Vol. 52, No. 3INFECTION AND IMMUNITY, June 1986, p. 671-6750019-9567/86/060671-05$02.00/0Copyright © 1986, American Society for Microbiology

Detection of Staphylococcal Membrane Receptors on Virus-InfectedCells by Direct Adhesin OverlayBARBARA A. SANFORD* AND MARY A. RAMSAY

Department of Microbiology, The University of Texas Health Science Center, San Antonio, Texas 78284

Received 13 August 1985/Accepted 19 February 1986

We partially characterized the interaction between 25I-labeled surface proteins of Staphylococcus aureus,obtained by thermal extraction, and purified plasma membranes from uninfected and influenza virusA/FM/1/47-infected Madin-Darby canine kidney cells. The radioactivity profile of surface-labeled proteins,derived from anion-exchange high-pressure liquid chromatography, showed a mixture of acidic polypeptides ina peak which contained approximately 5% of the total protein injected; adsorption with purified plasmamembranes reduced radioactivity by 5 to 7% and altered elution profiles. Using a direct adhesin overlayprocedure, we found that these surface-labeled proteins reacted with polypeptides located on the externalsurface of plasma membranes which were shared by both virus-infected and uninfected cells or were unique tovirus-infected cells. Our data may help explain the enhanced adherence of S. aureus to influenza virusA-infected cells in vitro.

In humans, superinfection with Staphylococcus aureus isa common and serious complication of influenza (9). Mulderand Hers (12) conducted an elegant histopathological studyon autopsy material obtained from numerous patients, in-cluding 142 patients who died from postinfluenzal staph-ylococcal infections of the respiratory tract. Cytologicalexamination of tissues from these patients clearly showedstaphylococcal adherence to tracheobronchial surfaces ofmucosa in areas infected by influenza virus. Subsequentexperiniental in vitro studies showed that influenza virus Ainfection of cells significantly promotes and enhances theadherence of S. aureus (6-8, 13, 16), presumably by gener-ating more receptors or new receptors or both on virus-infected cells (6, 7).The staphylococcal adhesins that mediate binding to virus-

infected host cells (in vitro) are protease- and trypsin-sensitive proteins that are distinct from the major cellsurface components of S. aureus (protein A, clumpingfactor, and teichoic acid) (7, 15). Heat treatrnent of S. aureusrenders the bacteria adherence negative, and the cell-free(thermal) extract obtained by this treatment is effective inblocking the adherence of staphylococci to both uninfectedand virus-infected host cells (7, 15). One purpose of thepresent study was to demonstrate that thermal extractscontain staphylococcal surface proteins and that these pro-teins are able to bind directly to plasma membranes obtainedfrom virus-infected cells. While staphylococcal membranereceptors present on host cells have not been defined,previous studies have indicated that the only virus-specificproteins located on the outer surface of the lipid bilayer ofvirus-infected cells, hemagglutinin and neuraminidase, donot act as receptors for staphylococci (7). Having observedthat host cell membrane receptors are protease-sensitive buttrypsin-resistant components on both virus-infected anduninfected cells (7), we undertook the present study tocharacterize the interaction of staphylococcal surface pro-teins with enzyme-treated plasma membranes and enzymedigests of the membranes by using a direct adhesin overlayprocedure.

* Corresponding author.

MATERIALS AND METHODS

Labeling of bacterial surface proteins. S. aureus 1071, aclinical isolate of low passage history (6), was grown in staticcultures in M199 medium (GIBCO Laboratories, GrandIsland, N.Y.) (lx 108 to 2 x 108 CFU of bacteria per 500 mlofmedium) at 37°C for 18 h. Staphylococci were harvested bycentrifugation, washed repeatedly in water, and freeze-dried.The surface proteins of strain 1071 were labeled as follows:100 mg (dry weight) of strain 1071 was labeled with 2 mCi of1251 (specific activity, 14.8 mCi of 1251 per ,ug of iodine) by thechloramine T method (5). The labeled bacteria (50 mg ofbacteria per 3 ml of phosphate-buffered saline [PBS], pH 7.2)were autoclaved (121°C, 15 lb/in2) for 15 min and pelleted bycentrifugation, and the supernatant (thermal extract) whichcontained surface-labeled proteins was dialyzed exhaustivelyagainst PBS and stored at 4°C. The thermal extract contained250 ,ug of total protein per ml, with a specific activity of 5,498cpm/,ug of protein.

Cell cultures. Madin-Darby canine kidney (MDCK) cellswere routinely maintained in Eagle basal medium (Auto PowBME; Flow Laboratories, Rockville, Md.) supplementedwith 10% fetal calf serum, 0.03% L-glutamine, and 10 jig ofgentamicin per ml. Cells were grown to confluency in150-cm2 tissue culture flasks (Coming Glass Works, Corn-ing, N.Y.) at 37°C in a 5% CO2 atmosphere. Cell monolayerswere infected with influenza virus A/FM/1/47 (H1N1) (6);control monolayers were sham inoculated with Hanks bal-anced salt solution (GIBCO).Plasma membrane purification. Plasma membranes were

obtained from intact MDCK cell monolayers (uninfected orvirus infected) before and after enzyme treatment. Washedcell monolayers were incubated with PBS, trypsin (DifcoLaboratories, Detroit, Mich.), or repurified protease (typeVI; Sigma Chemical Co., St. Louis, Mo.) at a concentrationof 1 mg of enzyme in PBS per 5 x 108 MDCK cells for 15 minat 37°C. The protease treatment was sufficient to causecomplete detachment of cells from the plastic. Untreatedand enzyme-treated cells were repeatedly washed withHanks balanced salt solution, and the plasma membraneswere stabilized with fluorescein mercuric acetate accordingto the procedure of Carlsen and Till (3). Purified plasmamembranes were obtained by the dextran-polyethylene gly-

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col aqueous two-phase system described by Brunette andTill (2). Membranes were stored at 4°C until they were usedin the direct binding or direct adhesin overlay assays.

Preparation of protease digests from MDCK cells. IntactMDCK cell monolayers (total number of cells, 1 x 109 to 2x 109) that were uninfected or virus infected were rinsed andincubated with PBS (10 ml per 150-cm2 tissue culture flask)at 37°C for 15 min. Like PBS supernatants were pooled. Toeach flask was added 10 ml of protease (100,ug of enzymeper ml of PBS) at 37°C for 15 min. The protease digests werepooled and together with pooled PBS supernatants weredialyzed (molecular weight cutoff, 6,000 to 8,000) exhaus-tively against water until they were salt free. Dialysates wereconcentrated to a final volume of 200,ul. The supematantswere stored at 4°C until they were used in the direct adhesinoverlay assay.

Direct binding assay. A 0.5-ml volume of I251-labeledsurface proteins from S. aureus 1071 (specific activity, 5,498cpm/nLg of protein) was reacted with 40 mg (wet weight) ofplasma membranes obtained from either uninfected or virus-infected cells for 2 h at ambient temperature. Membraneswere pelleted by centrifugation; unadsorbed and adsorbedsurface-labeled proteins were filtered (0.22,um), and a 200-,ulvolume was applied to a Protein Pak DEAE-5PW column(7.5 mm by 7.5 cm; Waters Associates, Inc., Milford, Mass.)in conjunction with a gradient liquid chromatography system(Du Pont Co., Wilmington, Del.) which included a series8800 gradient controller, model 860 absorbance detector,and a model 8800 chromatographic pump module. Thermalextracts were eluted under the following conditions: 20-mingradient (exponent = -3) from 0.02 M Tris hydrochloridebuffer (pH 8.0) to 0.02 M Tris hydrochloride buffer (pH 8.0)containing 0.5 M NaCl; flow rate, 1 ml/min; monitored atA285; 200-plI fractions collected and counted in a model 310gamma counter (Beckman Instruments, Inc., Fullerton,Calif.). A model Sp4270 chromatography integrator with a5-by-7 dot matrix printer (Spectra-Physics, San Jose, Calif.)was used to integrate and report data.

Direct adhesin overlay. Duplicate samples of untreated andenzyme-treated plasma membrane preparations were heatedat 100°C for 5 min (5 mg [wet weight] of membranessuspended in 40 pul of sample buffer) and subjected toelectrophoresis at a constant current of 120 V on a 5%stacking-10% resolving sodium dodecyl sulfate (SDS)-polyacrylamide gel according to the method of Laemmli (11).Protein profiles were detected for one set of samples bystaining with Coomassie brilliant blue R. The second set ofelectrophoresed membrane proteins were not stained butwere electroeluted out of the gel onto untreated nitrocellu-lose paper (NCP) by electrophoresis at a constant current of20 mA for 18 h in transfer buffer (14% glycine, 0.3% Tris,20% methanol). In order to quench unbound sites on thefilter, the NCP was soaked for 1 h in 1% bovine serumalbumin in the TSGAN buffer described by Cohen andFalkow (4), which consisted of 50 mM Tris hydrochloride(pH 7.5), 0.15 M NaCl, 0.25% gelatin, 0.15% sodium azide,and 0.1% Nonidet P-40. In the direct adhesin overlay pro-cedure the NCP was then rinsed with TSGAN buffer,reacted with 50 ml of TSGAN buffer containing 3 ml of'25I-labeled staphylococcal surface proteins (specific activ-ity, 3,385 cpm/lpg of protein per 4 pul), and incubated atambient temperature for 18 h. The NCP was rinsed, soakedin TSGAN buffer for 30 min at ambient temperature, rinsedin TSGAN buffer again, and dried in air. The overlays wereexposed to Kodak X-Omat AR film with a Du Pont Quanta-III CK intensifying screen at -70°C for 9 days. Standard

molecular weight markers were included in both original slabgels. Following autoradiography, the presence of markersand membrane components was visualized on the NCP bystaining with 0.5% Ponseau S in 7.5% trichloroacetic acidand destaining in 2% acetic acid.

In a subsequent experiment, the protease digests and PBScontrol supernatants (10 pul of test material plus 10 pul ofsample buffer) were subjected to SDS-polyacrylamide gelelectrophoresis and electroelution onto NCP as describedabove. A direct adhesin overlay was performed by using1251I-labeled staphylococcal surface protein (specific activity,5,498 cpm/pug of protein) as described above.

RESULTSFigure 1 shows the results of an experiment done to

demonstrate that surface-labeled proteins present in a ther-mal extract of S. aureus did, in fact, bind directly to purifiedplasma membranes obtained from uninfected and influenzavirus A-infected MDCK cell monolayers. Thermal extractsbefore and after adsorption with plasma membranes weresubjected to analysis by anion-exchange high-pressure liquidchromatography. The unadsorbed extract demonstrated twopeaks (Fig. 1A); the major peak (fractions 60 through 80) hada retention time of 19.9 min and contained 94% of the totalinjected protein. However, a heterogeneous population ofsurface-labeled proteins was present predominantly in frac-tions 35 through 60, with minor trailing into fraction 70. Theunlabeled proteins present in the large acidic peak eitherwere not surface proteins of S. aureus or, if they werepresent on the cell surface, were not accessible to labelingwith the isotope under the experimental conditions used.

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FIG. 1. Anion-exchange high-pressure liquid chromatographyprofiles of 251I-labeled surface proteins obtained from S. aureus 1071and tested unadsorbed (A) or following adsorption with purifiedplasma membranes obtained from MDCK cells that were notinfected (B) or were infected with influenza virus A/FM/1/47 (C).AUFS, Absorbance units, full scale.

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STAPHYLOCOCCAL MEMBRANE RECEPTORS 673

Following adsorption with purified membance profile of the thermal extract sldecrease in the major protein peak (Fig. 1ingly, adsorption had a less pronounced eactivity of surface-labeled proteins (Fig.was decreased 5 to 7% when the proteiwith virus-infected or uninfected host celWhile the experiment described abo

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ibranes, the absor- membranes, it was not possible to determine whether thehowed a dramatic adhesins were binding to the outer or inner surface of theLB and C). Surpris- membranes, nor was it possible to determine whether theeffect on the radio- adhesins were binding to a single membrane receptor or1B and C), which multiple receptor molecules. In order to address these twoins were adsorbed questions, surface-labeled staphylococcal proteins were11 membranes. used as receptor probes in a direct adhesin overlay proce-yve showed direct dure. Intact MDCK cell monolayers that were uninfected orproteins to plasma virus infected were pretreated with either protease or trypsin

in an effort to remove the putative staphylococcal receptorsbefore the cells were disrupted and the plasma membraneswere purified. Untreated and enzyme-treated membrane

e f preparations were subjected to SDS-polyacrylamide gelelectrophoresis, transferred to NCP, incubated with surface-labeled staphylococcal proteins, and visualized by autoradi-ography. Figure 2 shows the protein profiles of uninfectedand virus-infected membranes before and after enzyme

- 92.5 treatment, as detected by staining and by autoradiography.-66.2 The radiolabeled probe reacted with a major polypeptide

having a molecular weight of 54,000 which was present onlyin the virus-infected membranes (Fig. 2, lane j, solid arrow);however, proteolysis did not affect this polypeptide because

- 45 the reaction was also positive with protease-treated (lane k)and trypsin-treated (lane 1) membranes. The protease-

-t 31 sensitive, trypsin-resistant polypeptides (indicated by aster-isks on the autoradiographs) included one band withuninfected membranes (lane e) with a molecular weight of15,500 and three bands with virus-infected membranes (lane

- 21.5 k) having molecular weights of 50,000, 46,000, and 15,500.Figure 3 shows the results of the direct adhesin overlay

procedure in which polypeptides present in protease digests- 14.4 of intact uninfected and virus-infected cell monolayers were

separated by SDS-polyacrylamide gel electrophoresis, trans-ferred to NCP, and reacted with a receptor probe. In theautoradiograph four bands were detected with enzyme di-gests from uninfected cells (lane 2); these bands comigrated

k with four bands of the same molecular weights (35,000,34,000, 30,000 and 20,000) from enzyme digests of virus-infected cell cultures (lane 4). One unique but weaklyreactive band was visualized with digests from virus-infectedcells and had a molecular weight of 26,000.

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DISCUSSION

The initial steps that lead to an increased incidence ofstaphylococcal colonization and subsequent superinfectionin a host compromised by influenza virus are not wellunderstood. Recently, we reported that enhanced adherenceto virus-infected cells in vitro differs quantitatively andqualitatively between strains, that adherence-positive strainspossess thermolabile and thermostable adhesins, and thatmultiple adhesins mediate the binding of S. aureus to bothuninfected and virus-infected cells (15). In the present studywe used the surface-labeled proteins in a thermal extract ofone clinical strain of S. aureus as receptor probes to further

FIG. 2. Protein profiles of purified plasma membranes fromuninfected control MDCK cells that were untreated (lane a), prote-ase treated (lane b), or trypsin treated (lane c) and virus-infectedMDCK cells that were untreated (lane g), protease treated (lane h),or trypsin treated (lane i). Gels were stained with Coomassiebrilliant blue R. The autoradiographs show the reactions of 12511labeled staphylococcal surface proteins with polypeptide bands ofpurified plasma membranes from uninfected control MDCK cellsthat were untreated (lane d), protease treated (lane e), or trypsin

treated (lane f) and virus-infected MDCK cells that were untreated(lane j), protease treated (lane k), or trypsin treated (lane 1).Asterisks indicate bands that were absent in the membranes ofprotease-treated cells (lanes e and k) but were still present in themembranes of trypsin-treated cells (lanes f and 1). The arrowsindicate a 54,000-dalton polypeptide that was absent in uninfectedmembranes (lane d, open arrow) but present in virus-infectedmembranes (lane j, solid arrow) before and after enzyme treatment.The positions of molecular weight standards (103) are indicated.

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674 SANFORD AND RAMSAY

1 2 3 4 kd

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FIG. 3. Autoradiograph of a direct adhesin overlay, showing the

reaction of 'III-labeled staphylococcal surface proteins with the

control PBS supernatants from uninfected cells (lane 1) and virus-

infected cells (lane 3) and the protease digests from uninfected cells

(lane 2) and virus-infected cells (lane 4). A protease only control was

negative (data not shown). The positions of molecular weightstandards (103) are indicated. kd, Kilodaltons.

characterize the in vitro binding of staphylococci to

uninfected and influenza virus A-infected cells. Only 5 to 7%

of the surface-labeled proteins were found to bind directly to

purified membrane preparations obtained from uninfected

and virus-infected MDCK cells (Fig. 1). Our data suggest

that the labeled adhesins present in the thermal extract

represent a quantitatively minor group of proteins extracted

from the cell wall surface. It is interesting to speculate that

these adhesins, as characterized previously (6, 7), may be

type-specific antigens on the surface of the test organism.There are similarities in that multiple antigens may be

expressed by a single strain and they are released from the

cell surface by heat treatment, are sensitive to proteolytic

enzymes, and are present in small amounts (14).

We took advantage of the fact that staphylococcal recep-tors on uninfected and virus-infected cells are removed byprotease treatment but not by trypsin treatment (7). The

protein profiles of uninfected and virus-infected plasmamembranes are shown before and after enzyme treatment of

intact cells in Fig. 2. The differences and similarities in

patterns between uninfected and virus-infected membranes

were expected. It is well known that several hours after

infection with influenza virus the synthesis of cellular pro-

teins is progressively inhibited, with the subsequent appear-

ance of virus-specific polypeptides; while the viral structuralcomponents accumulate in the plasma membrane, there is

apparently no loss of host cell plasma membrane proteins(10, 17). In addition, it has been shown (1) that influenza

virus buds only from the apical surface of infected MDCKcells, whose composition differs from that of the basolateral

surface. Thus, any preparation of virus-infected membranesmust represent a mixture of polypeptides, some of which are

shared with uninfected cell membranes. A comparison of the

bands that were present in the stained gels with the bands

that reacted with the radiolabeled probe in the autoradio-graphs shows that the probe reacted with some, but by no

means all, of the membrane polypeptides available. A 54-

kilodalton probe-positive band was present in the virus-

infected membrane only (Fig. 2, lane j), which suggests that

the polypeptide was synthesized in the cells as a response to

virus infection; however, it appears that this major band wasnot located on the outer surface of the cell membranebecause it was resistant to both protease and trypsin treat-ment. The fact that some probe-positive bands differed intheir sensitivity to enzymatic treatment suggests that thecomponents are discrete entities. The probe-positive bandsremoved as a result of protease treatment were, of course, ofparticular interest. Each of the probe-reactive polypeptidesfrom virus-infected cell membranes comigrated withpolypeptides from uninfected cell membranes; that is, theywere not obviously virus-specific polypeptides. One prote-ase-sensitive polypeptide appeared to be shared byuninfected and virus-infected cells. In contrast, two of thepolypeptides appeared to be unique to virus-infected cellmembranes because they were protease sensitive while thecomigrating polypeptides from uninfected cell membraneswere protease resistant. It is possible that these polypeptidesrepresent host cell polypeptides whose synthesis is inducedupon virus infection. It is also possible that a probe-reactiveband may contain an aggregate of several polypeptides evenafter reduction and heating; identical molecular weights donot imply identical chemical structures.When protease digests of uninfected and virus-infected

monolayers were tested in the direct adhesin overlay proce-dures, we detected four comigrating bands (three of whichwere enhanced in digests from virus-infected cells) and onevery weak band that was unique to the virus-infected cellmonolayers. From these data it was not possible to ascertainwhether any of these bands were part of a single polypeptidewhich was cleaved by proteolysis or by SDS solubilizationor both before being subjected to SDS-polyacrylamide gelelectrophoresis. However, our data suggest that these bandsrepresent multiple receptor molecules.

In summary, we present evidence which suggests that thedirect adhesin overlay procedure is potentially useful foridentifying receptor candidates responsible for promotingthe adherence of S. aureus to uninfected and virus-infectedcells. The procedure was optimized by using purified plasmamembranes rather than crude whole cell lysates in whichreceptors present in small amounts might not be detectable.Furthermore, it was possible to distinguish probe-reactivepolypeptides that were located on the external surface ofplasma membranes by taking advantage of receptor sensitiv-ity to enzyme treatment.

ACKNOWLEDGMENT

This work was supported by Public Health Service grant A117242from the National Institutes of Health.

LITERATURE CITED1. Brock, C. J., and M. J. A. Tanner. 1982. Structure and synthesis

of integral membrane proteins, p. 75-130. In D. Chapman (ed.),Biological membranes. Academic Press, Inc., New York.

2. Brunette, D. M., and J. E. TUI. 1971. A rapid method for theisolation of L-cell surface membranes using an aqueous two-phase polymer system. J. Membr. Biol. 5:215-224.

3. Carlsen, S. A., and J. E. Till. 1975. An improved method for theisolation of surface membranes from cells in monolayer culture.Can. J. Biochem. 53:106-108.

4. Cohen, M. L., and S. Falkow. 1981. Protein antigens fromStaphylococcus aureus strains associated with toxic-shock syn-drome. Science 211:842-844.

5. Daugharty, D., D. T. Warfield, and M. L. Davis. 1972. Solid-phase radioimmunoassay of total and influenza-specific immu-noglobulin G. Appl. Microbiol. 23:360-367.

6. Davison, V. E., and B. A. Sanford. 1981. Adherence of Staph-

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7. Davison, V. E., and B. A. Sanford. 1982. Factors influencingadherence of Staphylococcus aureus to influenza A virus-infected cell cultures. Infect. Immun. 37:946-955.

8. Fainstein, V., D. M. Musher, and T. R. Cate. 1980. Bacterialadherence to pharyngeal cells during viral infection. J. Infect.Dis. 141:172-176.

9. Glasgow, L. 1972. Interaction of viruses and bacteria in host-parasite relations. N. Engl. J. Med. 287:42-43.

10. Hay, A. J. 1974. Studies on the formation of the influenza virusenvelope. Virology 60:398-418.

11. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

12. Mulder, J., and J. F. P. Hers. 1972. Influenza, p. 113-165.Wolters-Noordhoff Publishing, Groningen, The Netherlands.

13. Musher, D. M., and V. Fainstein. 1981. Adherence of Staphy-

lococcus aureus to pharyngeal cells from normal subjects,smokers, staphylococcal carriers and patients with viral infec-tions, p. 1011-1016. In J. Jeljaszewicz (ed.), Staphylococci andstaphylococcal infections. Gustav Fisher Verlag, New York.

14. Oeding, P. 1983. Taxonomy and identification, p. 1-31. InC. S. F. Easmon and C. Adlam (ed.), Staphylococci and staph-ylococcal infections, vol. 1. Academic Press, Inc., New York.

15. Sanford, B. A., V. E. Davison, and M. A. Ramsay. 1986.Staphylococcus aureus adherence to influenza A virus-infectedand control cell cultures: evidence for multiple adhesins. Proc.Soc. Exp. Biol. Med. 181:104-111.

16. Selinger, D. S., W. P. Reed, and L. C. McLaren. 1981. Model forstudying bacterial adherence to epithelial cells infected withviruses. Infect. Immun. 32:941-944.

17. Skehel, J. J., A. J. Hay, and M. D. Waterfield. 1980. Influenzavirus, p. 647-681. In H. A. Blough and J. M. Tiffany (ed.), Cellmembranes and viral envelopes, vol. 2. Academic Press, Inc.,New York.

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