Vol. 27, No. 1INFECTION AND IMMUNITY, Jan. 1980, p. 6-140019-9567/80/01-0006/09$02.00/0

Demonstration of Specific Binding Sites for Human SerumAlbumin in Group C and G Streptococci

ERLING B. MYHRE* AND GORAN KRONVALLDepartment ofMedical Microbiology, University of Lund, Lund, Sweden

A total of 297 bacterial strains belonging to 27 species was tested for quantitativeuptake of radiolabeled human serum albumin. Specific binding sites with highaffinity for human serum albumin were found exclusively in group C and Gstreptococci. The albumin binding was found to be a time-dependent, saturable,and displaceable process which obeyed simple kinetic equations. Scatchard anal-ysis revealed that human serum albumin bound to a homogeneous population ofreceptors with an affinity in the order of 10' liters/mol and that the averagebacterial cell carried more than 80,000 binding sites. The albumin receptor is aheat-stable component susceptible to proteolytic digestion. It has a surfacelocalization separate from the receptors for immunoglobulin G, fibrinogen, aggre-gated ,82-microglobulin, and haptoglobin. In individual strains, albumin reactivitywas also detected independently of these other types of interactions with humanproteins.

Streptococci are capable of highly specific in-teractions with human proteins. These interac-tions are mediated by binding structures on thebacterial surface. At present, four different typesof reactivities are known. First, group A, C, andG streptococci can react in a nonimmune waywith the Fc portion of immunoglobulin mole-cules (14, 20-22). Second, most group A, C, andG streptococci can bind aggregated fl2-micro-globulin (15, 16). A third type of reactivity is theuptake of fibrinogen by group A, C, and G strep-tococci (G. Kronvall, C. Schonbeck, and E.Myhre, Acta Pathol. Microbiol. Scand. Sect. B.,in press; 7, 29). Finally, strains carrying the T4antigen can combine with haptoglobin (13).These interactions between mammalian pro-teins and bacteria are important phenomena fortwo main reasons. Uptake of host-derived pro-teins might be of biological significance throughmodification of the host-parasite relationship.Furthermore, bacteria possessing specific bind-ing structures can be used as laboratory reagentsfor absorption procedures and for isolation ofproteins.

In recent studies of absorption of human se-rum samples with streptococci, we observed thatcertain strains removed considerable amounts ofserum albumin (17). The present investigationconfirms this observation and demonstrates spe-cific binding sites for human serum albumin ingroup C and G streptococci. The present seriesof experiments show that these binding siteshave a surface localization separate from thereceptors for four other human proteins. Thus,a new type of reactivity can be added to the listof bacterial interactions with host proteins.

6

MATERIALS AND METHODS

HSA. HSA was isolated by a two-step procedurefrom pooled serum containing serum samples from 58blood donors. Two-milliliter portions of serum wereseparated by using preparative block electrophoresisperformed in a 0.6% agarose gel with a 0.0075 MVeronal buffer (10). The albumin fraction was furtherpurified by gel filtration on a G-100 Sephadex column(1.5 by 95 cm) eluted at 40C with phosphate-bufferedsaline (PBS, 0.12 M NaCl, 0.03 M phosphate, pH 7.2)containing 0.02% sodium azide. The albumin peak waspooled and concentrated by using collodion filters(Satorius membrane filter GmbH, Gottingen, Ger-many). Immunoelectrophoresis of HSA obtained bythe two-step procedure showed a single precipitationarc when tested at a concentration of 2 mg/ml againstrabbit anti-HSA and anti-human serum proteins(Dakopatts, Copenhagen, Denmark). Albumin con-centrations were measured spectrophotometrically byusing an absorbance (EIRm) value at 280 nm of 5.31(8). The molecular weight of HSA was taken to be66,000 (24).

Bacterial strains. A total of 297 strains belongingto 27 different bacterial species were included in thestudy (Table 1). The following strains were kindlyprovided by 0. Holmberg, National Veterinary Insti-tute, Stockholm, Sweden: Streptococcus zooepidemi-cus, S. equii, S. dysgalactiae, fl-hemolytic bovinegroup G streptococci, group M, N, P, and U strepto-cocci, S. uberis, and Corynebacterium pyogenes. Al-pha-hemolytic group G streptococci were obtainedfrom R. Gudding, National Veterinary Institute, Oslo,Norway. All other strains were obtained consecutivelyfrom clinical specimens sent to the Clinical Microbi-ology Laboratory, University Hospital, Lund, Sweden.Serogrouping of streptococci was performed with thecoagglutination method (4). Streptococci, pneumo-cocci, C. pyogenes, and Brahamella catarrhalis weregrown in Todd-Hewitt broth. Staphylococci, Esche-

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ALBUMIN BINDING TO STREPTOCOCCI 7

richia coli, Klebsiella, Proteus, and Pseudomonasstrains were cultured in tryptone broth. A Todd-Hew-itt broth fortified with X and V factors and a brothcontaining peptone, yeast extract, hemin, starch, andvitamins were used for growing Haemophilus influ-enzae and Neisseria gonorrhoeae, respectively.

Radiolabeling. A 200-p1 amount of HSA (1 mg/milin 0.15 M phosphate buffer, pH 7.5) was labeled with0.2 mCi of '25I (code no. IMS 30, RadiochemicalCentre, Amersham, England) by mild electrolysis asdescribed by Rosa et al. (25) and modified for labelingof immunoglobulins (9). Incorporation of "I into theprotein was estimated by paper chromatography (9).Free, noncovalently bound isotope was removed byextensive dialysis against PBS containing 0.02% so-dium azide and 0.05% Tween 20 (PBSA-Tween). Thelabeled preparation was then gel filtered on the G-100Sephadex column used for purification of HSA. Themonomeric albumin fraction was pooled and used inall experiments. The specific activity of the pooledfraction was 0.7 mCi/mg of protein. Labeled HSA wasmixed with normal human serum and with unlabeledHSA and analyzed by immunoelectrophoresis as de-scribed above. Autoradiographic analysis of the slidesshowed that the radioactivity was associated only withthe albumin precipitate. Commercially available hu-man haptoglobin (Hoechst, Frankfurt, Germany) andhuman polyclonal immunoglobulin G (IgG), fibrino-gen, and aggregated,82-microglobulin obtained as pre-viously described (15, 16) were also iodinated by mildelectrolysis.HSA-binding assay. Overnight broth cultures of

bacterial strains were washed twice and suspended inPBSA-Tween buffer to 109 bacteria per ml, as calcu-lated from optical density values at 520 nm on dilutedsamples in a Beckman CP-1 colorimeter. Binding stud-ies were performed at room temperature in triplicatesin polystyrene test tubes (12 by 70 mm; AB Cerbo,Trollhattan, Sweden) by mixing 0.8 ug of radiolabeledHSA with 2 x 108 bacterial organisms in a final volumeof 225 pl. After 1 h, 2 ml of cold PBSA-Tween bufferwas added, and the bacteria were deposited by cen-trifugation at 1,800 x g for 10 min. The supernatantwas sucked off, and the radioactivity of the bacterialpellet was determined in a gamma counter (LKB RackGamma 1270, Bioteck, Stockhlom, Sweden). The al-bumin uptake was expressed as the percentage of totalradioactivity added. The reproducibility of the assaysystem was assessed by testing 10 different brothcultures of a highly reactive strain. The observedbinding levels (arithmetic mean of triplicate samples)showed that determinations could be performed withan imprecision of 4.3% (coefficient of variation). Thebinding to test tubes in the absence of bacteria wasless than 1%.

Uptake levels at pH values ranging from 3.0 to 8.0were determined with bacteria suspended in 0.1 Mcitrate and phosphate buffers containing 0.05% Tween20. Binding studies were also performed with 0.8-, 5-,10-, and 15-, 20- and 25-,ug quantities ofHSA and heat-killed bacteria (2 x 108 organisms as counted in aPetroff-Hauser Chamber (19) in a final volume of 500,.l. Heat treatment at 800C for 5 min did not affect theHSA binding capacity. A control sample containing 1mg of unlabeled HSA was included to estimate the

nonspecific uptake, presumed to be the same for allsamples. Specific binding was calculated by subtract-ing from the radioactive uptake the amount that couldnot be displaced by unlabeled HSA.

Kinetics studies. All experiments were performedin triplicate at room temperature with 0.8 ,g of radio-labeled HSA and 2 x 108 heat-killed group G strepto-cocci (G 148). Binding assays were performed withincubation times ranging from 10 s to 90 min. Furtheruptake of labeled HSA was prevented by addition of400 pl of ice-cold unlabeled HSA (2 mg/mil in PBSA-Tween buffer). For dissociation studies, bacteria werefirst reacted with labeled HSA in a final volume of 50id. After 1 h the samples were diluted 60 times withPBSA-Tween buffer, and after further 10, 20, 30, 40,50, and 60 min, the amount of HSA still bound to thepellet was determined. Corrections were made fornonspecific binding. Rate constants were calculated asdescribed by Cuatrecasas and Hollenberg (6). Theassociation rate was computed by using the secondorder equation k, = 2.303(1/t)*[1/(a - b)]*log[b(a -

x)/a(b - x)]. The first order equation k2 = 2.303 (1/t) -log(x/x - a) was used for calculation of the disso-ciation rate. In these equations a is the concentrationof albumin, b is the concentration of receptor, and x isthe concentration of albumin-receptor complex at thetime t. The association constant ka was calculatedfrom the equation ka = ki/k2.

Pepsin and trypsin digestion. Increasingamounts of pepsin and trypsin were added to 109 heat-killed bacteria suspended in 1.0 ml of 0.1 M acetatebuffer (pH 4.5) and in 1.0 ml of 0.15 M phosphatebuffer (pH 7.5), respectively. After 1 h of incubationat 37°C the reaction was stopped by addition of 100p1 of 1 M tris(hydroxymethyl)aminomethane base tothe peptic digest and 1 ml of trypsin inhibitor solution(1 mg/mil in PBS) to trypsin-treated bacteria. Thebacteria were washed and suspended in PBSA-Tweenbuffer and then tested for albumin binding. Pepsin,trypsin, and trypsin inhibitor were purchased fromSigma Chemical Co., St. Louis, Mo. (catalog no. P7012, T 8253, and T 9128).

Inhibition assay. Increasing amounts of humanserum albumin, fibrinogen, polyclonal IgG, and normalhuman serum were mixed with 0.8 jig of labeled HSA,and the uptake of labeled material to 2 x 10' bacteriawas determined.

RESULTS

Quantitative binding of HSA. A total of297 bacterial strains representing 27 differentgram-positive and -negative species were testedfor the binding of HSA (Table 1). Binding ca-pacities were determined by direct measurementof uptake of 0.8 ,ug of radiolabeled HSA to 2 x108 bacterial organisms. Significant binding wasobserved only with group C and G streptococci(Fig. 1). All four recognized group C streptococ-cal species were included. Fifteen human S.equisimilis strains were all positive with bindinglevels of 22 to 61%. The majority of 15 nonhumanS. zooepidemicus strains showed a low but def-inite albumin binding. S. equii strains demon-

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8 MYHRE AND KRONVALL

strated the lowest reactivity with only one ofseven strains positive, binding 14%. S. dysgalac-tiae, a-hemolytic bovine streptococci, presenteda continuous spectrum of uptake levels rangingfrom 2 to 63%. Group G streptococci of humanand bovine origin differed in albumin reactivity.Fifteen fl-hemolytic human group G streptococciwere reactive with binding levels of 32 to 72%.The highest uptake levels were noted with fi-hemolytic bovine group G streptococci. Thesestrains formed a homogeneous group binding ca.90% of added albumin. In contrast, a-hemolyticbovine group G streptococci were all negative.Binding studies performed with a group G

streptococcus (G 148) suspended in citrate andphosphate buffers with pH values ranging from3.0 to 8.0 revealed that the albumin uptake was

TABLE 1. Capacity of27 bacterial species to bindHSAa

% Albumin

Bacterial species bound No. ofstrains

Mean Range

Group A streptococci 4 2-8 15Group B streptococci 1 1-2 10Group C streptococci

S. equisimilis 50 22-63 15S. zooepidemicus 15 5-26 15S. equii 6 4-15 7S. dysgalactiae 27 2-62 15

Group D streptococci 1 1-2 17Group G streptococci

fl-Hemolytic human 50 32-72 15isolates

,/-Hemolytic bovine 91 88-92 15isolates

a-Hemolytic bovine 3 2-5 12isolates

Group M streptococci 3 2-3 6Group N streptococci 2 1-8 11Group P streptococci 8 3-13 10Group U streptococci 4 3-8 14S. uberis 5 4-6 2Staphylococcus aureus 2 1-2 15Staphylococcus epidermidis 2 1-3 10Staphylococcus 2 1-4 10

saprophyticusDiplococcuspneumoniae 1 1-2 10Corynebacteriumpyogenes 3 2-3 3Brahamella catarrhalis 2 14 10N. gonorrhoeae 1 1-2 10H. influenza 2 2-3 10E. coli 1 1-2 10Klebsiella spp. 1 1-2 10Proteus spp. 1 1-2 10Pseudomonas spp. 2 1-4 10

a Binding capacities are expressed as the percentuptake of 0.8 jug of radiolabeled albumin to 2 x 105bacterial organisms. Arithmetic mean values ofuptakelevels of individual strains were tested in triplicate.

INFECT. IMMUN.

dependent on the hydrogen ion concentration. Amaximal uptake of 84% was seen at pH 4.5.Considerably lower levels of binding occurredboth at lower and higher pH values. Similardifferences were noted when bacteria coatedwith HSA were resuspended in these buffers.Less than 10% of bound HSA were dissociatedat pH 4.5 to 6.5. Resuspension at pH 3.0 and 8.0produced a 70 and 20% elution of bacteria-asso-ciated HSA.Binding as a function of HSA concentra-

tion. Binding assays performed with 2 X 10'group G streptococci (G 148) showed that theHSA uptake is a saturable process (Fig. 2A). Acorrelation was observed between the bound andthe added albumin fraction at concentrationsbelow 15 ,ug/500 pi. At higher concentrations aplateau was noted. Thus a suspension of 2 x 10'bacterial organisms (G 148) are capable of bind-ing more than 5 ,ug of HSA.Binding as a function of the number of

bacteria. Uptake levels were recorded with 106to 109 bacteria (G 148) in the assay system (Fig.2B). A steady increase in the bound fraction wasobserved over the whole range studied. With 2x 108 bacteria in a final volume of 300 p1, a 50%binding was obtained, and by increasing theamount of bacteria to 109, a nearly 70% uptakewas achieved.Kinetic studies. The kinetics of the albumin

binding was explored by mixing small quantitiesof radiolabeled HSA with sufficient amount ofbacteria to ensure an excess of binding sites.

(i) Association kinetics. Early associationevents could not be resolved merely by separat-ing the components by centrifugation. The bind-ing step was therefore terminated by addition ofunlabeled HSA in vast excess. Displacement ofbound labeled HSA was minimized by rapidcooling of the mixture before centrifugation.Studies performed with this technique demon-strated a time-dependent process with rapid as-sociation of the components. A typical experi-ment using a human group G streptococcus (G148) is shown in Fig. 3A. After 1, 2, and 5 minuptake levels of 36, 46, and 61%, respectively,were noted. After 10 min only modest increasesin the uptake were seen and after 20 min a stateof equilibrium was reached. Regression analysisof these data with an exponential model (y =aebx) showed a correlation with an r2 value of0.96. The binding data were used to determinethe rate of association. The association rate con-stant for the strains G 148 was found to be 8.5X 103M-' S-

(ii) Dissociation kinetics. The dissociationprocess was studied by first reacting radiolabeledHSA with heat-killed group G streptococci (G148) in a minimal final volume and then diluting

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ALBUMIN BINDING TO STREPTOCOCCI 9

str str str.equisimilis zooepidemicus equii

str.dysgalactiae

human bovinestrains strains

FIG. 1. Binding ofHSA to 52 group C streptococci (streptococcal species equisimilis, zooepidemicus, equii,and dysgalactiae) and to 30 ,8-hemolytic group G streptococci ofhuman and bovine origin. Radiolabeled HSA(0.8 pg) was added to 2 x 108 bacteria, and the uptake is expressed as the percentage of added radioactivity.

the incubation mixture 60 times with buffer.Complexed HSA will dissociate until a new equi-librium is reached. In our experiments the dis-sociation was followed for 1 h by determiningthe amount of HSA still bound to bacteria atselected time intervals (Fig. 3B). Regressionanalysis of the data showed that a straight line(r2 = 0.98) could be constructed for the semilog-arithmic plot. Thus the dissociation process fol-lows first-order kinetics. The dissociation is slow,and the half-time of the albumin-receptor com-

plex determined by this technique is ca. 120 min.This corresponds to a dissociation rate constantof 9.6 x 10-5 s-.Digestion with proteolytic enzymes. The

susceptibility of the albumin binding componentto pepsin and trypsin was determined by incu-bation of 109 heat-killed bacteria with 0.1- to1,000-pg quantities of enzymes and then testingthese suspensions for albumin binding. Severalgroup C and G streptococci were tested withsimilar results. The data obtained with the hu-man group G strain G 148 is plotted in Fig. 4.The binding component is sensitive both to pep-sin and trypsin digestion. Digestion with 10 to20 ,ug of enzyme reduced the binding capacity by50%, and use of 100 ,ug of enzyme produced an

almost complete loss of reactivity. The bacterialsuspensions were also tested for binding of ra-

diolabeled human IgG. The immunoglobulin re-

ceptor was rather resistant to both pepsin andtrypsin treatment. The uptake of human IgG

was not affected by enzyme amounts less than500 ,jg, and digestion with 1,000 ,ug of pepsin andtrypsin reduced the bound fraction from 85% toonly 65 and 40%, respectively.Relationship to other streptococcal reac-

tivities. In a series of experiments purified hu-man serum proteins with affinity for streptococ-cal surface structures were tested for their ca-

pacity to inhibit the uptake of radiolabeled HSAto a group G streptococcus. Addition of up to500 ,ug of IgG and fibrinogen to the test systemhad no effect on albumin binding (Fig. 5). Theseamounts were sufficient to saturate the IgG andthe fibrinogen receptors on the test organisms.Because no blocking or steric hindrance was

observed, it seems justified to conclude that thebinding sites for serum albumin are structurallyseparated from the receptors for IgG and fibrin-ogen. Addition of 10 ,tg of unlabeled HSA pro-duced half maximal inhibition, and employmentof 100 ,ug resulted in complete inhibition of thespecific uptake of labeled HSA. Dilutions ofhuman serum were tested and found inhibitoryat concentrations corresponding to the inhibi-tion obtained with isolated HSA in the absenceof other plasma proteins. In a second series ofexperiments, 60 group C and G streptococcalstrains were tested for binding of radiolabeledHSA, human IgG, fibrinogen, haptoglobin, andaggregated f82-microglobulin. In individualstrains, albumin reactivity was detected inde-pendently of the capacity to bind any other

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10 MYHRE AND KRONVALL

albumin bound at saturation. The slope of thelines reflect the effective association constantbetween the albumin molecules and the recep-tors. From Fig. 6 it is evident that the two groupG streptococci tested have similar binding ca-pacities, but differ in affinity. Based on the as-sumption that one albumin molecule interactswith one receptor, the average number of bind-ing sites per bacterial cell are 84,000 for G 148and 82,000 for DG 6. The linearity of the Scat-chard analysis suggests that albumin binds to ahomogeneous population of receptors. The as-

5 10 15 20 25jig human serum albumin added

FIG. 2. (A) Uptake ofHSA (micrograms) to 2 x 108organisms of a human group G streptococcus (G 148)tested with 0.8- to 25-pg quantities of labeled HSA ina final volume of 500 IL. (B) Binding of HSA as a

function of the number of test organisms. Radiola-beled HSA (0.8 ug) was mixed with bacterial suspen-sions containing from 10o to 108 bacteria (G 148).

protein preparation. In strains binding several ofthe test proteins, no statistical correlation was

found between the uptake of albumin and thelevels of the other reactivities.Determination of number of albumin re-

ceptors and affinity constants. A humangroup G streptococcus (G 148) and a bovinegroup G strain (DG 6) were tested for binding ofvariable amounts of radiolabeled HSA. Bound(b) and free (f) fractions were determined, andthe data was plotted as described by Scatchard(26), using the relationship b/f= nK - b (Fig.6). Linear regression lines were drawn for theplots (r2 = 0.99). The intercept of these lineswith the x-axis gives the value n, the amount of

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ALBUMIN BINDING TO STREPTOCOCCI

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sociation constants derived from the plots are

5.8 x 106 and 1.7 x 107 liters/mol for G 148 andDG 6, respectively. Association constants can

also be calculated from the rate constants ofassociation and dissociation. Based on the ki-netic data, the association constant for strain G148 is 8.9 x 107 liters/mol. This value is about10 times higher than the value derived from the

Scatchard plot. The discrepancy might reflectthe differences in test circumstances. The kineticexperiments were performed with an excess ofbacterial receptors in contrast to the Scatchardanalysis, which was carried out with an excessof albumin molecules. Thus, the effective asso-ciation constant might depend on the degree ofsaturation of the receptor. Small quantities of

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12 MYHRE AND KRONVALL

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FIG. 6. Scatchard plots of binding experiments performed with 5 to 25 jtg of labeled HSA. A human groupG strain (G 148[aJ) and a bovine f3-hemolytic group G strain (DG 6[b]) were studied.

ligand will then bind with higher affinities thanamounts which saturate the streptococcal bind-ing sites.

DISCUSSIONThe present studies describe the presence of

binding sites for HSA on the surface of group Cand G streptococci. The albumin uptake is asaturable process indicating existence of a lim-ited number of binding sites on the bacterialsurface. Rate constants for association and dis-sociation can be measured, and the kinetic datawas found to obey simple rate equations. Thus,this new type of streptococcal reactivity seemsindeed to represent a highly specific interaction.The bacterial structure responsible for the

HSA binding was not identified, but some char-acteristic features were observed. Heat treat-ment of bacteria at 80'C for 5 min did not affectthe reactivity. In contrast, digestion with pepsinat suboptimal pH and with trypsin at pH 7.5resulted in a complete loss of specific bindingactivity. Several group C and G streptococcihave been subcultured weekly in our laboratoryfor more than 1 year without loss of albuminreactivity. Thus the albumin receptor appearsto be a heat-stable component which is either aprotein itself or connected to the bacterial sur-face by a protein structure. Human IgG andfibrinogen had no inhibitory effect on the uptakeof labeled HSA to a streptococcal strain carrying

binding sites for all three proteins. Receptors forhuman IgG and aggregated 8l2-microglobulinwere found to differ in sensitivity to trypsindigestion. Furthermore, in individual streptococ-cal strains HSA reactivity was detected inde-pendently of the capacity to interact with IgG,fibrinogen, aggregated 8l2-microglobulin, andhaptoglobin. These observations indicate thatthe streptococcal surface possesses specific bind-ing sites for HSA and that these binding siteshave a molecular localization separated from thereceptors for other human proteins.W. A. Simpson, I. Ofek, and E. H. Beachey

have recently demonstrated an interaction be-tween albumin and lipoteichoic acid (LTA) ex-tracted from streptococci (Clin. Res. 27:357A,1979). This interaction involves the lipid-con-taining portion ofLTA which combines with thefatty acid binding sites on the albumin molecule.Although LTA is a possible candidate for albu-min binding to intact bacterial cells as describedin the present paper, we consider this possibilityrather unlikely. First of all, LTA is a regularcomponent with a uniform structure found inmost gram-positive bacteria, including strepto-cocci (12,31). The restriction of albumin reactiv-ity to group C and G streptococci is difficult toreconcile with this fact. Second, LTA is clearlyamphipathic with a small hydrophobic glyco-lipid portion associated with the bacterialplasma membrane and a long polar glycerylphosphate chain extending into the cell wall (12,

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ALBUMIN BINDING TO STREPTOCOCCI 13

31). The lipid portion of LTA which showedaffinity for albumin seems to be deeply buriedin the bacterial cell wall and hardly accessible atthe cell surface. Beachey et al. have, however,presented evidence suggesting a surface locali-zation of the lipid part of LTA in group Astreptococci (2, 23). In the present experimentsgroup A streptococci demonstrated only a lowbackground binding seen with nearly all otherbacteria tested. In our previous study we notedsome absorption of albumin by two group Astrains (17). The absorption experiments wereperformed with about 100 times the number oforganisms used in the present test system. Thegreat number of bacteria used favors interac-tions of low avidity and affinity.Albumin reactivity was demonstrated in all

four group C species and in both human andbovine fl-hemolytic group G streptococci (Fig.1). This observation underlines earlier describedsimilarities between these bacterial species asrevealed in immunoglobulin reactivity. Group Cand G streptococci carry the same type of Fcbinding structures, which differs from that pres-ent on group A streptococci (21, 22). Humangroup C and G streptococci therefore seem to bemore closely related to each other than to otherstreptococcal species. The albumin receptor mayhave evolved late in the evolution of bacteriaand may have then been preserved through thefurther separation ofgroup C and G streptococci.

In the present investigation estimates weremade of the affinity constant for the albuminbinding, and the number of binding sites ondifferent bacterial strains was calculated. Whennew types of interactions are described, attemptsshould be made to determine these parameters.High-avidity bindings tend to be more specificthan low avidity interactions. It is possible thatdifferent types of interactions can be related tothe degree of avidity. For instance, adherence ofbacteria to host cells might require only low-avidity receptors as several adjacent bindingsites could be engaged simultaneously resultingin additive effects. Higher avidities may beneeded for interactions with single moleculesmasking determinants on the host cell surface.

Protein A carrying staphylococci have beenused in the laboratory for selective absorption ofimmunoglobulins (1, 18, 28). Albumin bindingstreptococci might serve similar purposes. Be-cause streptococcal receptors for albumin, im-munoglobulins, fibrinogen, and haptoglobin oc-cur independently, strains binding one or moreof these plasma proteins can be selected forparticular use. Streptococci conserved by heattreatment as described in this study can bestored over several months without loss of al-bumin binding capacity. The uptake of albumin

is a reversible process, and absorbed albumincan be recovered by elution with either acidbuffers or with dissociating agents like isothio-cyanate. The high capacity and avidity ofbovinegroup G strains make them particularly suitablefor specific albumin binding applications.The biological significance of uptake ofhuman

proteins by bacterial surface structures has notbeen determined. Schistosoma mansoni, amammalian parasite residing in the intestinalblood vessel system of the host, are capable ofspecific binding of host proteins like blood groupsubstances and histocompatibility antigens to itssurface (5, 27). Receptors with specificity for IgG(Fc) and human 832-microglobulin has recentlybeen identified on the tegumental surface of theparasite (11, 30). Acquisition ofhost-derived pro-teins seems to enable the parasite to evade im-mune response of the host and thus to escapesubsequent destruction (3, 30). In analogy, up-take of human serum proteins by streptococcicould provide a mechanism to resist normaldisposal. Pathogenic bacteria have often severallines of defense, and binding of host proteinsmight be one of them. The present study addsanother type of human protein binding withsuch potential capacities.

ACKNOWLEDGMENTSThis project was supported by the Swedish Medical Re-

search Council, grant 5210.We thank Elisabeth Nilsson and Ake Christensson for

excellent technical assistance and Eva Carlquist for secretarialhelp.

LITERATURE CITED

1. Ankerst, J., P. Christensen, L. Kjellen, and G. Kron-vall. 1974. A routine diagnostic test for IgA and IgMantibodies to rubella virus: absorption of IgG withStaphylococcus aureus. J. Infect. Dis. 130:268-273.

2. Beachey, E. H., and I. Ofek. 1976. Epithelial cell bindingof group A streptococci by lipoteichoic acid on fimbriaedenuded ofM protein. J. Exp. Med. 143:769-771.

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