagents. All chemicals were obtained from Sigma-Aldrich orBDH Chemicals unless otherwise indicated.

Bacterial strains and plasmids. The S. aureus strains and plasmids used in thisstudy are listed in Table 1.

Bacterial storage and growth conditions. S. aureus was stored in trypticase soybroth with glycerol (15% [vol/vol]) at �80°C. Cultures were inoculated fromstocks into 10 ml of medium contained in 35-ml glass universal containers. S.aureus was grown in Todd-Hewitt broth (THB; Difco) or used peritoneal dialy-sate for 15 to 18 h under constant rotation at 37°C in air. Escherichia coli strainDH5� was cultured in Luria-Bertani medium under constant rotation at 37°C inair. Antibiotics were incorporated into media, where appropriate, at the follow-ing concentrations: erythromycin, 10 �g/ml; tetracycline, 2 �g/ml; and chloram-phenicol, 10 �g/ml.

Used peritoneal dialysis fluid (hereafter termed dialysate) was obtained on ananonymous basis from patients receiving outpatient care at the Oxford RegionalRenal Unit. Sterile, antibiotic-free dialysate from five different patients waspooled, aliquoted, and stored at �20°C. This served as the stock throughout thestudy. Sterility was checked by plating 100 �l of dialysate onto 5% horse bloodagar, which was incubated at 37°C in air for 24 h. The presence of antibiotics wasdetermined by pipetting 20 �l of dialysate onto a lawn of S. aureus NCTC 6571on Diagnostic Sensitest agar. The plate was incubated at 37°C in air for 24 h, andany inhibition of growth was taken to indicate the presence of antibiotics.

Removal of immunoglobulins from dialysate. Immunoglobulins were removedfrom dialysate using a protein L column (Actigen), which binds all immunoglob-ulin classes and proteins complexed to them. The dialysate was passed twicethrough the column, and Western immunoblotting demonstrated depletion ofimmunoglobulin (data not shown).

Adhesion of S. aureus to purified human proteins. THB or dialysate wasinoculated with bacterial strains and incubated overnight under constant rotationat 37°C in air. Bacteria were harvested by centrifugation and washed three timesin sterile phosphate-buffered saline (PBS). Adherence of S. aureus to purifiedhuman fibronectin or fibrinogen (Calbiochem) at a concentration of 10 �g/mlwas assessed using a standardized microtiter plate assay, as previously described(35). The fibrinogen used was purified to remove contaminating fibronectin (22,34). Adherent bacteria were detected by staining with crystal violet (0.5% [vol/vol]), and the optical density was measured using an enzyme-linked immunosor-bent assay plate reader. Each isolate was tested in quadruplicate in an individualassay, and each experiment was performed three times. All assay plates includeda positive control (S. aureus strain Newman for fibrinogen and 8325-4 for fi-bronectin adherence assays) and PBS without bacteria as a negative control. Theoptical density at 405 nm used in the analysis was the mean value for a givenstrain minus the background optical density at 405 nm taken from the reading forthe negative control on the same plate. Data were analyzed using the Statviewversion 4.5 software package (Abacus, Berkeley, Calif.). Comparison of the meancount between bacterial strains or growth conditions was performed using anunpaired t test.

Transmission electron microscopy. Bacterial strains were grown overnight ineither THB or dialysate at 37°C in air, harvested by centrifugation, and washedthree times with PBS. Transmission electron microscopy of bacterial isolates wasperformed as previously described (49).

SDS-PAGE and Western immunoblotting. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) and Western immunoblotting were usedto examine three types of sample: (i) cell wall-associated proteins released bylysostaphin treatment, as previously described (35); (ii) host proteins in 20�concentrated dialysate; and (iii) host proteins associated with the bacterial cellwall after overnight incubation in dialysate. The last were removed from a 10-mlculture by washing the pellet three times in PBS and then boiling it for 2 min in

an equal volume of SDS sample buffer. Proteins were separated by SDS-PAGEusing a 7.5 to 10% acrylamide gradient and standard methods (18) and thentransferred electrophoretically to polyvinylidene difluoride Western-blottingmembrane (Boehringer Mannheim) by the semidry transblot system (Bio-Rad).Human proteins were detected using rabbit polyclonal antibody against fibronec-tin (F3648; Sigma), fibrinogen (catalog no. 341552; Calbiochem), or IgG, IgA,and IgM (A0190; Dako) followed by alkaline phosphatase-labeled goat anti-rabbit antibody (Chemicon) for fibronectin and immunoglobulin or alkalinephosphatase-labeled protein A (Dako) for fibrinogen. Antibody was visualizedusing the AP Conjugate Substrate kit (Bio-Rad) as instructed by the manufac-turer. Bacterial FnBPs, ClfA, and ClfB were detected by immunoblotting usingpolyclonal antibodies as previously described (11, 21, 24, 28).

Transformation. E. coli cells were transformed following CaCl2 treatment(38). S. aureus was electroporated as previously described (30) using S. aureusRN4220 as the initial recipient for plasmids prior to electroporation into therequired background.

Manipulation of DNA. DNA manipulations were performed by standard pro-cedures (38). Chromosomal DNA was extracted using a Puregene DNA extrac-tion kit (Gentra Systems), with the modification that 30 �g of lysostaphin(Ambi)/ml was added at the cell lysis step. Plasmid DNA for cloning was purifiedfrom E. coli using WizardPlus minipreps (Promega Corp.) and from S. aureususing Qiagen midipreps with the addition of lysostaphin (30 �g/ml) in the cellresuspension buffer. Restriction enzymes were purchased from New EnglandBioLabs or from Boehringer Mannheim and were used as recommended by thesuppliers.

PCR amplification of the spa gene from S. aureus. Using a sequence in theGenBank database (accession number J01786), oligonucleotide primers weredesigned to amplify the full-length spa gene encoding S. aureus protein A. Thenucleotide sequences of the primers used were as follows: 5�-CGGGATCCTCGAAATAGCGTGATTTTGC-3� (forward) and 5�-CGGGATCCGCACTGAGCAACAAAAGATG-3� (reverse); the underlined regions indicate the recogni-tion sites for the restriction enzyme BamHI. The PCR mixtures contained 100pmol of each primer, 10 ng of template DNA (strain 8325-4), 200 �M de-oxynucleoside triphosphate, reaction buffer (1�), 1.5 mM MgCl2, and 2.5 U ofPfu polymerase in a volume of 50 �l. Amplifications were carried out in a DNAthermal cycler (Peltier Thermal Cycler) under the following conditions: 30 cyclesof 94°C for 1 min, 65°C for 1 min, and 72°C for 3 min followed by a 10-minincubation at 72°C. Aliquots were analyzed on a 0.8% agarose gel, and theremaining DNA was extracted using the Wizard PCR purification kit (Promega)as instructed by the manufacturer.

Phage transduction. The spa::Tcr mutation was transduced from DU5873 (S.aureus Newman spa::Tcr) to S. aureus strain V8 by phage 85-mediated transduc-tion (1). Transductants resistant to 2 �g of tetracycline/ml were selected. Thegenetic background of transductants was verified by pulsed-field gel electro-phoresis using established methodology (36). Genotypic verification was per-formed by the demonstration of an appropriate increase in PCR product sizefollowing amplification of the spa gene. The lack of adherence to myeloma IgG1at 10 �g/ml was confirmed by microtiter adherence assay.

Construction of plasmid pSPA, expressing protein A. The S. aureus-E. colishuttle plasmid pCU1 (2) was used to clone the spa gene in E. coli and tosubsequently express protein A in S. aureus. Plasmid DNA and the PCR-ampli-fied spa gene were digested overnight at 37°C using BamHI and the purifiedDNA ligated together using T4 ligase. E. coli strain DH5� was transformed withthe ligation product. This plasmid (pSPA) was electroporated into S. aureusRN4220 and then into strain Wood. Adherence of transformants to myelomaIgG1 at 10 �g/ml was confirmed by microtiter adherence assay.

TABLE 1. S. aureus strains used in this study

Strain Relevant genotype Properties Source or reference

8325-4 NCTC 8325 cured of prophages 29Newman High level of fibrinogen-binding protein ClfA 5DU5873 �spa::Tcr Mutant strain of Newman defective in protein A 24DU5917 cps::Tn917 Emr Mutant strain of Newman defective in capsular polysaccharide 39V8 Strain from which serine protease was first isolated 4SP20 �spa::Tcr Mutant strain of V8 defective in protein A This studyWood Naturally deficient in protein A 17SP21 Wood (pSPA) Cmr Wood complemented with multicopy plasmid expressing protein A This study

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RESULTS

Adherence of S. aureus to solid-phase fibronectin andfibrinogen is lost following growth in dialysate. The adher-ence of S. aureus to fibronectin and fibrinogen was examinedfollowing overnight growth in THB and dialysate. This wasperformed using laboratory isolates known to have an appro-priate adhesive phenotype for a given protein; hence the use ofNewman in place of 8325-4 for the fibrinogen assay. The ad-herence of S. aureus strains 8325-4, V8, and Wood to fibro-nectin was markedly reduced following growth in dialysatecompared with that following growth in THB (P � 0.001 foreach strain) (Fig. 1a). Bacterial adherence to fibrinogen aftergrowth in dialysate was markedly reduced for S. aureus strainsNewman and V8 (P � 0.001 for both strains); the adherence ofS. aureus strain Wood was decreased to a lesser extent (Fig.1b).

The loss of adherence to fibronectin and fibrinogen follow-ing growth in dialysate could be due to decreased expression orincreased degradation of bacterial adhesins. Alternatively, theligand binding sites of the FnBPs and fibrinogen-binding pro-teins could become blocked through saturation with their tar-get protein or by steric hindrance from other proteins associ-

ated with the cell wall. The following strategies were used todefine the mechanisms responsible.

FnBPs and fibrinogen-binding proteins are expressed on thesurface of S. aureus following growth in dialysate. Westernimmunoblotting of cell wall-associated proteins was used toexamine the possibility that surface-expressed adhesins wereaffected through contact with or growth in dialysate. Equalnumbers of bacterial cells were examined following growth inused dialysate and THB. Full-length ClfA, ClfB, and FnBPAwere demonstrated in the cell wall extracts from Newman(ClfA and ClfB) and 8325-4 (FnBPA) grown under both cul-ture conditions (Fig. 2). Thus, alteration in expression of ClfA,ClfB, and FnBPA or subsequent enzymatic degradation doesnot appear to explain the reduction in adherence to fibronectinand fibrinogen.

S. aureus grown in THB but preincubated in dialysate failsto adhere to fibronectin and fibrinogen. S. aureus was grownovernight in THB, washed, and then incubated in 10 ml ofdialysate at 37°C for 1 h prior to adherence assays. The adher-ence of preincubated bacteria mirrored that following over-night growth in dialysate, with a significant reduction in theadherence of 8325-4, V8, and Wood to solid-phase fibronectin

FIG. 1. Functional blocking of S. aureus adhesins following growth in dialysate. The adherence of S. aureus strains to fibronectin (a) andfibrinogen (b) was assessed by microtiter plate assay following growth in dialysate and THB. The data are shown as the mean standard errorof the mean. OD405, optical density at 405 nm.

FIG. 2. Surface expression of S. aureus adhesins. Western immunoblots demonstrate the cell wall-associated proteins ClfA, ClfB (strainNewman), and FnBPA (strain 8325-4) extracted from bacteria following growth in dialysate or THB.

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(P � 0.001 for all strains) and of Newman and V8 to fibrinogen(P � 0.001 for both strains) (data not shown). This suggeststhat reduction in adherence resulted from the interaction be-tween bacterial adhesins and human proteins or other compo-nents of the dialysate.

S. aureus grown in dialysate becomes associated with hostproteins. Western immunoblotting confirmed the presence offibronectin, fibrinogen, and immunoglobulins in dialysate (Fig.3), the fibrinogen observed appearing to have degraded to alower apparent molecular mass. Strain Newman was examinedby transmission electron microscopy following growth in THBand dialysate. Bacteria grown in dialysate acquired a surfacecoat that was not present on bacteria grown in THB (Fig. 4).This coat did not represent either enhanced visualization orproduction of capsular polysaccharide, since NewmanDU5917, a strain deficient in capsular polysaccharide expres-sion, also acquired a surface coat during growth in dialysateand its fibrinogen-binding properties were affected in a similarmanner (data not shown). Possible explanations for the ap-pearance of the coat include expression of a surface protein bybacteria grown in dialysate but not by those grown in THB orattachment of components derived from the dialysate. Thelatter is consistent with the observation that S. aureus failed toadhere to fibronectin and fibrinogen following pretreatmentwith dialysate, leading to the hypothesis that host proteins weremajor components of the surface coat. This was confirmed byWestern immunoblots of material removed from the surface of8325-4 by boiling in SDS following overnight growth in dialy-sate. Fibronectin, immunoglobulins, and fibrinogen were alldetected (Fig. 3).

Taken together, these results suggest that saturation of li-gand binding sites by target proteins in dialysate was the most

likely explanation for the reduction in bacterial adherence tofibronectin and fibrinogen. However, this does not explain theobservation that adherence of strain Wood to fibrinogen fol-lowing growth in dialysate was affected to a much lesser extentthan that for other strains tested. Wood is naturally deficient inprotein A (17), raising the possibility that the reduction inbacterial adherence to fibrinogen was due to an interactionbetween fibrinogen-binding proteins and protein A followingcontact with host proteins.

Protein A binds immunoglobulin in dialysate and preventsadherence of S. aureus to solid-phase fibrinogen. A panel ofisogenic mutants (defective in protein A) and strain Wood(naturally deficient in protein A) complemented with a multi-copy plasmid carrying the spa gene were constructed. Inacti-vation of spa in V8 and Newman resulted in an increase inadherence to fibrinogen compared with the wild type followinggrowth in dialysate (P � 0.001 for both strains) (Fig 5).Complementation of Wood with the pSPA plasmid resulted ina reduced adherence to fibrinogen compared with the wild typefollowing growth in dialysate (P � 0.001) (Fig. 5). We con-firmed that adherence to solid-phase fibrinogen after growth inTHB was not affected by the introduction of spa mutations orplasmid pSPA (data not shown). Dialysate depleted of immu-noglobulin was used as a growth medium for Newman, V8, andWood. Growth in this medium resulted in an increase in ad-herence to fibrinogen for Newman and V8 compared with thatfollowing growth in nondepleted dialysate (P � 0.001), whileadherence for Wood was not significantly affected (P 0.05)(Fig. 5). These data indicate that immunoglobulins bound toprotein A prevent the interaction between the S. aureus fibrin-ogen-binding proteins and human fibrinogen.

FIG. 3. Fibronectin, fibrinogen, and immunoglobulins are present in dialysate and become associated with the surface of S. aureus. Westernimmunoblotting was used to detect the presence of human proteins in dialysate concentrated 20-fold and in the surface-associated layer acquiredby S. aureus following growth in dialysate. Purified human proteins were used as positive controls. Lanes 1, 2, and 3, purified fibronectin, dialysate,and surface coat, respectively, probed with anti-fibronectin antibodies; lanes 4, 5, and 6, purified fibrinogen, dialysate, and surface coat, respectively,probed with anti-fibrinogen antibodies; lanes 7, 8, and 9, purified immunoglobulins (classes IgG, IgM, and IgA), dialysate, and surface coat,respectively, probed with antibodies to IgG, IgM, and IgA.



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DISCUSSION

The study of S. aureus following growth in laboratory brothin vitro has led to the identification of numerous putativevirulence determinants. The challenge now is to determinehow they function in vivo and to define which are important inhuman disease. The findings of this study add to existing evi-dence that in vitro expression and/or function of S. aureusfactors, such as those involved in metabolism and the globalregulator agr, may not mirror that in vivo or following growthin ex vivo media (9, 10, 51).

The rationale for using peritoneal dialysate from individualswith chronic renal failure as a growth medium for S. aureusrests primarily on the fact that many of the host componentsfound in blood or plasma are present in dialysate at lowerconcentrations. Although this medium is relatively ill defined,its use makes it possible to study adhesins in a way that wouldprove difficult using blood or blood products which wouldcause S. aureus to become highly aggregated, while providingan environment that is closer to in vivo conditions than con-ventional media.

Bacterial adherence to solid-phase fibronectin was not pre-served following growth in dialysate, a result that we haveascribed to saturation of FnBP ligand binding sites. Human

fibronectins are present in soluble form in plasma and in manyextracellular matrices, where they exist as polymers. It is pos-sible, given our findings, that S. aureus in the bloodstream doesnot interact efficiently with extracellular matrix fibronectin andmay therefore interact poorly with host cells such as endothe-lium in vivo. However, the acquisition of a surface coat of hostproteins may confer other benefits. For example, immunolog-ical recognition and/or clearance of bacteria by phagocytesmay be impeded. The idea that FnBPs interact with host de-fenses in vivo gains credibility from a study reporting thatrecombinant FnBPA interacts with integrin �5�1 via a fi-bronectin bridge to mediate adhesion and costimulatory sig-nals to T lymphocytes (26).

Our observation that adherence to solid-phase fibrinogenwas reduced after growth in dialysate can also be explained inpart by functional blocking by fibrinogen acquired from dialy-sate. Fibrinogen is a soluble plasma protein, but unlike fi-bronectin, it is not a component of extracellular matrices. Wespeculate that the relevance of our observation to the in vivosituation may relate more to the biological imperative for S.aureus to become masked with host protein than the inabilityto bind further fibrinogen once coating has occurred. Bindingof fibrinogen to the M protein of Streptococcus pyogenes blockscomplement via the alternative pathway and inhibits the dep-osition of C3b, leading to reduced phagocytosis by polymor-phonuclear leukocytes (14, 50). It is possible that S. aureus cellsurface-associated fibrinogen is similarly antiphagocytic. It isalso possible that S. aureus coated with fibrinogen can interactwith platelets, since fibrinogen binds to platelets during theformation of thrombus. This could result in bacterial integra-tion into a clot, which then becomes impacted in the microcir-culation, where proliferation can occur prior to spread andseeding. S. aureus also appears to interact with platelets di-rectly, since a mutant defective in ClfA has been shown to havereduced ability to bind human platelets in vitro (40) while

FIG. 4. S. aureus grown in dialysate acquires a surface coat. Trans-mission electron microscopy of S. aureus strain Newman cultured over-night in THB (a) and dialysate (b). The bacteria acquire an electron-dense surface layer during growth in dialysate. Magnification, �48,000.

FIG. 5. Expression of protein A and its interaction with immuno-globulins interfere with bacterial adherence to fibrinogen followinggrowth in dialysate. Shown are adherence of wild-type (WT) S. aureusstrains Newman, V8, and Wood; Newman and V8 mutants defective inprotein A (spa�); and Wood complemented with a multicopy plasmidexpressing protein A (spa�) following growth in dialysate (wild-typeWood is naturally deficient in protein A). Also shown is the adherenceof wild-type Newman, V8, and Wood following growth in IgG-depleteddialysate. The data are shown as the mean standard error of themean. OD405, optical density at 405 nm.



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extracellular fibrinogen-binding protein (Efb) has been dem-onstrated to inhibit platelet aggregation (31).

Our study also demonstrated an interaction between fibrin-ogen adherence and immunoglobulins bound to protein A.The mechanism for this is unclear, but it could be due to sterichindrance if the two adhesins are closely related on the bac-terial cell surface. The biological relevance of this interactionalso remains unclear. Our findings indicate that S. aureus ad-hesins may participate in complex interactions in the presenceof host proteins, suggesting that the study of a single adhesin inisolation may represent an oversimplification of events in vivo,as may examining more than one adhesin in the absence ofhost proteins.

ACKNOWLEDGMENTS

This work was funded by a Baxter Healthcare Extramural Grant.We thank the patients and nurses at the Oxford Regional Renal

Unit, who provided dialysate.Ruth C. Massey and Shobana R. Dissanayeke contributed equally to

this work.

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