Vol. 57, No. 8INFECTION AND IMMUNITY, Aug. 1989. p. 2397-24040019-9567/89/082397-08$02.00/0Copyright © 1989, American Society for Microbiology

Ultrastructural Localization of the Fibrinogen-Binding Domain ofStreptococcal M Protein

MILOS RYC,'t EDWIN H. BEACHEY,' 2 AND ELLEN WHITNACK .2*

University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163,1 and Veterans AdministrationMedical Center, Memnphis, Tennessee 381042

Received 6 February 1989/Accepted 5 May 1989

Binding of fibrinogen to the M protein located on the surface fibrillae of group A streptococci impedesdeposition of complement and thus contributes to the virulence of these organisms. We investigated this bindingby electron microscopy using postembedding immunogold labeling. Both fibrinogen and its D fragment formeda distinct dense layer in the surface fibrillae, separated by 10 nm from the compact part of the cell wall.Labeling the sections with anti-fibrinogen or anti-fragment D showed that the fibrinogen-binding region laywithin a 25-nm segment of the fibrillae beginning approximately 30 nm from the inner surface of the cell wall.The outer surface of the fibrinogen layer could be labeled with antibody to the amino-terminal half of type 24M protein, indicating that the fibrillar tips remained exposed after fibrinogen binding. The degree of labelingwith anti-fibrinogen, determined by gold particle counting, was the same whether the bacterial cells had beenincubated with purified fibrinogen or whole plasma. These results indicate that the fibrinogen-binding regionlies in the distal (amino-terminal) half of the M protein molecule but excludes the most distal portion, whichis the site of epitopes that interact with opsonic anti-M antibody, and that plasma proteins other thanfibrinogen, a number of which are known to bind to group A streptococci, do not interfere with fibrinogenbinding.

Binding of human fibrinogen to group A streptococci wasfirst reported over 50 years ago, when Tillet and Garner (37)described agglutination of hemolytic streptococci by plasmaand part;ally purified fibrinogen. Using 125I-labeled fibrin-ogen, Kronvall et al. (21) found that each of the group Astreptococcal strains examined bound human fibrinogen.Most group A streptococci also bind fibrinogens of otheranimal species (24, 30). Runehagen et al. (32) demonstratedthat fibrinogen binds via the D domain.

Kantor and Cole (17) and Kantor (16) identified thefibrinogen-binding factor as M protein, a component of thesurface fibrillae of group A streptococci responsible for theirresistance to opsonization and phagocytosis (11, 25). Otherssubstantiated this suggestion (6, 12, 13, 15, 20, 23, 39, 40). Inour studies (40), 3H-labeled human fibrinogen bound to type24 streptococci with high affinity (KC,, 5 nM). Binding wascompletely inhibited by antisera against the pepsin-extractedamino-terminal half of type 24 M protein (pep M24) but notby antisera to other components of the surface fibrillae.Moreover, pep M24 precipitated fibrinogen (40) and reactedwith its D fragment on Western blots (immunoblots) (41).Fibrinogen and its D fragment, when bound to the M proteinon the streptococcal surface, blocked C3 deposition andthereby enhanced the antiopsonic function of M protein.Bound fibrinogen also blocked certain opsonic cross-reac-tions between M serotypes (43). The binding of fibrinogen tostreptococci is therefore a virulence mechanism and maycontribute to the type specificity of streptococcal immunity.

Streptococcal M proteins are single polypeptide chainswith molecular masses of 40 to 80 kilodaltons (9). Thecarboxy termini appear to be anchored in the cell membraneby a hydrophobic sequence of 19 amino acids (14) that maybe common to all serotypes (34). Next is a proline- and

* Corresponding author.t Visiting scientist from the Institute of Hygiene and Epidemiol-

ogy, Prague, Czechoslovakia; died, 17 February 1989.

glycine-rich region that traverses the cell wall (28). Accord-ing to the current model, the remainder of the M proteinmolecules project from the cell wall as noncovalent dimersthat are wound about each other in an a-helical coiled-coilconfiguration, excluding the 10 to 20 amino-terminal residues(10, 29). The M protein is a major component of the hairlikesurface fibrillae that in electron micrographs appear as afuzzy layer about 50 nm thick (29).

Ultrastructural visualization of fibrinogen bound to strep-tococci has been reported by several laboratories (13, 38,40-42). These electron micrographs show enhanced densityof the surface fibrillae, which lose their hairlike appearanceand become a dense, homogeneous layer. In the presentstudy, we took advantage of the improved localizationafforded by low-temperature embedding and postembeddingimmunogold labeling to analyze the ultrastructural locationof bound fibrinogen and its D fragment on the streptococcalsurface. We present evidence that the region of M proteinthat binds the D domain of fibrinogen lies within a 25-nmsegment of the fibrillae that begins approximately 30 nmfrom the inner surface of the cell wall and excludes theamino-terminal tips of the fibrillae.

MATERIALS AND METHODS

Streptococci. Group A streptococci (M serotype, 24[Vaughn strain]) were blood passed to a high level ofresistance to phagocytosis and stored at -80°C. The bacteriawere grown to early log phase in Todd-Hewitt broth (DifcoLaboratories, Detroit, Mich.) supplemented with 20% nor-mal rabbit serum (GIBCO Laboratories, Grand Island,N.Y.), harvested by centrifugation, killed by UV irradiationfor 3 min, and washed in phosphate-buffered saline (PBS;0.15 M NaCl, 0.02 M phosphate [pH 7.4]).

Reagents and antisera. Human fibrinogen (grade L; Kabi-Vitrum, Stockholm, Sweden) was dissolved in PBS at aconcentration of 10 FiM, dialyzed against the same buffer,and stored at -80°C. Purified, lyophilized fragment DI

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(hereinafter referred to as fragment D; Diagnostica Stago,Asnieres, France) was reconstituted with distilled water to aconcentration of 1.43 mg/ml (16 FiM) and stored at -20°C.Rabbit anti-human fibrinogen (Organon Teknika, Malvern,Pa.) and rabbit anti-human fragment D (Diagnostica Stago)were absorbed with 1 volume of packed type 24 streptococ-cal cells for 2 to 3 h at 37°C and then overnight at 4°C.Antibody to type 24 M protein was raised in rabbits byimmunization with a 30-kilodalton purified peptic fragmentrepresenting the amino terminus of the molecule (pep M24)and assayed as previously described (2); the titer in anenzyme-linked immunosorbent assay was 1:25,600. Normalrabbit serum was obtained from GIBCO. Goat anti-rabbitimmunoglobulin G (IgG) adsorbed to colloidal gold (10-nm-diameter particles; AuroProbe EM, GAR G10) waspurchased from Janssen Life Sciences Products, Piscat-away, N.J. Fresh human serum and plasma were obtainedfrom a donor who lacked antibody to type 24 M protein asdetermined by enzyme-linked immunosorbent assay and bygrowth of type 24 streptococci in the donor's blood. Heat-inactivated serum was treated at 56°C for 30 min.Treatment of streptococci. Sedimented bacteria were incu-

bated with 10 FM human fibrinogen, 16 FiM fragment D,fresh human plasma containing heparin at 8 U/ml, or freshhuman serum. The ratio of test fluid to bacteria was 100 p.l/3x 108 CFU. Incubations were performed at ambient temper-ature for 30 min.

Fixation and embedding of bacteria for electron micros-copy. Untreated control bacteria and bacteria incubated withvarious plasma proteins (see above) were washed twice inPBS and lightly fixed with 0.5% glutaraldehyde (EM grade;Polysciences Inc., Warrington, Pa.) for 30 min at roomtemperature, washed twice in PBS, and enrobed in 2% agar(BBL Microbiology Systems, Cockeysville, Md.). Smallcubes were dehydrated with 30% ethanol for 30 min at 0°Cand then with 50% ethanol for 60 min at -20°C, 70% ethanolfor 60 min at -35°C, 95% ethanol for 60 min at -35°C, andabsolute ethanol twice for 60 min each time at -35°C.Samples were embedded in Lowicryl K 11 M, a low-viscosity polar medium (Polysciences Inc.) (1, 5) at -35°C asfollows: Lowicryl-ethanol at a 1:2 ratio for 60 min, Lowicryl-ethanol at a 2:1 ratio for 60 min, Lowicryl for 60 min, andfresh Lowicryl overnight. Samples were then placed in freshLowicryl and polymerized by indirect UV irradiation (360nm) for 2 to 3 days at -35°C, followed by further polymer-ization at room temperature for 3 days. Ultrathin sectionswere cut with a diamond knife on a Reichert Ultracut Eultramicrotome and mounted on nickel grids with carbon-coated Formvar film. After immunolabeling, the sectionswere evaluated with a JEOL 1200 EX electron microscopeoperating at 60 kV.

Immunolabeling. The sections were incubated at roomtemperature on drops of the following reagents: 1% bovineserum albumin (Sigma Chemical Co., St. Louis, Mo.) in PBSfor 10 min; rabbit antiserum (antibody 1) diluted to variousdegrees in PBS for 60 min; 1% bovine serum albumin in PBSthree times for 10 min each time; goat anti-rabbit IgGadsorbed to colloidal gold (antibody 2) diluted 1:20 in PBSfor 60 min; PBS three times for 10 min each time; double-distilled water three times for 10 min each time. The sectionswere then contrasted with saturated uranyl acetate for 5 minand lead citrate (31) for 5 s. All antisera 1 were used in aseries of dilutions (1:10, 1:20, 1:50, 1:100, and 1:200); forfinal evaluation, the highest dilution that gave maximalspecific immunolabeling with minimal nonspecific back-ground labeling was used. Controls were treated in a similar

way by using normal rabbit serum instead of antiserum 1 orlabeled antiserum 2 alone. All of the controls exhibitednegligible nonspecific labeling (<5 gold particles per cell).Measurements. All measurements were performed on en-

larged electron micrographs with a magnifying lens equippedwith a built-in scale marked in 0.1-mm divisions (Zeiss, Jena,German Democratic Republic). Only antitangentially sec-tioned cell surfaces were evaluated. The distance of the goldparticles from the inner surface of the cell wall (mean +standard deviation [SD]) was calculated on the basis of 200individual measurements; only gold particles external to theinner surface of the cell wall were counted. As an indicatorof accessible fibrinogen epitopes, the number of gold parti-cles in 100 cell wall segments, each 0.2 ,um long, wascounted.

RESULTS

Ultrastructural morphology of the streptococcal surfacebefore and after treatment with fibrinogen, plasma, or serum.Lowicryl-embedded group A streptococci exhibited cellsurface morphology comparable to that of streptococci em-bedded in conventional media (35). In our strain (Fig. la),the cell wall was composed of two distinct layers, a 5-nminner layer of higher electron density and a 10-nm outer layerof lower density. These two layers (the compact part of thecell wall) were covered with fibrillae ca. 46 nm long. Thus,the whole cell wall (compact part plus fibrillae) measuredapproximately 61 nm.

Incubation of streptococci with fibrinogen resulted information of a distinct electron-dense layer on the surface ofthe bacteria (Fig. lb). This layer, the thickness of which was40 nm, was rather sharply delineated on both the outer andinner surfaces and was separated from the surface of thecompact part of the cell wall by a gap of low electron densitythat measured 10 nm. The outer surface of the dense layerwas 65 nm from the compact part of the cell wall, comparedwith 61 nm for the tips of the fibrillae on untreated bacteria;thus, the fibrinogen layer did not substantially exceed thelength of the fibrillae. When streptococci were incubatedwith the D fragment of fibrinogen, the layer so formed wassomewhat narrower (33 nm) and less electron dense (Fig.lc). This layer was also separated from the compact part ofthe cell wall by a 10-nm gap.

Streptococci incubated with whole plasma were coveredwith an electron-dense layer similar to that of bacteriaincubated with fibrinogen alone (Fig. ld). Its thickness was45 nm and it, too, was separated from the compact part ofthe cell wall by a 10-nm interspace. The distance of theexternal surface of the plasma layer from the compact part ofthe cell wall exceeded the length of the fibrillae (55 versus 46nm).

Streptococci incubated with fresh human serum also ex-hibited a surface layer approximately 46 nm thick, but itselectron density was substantially lower than that of fibrin-ogen- or plasma-treated bacteria (Fig. le). The serum layerwas not sharply limited on its external surface and was notdistinctly separated from the compact part of the cell wall.

Immunolabeling of fibrinogen epitopes. Immunogold label-ing of fibrinogen-coated streptococci with anti-fibrinogenserum revealed fibrinogen epitopes mostly in the electron-dense fibrinogen layer on the surface of streptococci. How-ever, gold particles were also found both external to thatlayer and in the interspace between it and the compact partof the cell wall (Fig. 2a). The mean distance of gold particlesfrom the inner surface of the wall (38.8 nm; Table 1)

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FIG. 1. Type 24 group A streptococcal cells incubated withbuffer (a), fibrinogen (b), fragment D of fibrinogen (c), whole plasma(d), or serum (e). The compact part of the cell wall, between thearrow tips in panel c, comprises an inner dense layer and an outer,slightly thicker, lucent layer delimited by a faint, thin, dense line. Incontrol organisms (a), the fibrillar layer has its normal fuzzyappearance. In panels b, c, and d, treatment with fibrinogen,fragment D, or plasma resulted in increased density of the outer partof the fibrillar layer. After treatment with serum (e), the fibrillae

appeared fluffier compared with those shown in panel a, probablybecause of albumin binding (38). Bar, 0.2 p.m.

indicated that the fibrinogen bound to a region located in theperipheral two-thirds of the complete cell wall thickness,near the middle of the fibrillae. The high SD (Table 1) reflectsthe broad distribution of fibrinogen epitopes outside andinside the electron-dense layer. Labeling of fibrinogen-treated bacteria with antiserum against the D fragment offibrinogen yielded identical results in terms of both theultrastructural location of epitopes (Fig. 2b) and statisticalevaluation (Table 1).

Fibrinogen has a trinodular structure, with the D domainsat the ends and the E domain in the center (26). Reasoningthat at least some of the fibrinogen molecules might bind to

D, M protein by only one D domain, particularly at the saturat-ing concentrations of fibrinogen used in this study, we

suspected that the uneven distribution of fibrinogen epitopesmight have resulted from labeling of unbound D and Edomains at some distance from the bound D domain. Ac-cordingly, we performed a similar immunolabeling of strep-tococci incubated with the purified D fragment of fibrinogen.As expected, the labeling pattern was much more regular.Most gold particles were located in the electron-dense layerwhether anti-fibrinogen (Fig. 2c) or the anti-D fragment offibrinogen (Fig. 2d) was used for immunolabeling. Whenstreptococci treated with fragment D were incubated withanti-D serum, the mean distance of the gold particles fromthe inner surface of the cell wall was 43.1 nm, compared with38.8 nm for fibrinogen-coated streptococci (Table 1). The SDwas much lower than that of fibrinogen-treated bacteria (10.2versus 25.2 nm) and was, in fact, equal to the size of the goldparticles used (10 nm) or the size of the immunoglobulinmolecule (12 to 13 nm) (Table 1).

Location of pep M24 epitopes in relation to fibrinogen-binding sites. In previous studies, we showed that thebinding site for fibrinogen on streptococcal cells resides, atleast in part, in the pep M moiety of the surface M protein.Fibrinogen does not, however, impede opsonization of theorganisms by anti-pep M serum unless the bacterial cells are

exposed to it before addition of the antiserum, and even thend the inhibition is only partial, implying that pep M epitopes

remain exposed after fibrinogen is bound (41, 42). Accord-ingly, we examined the accessibility of pep M24 epitopes on

fibrinogen-coated streptococci.

TABLE 1. Distribution of fibrinogen epitopes on streptococciincubated with fibrinogen or its D fragment

e

Pretreatment of Treatment of Mean + SDstreptococci ultrathin sections distance (nm)"Fibrinogen Anti-fibrinogen 38.8 ± 23.1Fibrinogen Anti-fragment D 38.8 ± 25.2Fragment D Anti-fibrinogen 40.4 + 13.8Fragment D Anti-fragment D 43.1 + 10.2

" Distance of gold particles from the inner surface of the compact part of thecell wall.

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FIG. 2. Immunolabeling of fibrinogen or its D fragment epitopes (indirect immunogold technique). Streptococcal cells were incubated withfibrinogen before embedding and anti-fibrinogen (1:50) on section (a), fibrinogen before embedding and anti-fragment D (1:50) on section (b),fragment D before embedding and anti-fibrinogen (1:20) on section (c), or fragment D before embedding and anti-fragment D (1:200) on section(d). Bar, 0.2 ,.m.

Sections of untreated streptococci incubated with anti-pepM24 serum exhibited numerous pep M24 epitopes along thesurface fibrillae (Fig. 3a). The few pep M epitopes in thebacterial cytoplasm presumably represent sites of M proteinsynthesis. The mean distance of pep M24 epitopes from theinner cell wall surface (the distance of gold particles from theinner surface of the compact part of the cell) was 36.7 (SD,20.2) nm.

Labeling of fibrinogen-coated streptococci with anti-pepM24 serum clearly demonstrated that fibrinogen did notoccupy the whole pep M24 portion of the M protein moleculeand that after fibrinogen treatment of bacteria many pep Mepitopes remained accessible for reaction with anti-pep Mantibodies (Fig. 3b). Many pep M24 epitopes remained free,mostly on the surface of the fibrinogen layer and, to a lesserextent, under that layer. In the presence of a fibrinogen coat,the mean distance of the pep M24 epitopes was shifted from36.7 (SD, 20.2) to 50.7 (SD, 26.5) nm. Similarly, many pepM24 epitopes remained available after treatment of strepto-cocci with plasma (Fig. 3c); the free epitopes were locatedon both the inner and outer sides of the dense layer. Themean distance of the pep M24 epitopes from the inner cellwall (52.1 ± 23.6 nm) was shifted distally, as in bacteriatreated with fibrinogen alone.Comparison of the number of fibrinogen epitopes on strep-

tococci incubated with fibrinogen or plasma. A number of

blood proteins besides fibrinogen bind to group A strepto-cocci (reviewed in reference 33). In previous studies, wenoted that serum does not inhibit binding of fibrinogen tostreptococcal cells, indicating little interference by serumproteins with binding (40). We therefore counted fibrinogenepitopes on segments of cells incubated with fibrinogen orplasma (see Materials and Methods). Both the labelingpattern (Fig. 4a; compare Fig. 2a) and the average goldparticle count were virtually identical on cells incubated withfibrinogen or plasma. The mean ± SD gold particle counts of100 cell surface segments, each 200 nm long, from strepto-cocci incubated with serum (controls), fibrinogen, or plasmawere 0.1 ± 0.5, 11.9 ± 3.9, and 12.6 ± 4.2, respectively.These results support the inference that binding of otherblood proteins does not interfere with binding of fibrinogen.

Control streptococci treated with serum (Fig. 4b) exhib-ited negligible labeling, demonstrating the high specificity ofthe cell-labeling procedures used in these experiments.

DISCUSSION

The aim of these experiments was to analyze on anultrastructural level the binding of fibfinogen to an M-positive strain of group A streptococci and to visualize thesite of the interaction of fibrinogen with the M proteinmolecule... ,i1R,;,.........** S*^'.*.......*i;* .4 s : "'-iF , * ^ s l Wi e;tw i > .. f L:,

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FIG. 3. Immunolabeling of pep M24 epitopes (indirect immunogold technique). Streptococcal cells were incubated with buffer beforeembedding and anti-pep M24 (1:50) on section (a), fibrinogen before embedding and anti-pep M24 (1:50) on section (b), or whole plasma beforeembedding and anti-pep M24 (1:50) on section (c). Bar, 0.2 p.m.

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FIG. 4. Immunolabeling of fibrinogen epitopes (indirect immunogold technique). Streptococci were incubated with whole plasma (a) orserum (b) before embedding. Sections were incubated with anti-fibrinogen (1:20). Bar, 0.2 ,um.

In previous studies (40, 43), we obtained evidence that thefibrinogen-binding region of type 24 M protein lies within thedistal (amino-terminal) half of the M protein molecule.Treatment of type 24 streptococcal cells with dilute pepsin atsuboptimal pH shortened the surface fibrillae and reducedfibrinogen binding by 90%. pep M24, the large peptide of Mprotein removed from the cell surface by this procedure,precipitated fibrinogen and, when used at concentrationssufficient to exceed the zone of precipitation, inhibitedbinding of fibrinogen to intact cells by approximately 80%.We therefore expected to find that fibrinogen and its Mprotein-binding moiety, the D fragment, would bind to theouter half of the fibrillae, and this proved to be true.Streptococci treated with fibrinogen or its D fragment exhib-ited a distinct electron-dense layer in the outer part of thefibrillae, not extending beyond them. Separation of thefibrinogen or D fragment layer from the outer surface of thecompact part of the streptococcal cell wall by an electron-transparent interspace indicates that little, if any, fibrinogenbinds to the proximal part of the M protein molecule.Comparison of the length of the fibrinogen molecule (45 nm)(3) with the length of the fibrillae (here, 46 nm) indicates thatfibrinogen cannot be arranged perpendicularly to the strep-tococcal surface. It may, therefore, cross-link binding siteson different fibrillae via its two D domains. However, somefibrinogen molecules may be bound to M protein only by oneD domain and protrude either on the outer surface ofbacteria or into the interspace between the fibrinogen layerand the compact part of the cell wall. Alternatively, some ofthe fibrinogen epitopes on the outer surface of the fibrillar

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layer may be due to fibrinogen-fibrinogen interactions, assuggested by Havlfiek et al. (12).More precise localization of the fibrinogen-binding site

was achieved by incubating bacteria with the purified Dfragment of fibrinogen and labeling sections with anti-Dserum. In this case, the gold particles were found almostexclusively in the dense layer formed by the D fragment. Themean distance of epitopes from the inner surface of the cellwall (43.1 ± 10.2 nm) was almost exactly in the middle of theD fragment dense layer (25 to 58 nm from the inner surfaceof the wall; Fig. 5). By subtracting the average radius offragment D (4.5 nm; 3) from each side of the fragment Dlayer, the fibrinogen-binding region could be localized to a25-nm segment of the fibrillae. We conclude that the fibrin-ogen-binding region occupies a segment ofM protein startingnear the middle of its length and reaching almost, but notcompletely, to its amino terminus. The SD of measurementin these experiments was similar to the size of the goldparticles used for immunolabeling or the size of an IgGmolecule, indicating a high degree of precision in localizingthe binding region. Apparently, antibody 1 was, for the mostpart, perpendicularly bound to the plane of the section sothat the marker (colloidal gold) was close to the antibody-binding site. This assumption is, of course, good only forevaluating the results of the postembedding immunolabelingtechnique.The estimated length of the fibrinogen-binding segment is

severalfold greater than the diameter of fragment D. Thiscould mean that the estimate is, despite the considerationsgiven above, too large. Alternatively, there may be several

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FIG. 5. Scheme of the location of the fibrinogen-binding region on type 24 group A streptococcal surface fibrillae. For an explanation, seethe text.

43.1 + 10.2 nm(mean distance ± SD)

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points of attachment for fragment D along the type 24 Mprotein molecule, which has 35-residue sequence repeats inthe pep M moiety (27). However, we have not found an M24peptide smaller than 20 kilodaltons that both binds fibrinogenand inhibits binding of fibrinogen to M protein (unpublisheddata).

In previous studies, we noted that bound fibrinogen doesnot interfere with opsonization of the streptococci by anti-pep M24, suggesting that part of the M protein remainsexposed and accessible to the antibody after binding offibrinogen (42, 43). Although it was not possible to discernthe tips of the fibrillae projecting past the fibrinogen layer onthe electron micrographs, the calculated location of thefibrinogen-binding region (see above) supported this infer-ence. Further confirmation was obtained by immunolabelingfibrinogen- or plasma-treated streptococci with anti-pep M24serum. Many M protein epitopes remained free (Fig. 3b),mostly at the periphery of the fibrinogen layer. Free Mepitopes were also found, in smaller numbers, in the inter-space between the fibrinogen layer and the compact part ofthe cell wall. There are at least two possible explanations forthe labeling of the interspace. (i) Some fibrillae may not lieparallel to the plane of the section. Postembedding immuno-labeling demonstrates only epitopes accessible on the sur-face of the section (4), and thus, at least some pep Mepitopes found under the fibrinogen layer may representperipheral portions of the M protein molecule. (ii) Fibrillaemay vary in length; pep M epitopes found in the interspacemight represent the distal portions of the M protein moleculeduring its synthesis and transport through the cell wall.Group A streptococci are known to bind, besides fibrin-

ogen, albumin (22), IgG via its Fc portion (19), IgA myelomaprotein (7), haptoglobin (18), fibronectin (8, 36), and ferritin(33), raising the possibility that these proteins might interferewith the binding of fibrinogen by either direct competition orsteric hindrance. Evidence from our previous work (40)suggested that this does not occur; nonimmune serum at a50% concentration does not inhibit binding of [3H]fibrin-ogen. Furthermore, in a study using ferritin-labeled fibrin-ogen and gold-labeled albumin (38), it was found that albu-min bound to the fibrillae nearer to the cell wall than didfibrinogen. In the present study, we compared the number offibrinogen epitopes on streptococci incubated with purifiedfibrinogen with the number on streptococci treated withwhole plasma. This measurement derives its accuracy fromthe fact that only epitopes lying at the surface of the sectionare labeled in the postembedding immunolabeling procedure(4) so that the count of gold particles does not depend on thethickness of the section. As predicted, the numbers offibrinogen epitopes per segment of cell surface of fibrinogen-treated and plasma-treated streptococci were nearly identi-cal (see Results).

ACKNOWLEDGMENTS

We thank Loretta Hatmaker and Jeff Goode for excellent techni-cal assistance and David Hasty for preparing Fig. 5.

This work was supported by institutional funds from the U.S.Veterans Administration and by grants Al-10085 and Al-13550 fromthe National Institutes of Health. E.W. is a recipient of a ClinicalInvestigator Award from the U.S. Veterans Administration.

LITERATURE CITED1. Acetarin, J.-D., E. Carlemalm, and W. Villiger. 1986. Develop-

ment of new Lowicryl resins for embedding biological speci-mens at even lower temperatures. J. Microsc. (Oxford) 143:81-88.

2. Beachey, E. H., G. H. Stollerman, E. Y. Chiang, J. M. Seyer,and A. H. Kang. 1977. Purification and properties of M proteinextracted from group A streptococci with pepsin: covalentstructure of the amino terminal region of type 24 M protein. J.Exp. Med. 145:1469-1483.

3. Beijbom, L., U. Larsson, U. Kaveus, and H. Hebert. 1988.Structure analysis of fibrinogen by electron microscopy andimage processing. J. Ultrastruct. Mol. Struct. Res. 98:312-319.

4. Bendayan, M., A. Nanci, and F. W. K. Kan. 1987. Effect oftissue processing on colloidal gold cytochemistry. J. His-tochem. Cytochem. 35:983-996.

5. Carlemalm, E., W. Villiger, J. A. Hobot, J.-D. Acetarin, and E.Kellenberger. 1985. Low temperature embedding with Lowicrylresins: two new formulations and some applications. J. Microsc.(Oxford) 140:55-63.

6. Chhatwal, G. S., I. S. Dutra, and H. Blobel. 1985. Fibrinogenbinding inhibits the fixation of the third component of humancomplement on surface of groups A, B, C, and G streptococci.Microbiol. Immunol. 29:973-980.

7. Christensen, P., and V.-A. Oxelius. 1975. A reaction betweensome streptococci and IgA myeloma proteins. Acta Pathol.Microbiol. Scand. Sect. C 83:184-188.

8. Courtney, H. S., I. Ofek, W. A. Simpson, D. L. Hasty, and E. H.Beachey. 1986. The binding of Streptococcus pyogenes to soubleand insoluble fibronectin. Infect. Immun. 53:454-459.

9. Fischetti, V. A., K. F. Jones, and J. R. Scott. 1985. Sizevariations of the M protein in group A streptococci. J. Exp.Med. 161:1384-1401.

10. Fischetti, V. A., D. A. D. Parry, B. L. Trus, S. K. Hollingshead,J. R. Scott, and B. N. Manjula. 1988. Conformational charac-teristics of the complete sequence of group A streptococcal M6protein. Proteins Struct. Funct. Genet. 3:60-69.

11. Fox, E. N. 1974. M proteins of group A streptococci. Bacteriol.Rev. 38:57-86.

12. HavliUek, J., J. Pokorny, and H. Havlickova. 1987. Quantitativecharacter of fibrinogen uptake by M+ and M- variants ofStreptococcus pyogenies. Zentralbl. Bakteriol. Parasitenkd. In-fektionskr. Hyg. Abt. 1 Orig. Reihe A 265:1-11.

13. HavliUek, J., M. Ryc, J. Pokorny, H. Havlickova, and 0.Kuhnemund. 1985. Electron microscopic visualization of fibrin-ogen binding to group A streptococcus and its M protein-linkednature: competition with other plasma proteins, p. 79-81. In Y.Kimura, S. Kotani, and Y. Shiokawa (ed.), Recent advances instreptococci and streptococcal diseases. Reedbooks Ltd.,Bracknell, England.

14. Hollingshead, S. K., V. A. Fischetti, and J. R. Scott. 1986.Complete nucleotide sequence of type 6 M protein of the groupA streptococcus: repetitive structure and membrane anchor. J.Biol. Chem. 261:1677-1686.

15. Hryniewicz, W., B. Lipinski, and J. Jeljaszewicz. 1972. Nature ofthe interaction between M protein of Streptococcus pyogenesand fibrinogen. J. Infect. Dis. 125:626-630.

16. Kantor, F. 1965. Fibrinogen precipitation by streptococcal Mprotein. 1. Identity of the reactants and stoichiometry of thereaction. J. Exp. Med. 121:849-859.

17. Kantor, F. S., and R. M. Cole. 1959. A fibrinogen precipitatingfactor (FPF) of group A streptococci. Proc. Soc. Exp. Biol.Med. 102:146-150.

18. Kohler, W., and 0. Prokop. 1978. Relationship between hapto-globin and Streptococcus pyogenes T4 antigens. Nature(London) 271:373.

19. Kronvall, G. 1973. A surface component of A, C, and Gstreptococci with non-immune reactivity for immunoglobulin G.J. Immunol. 111:1401-1406.

20. Kronvall, G., L. Bjorck, E. B. Myhre, and L. W. Wannamaker.1979. Binding of aggregated ,B-microglobulin, lgG, IgA, andfibrinogen to group-A. -C, and -G streptococci with specialreference to streptococcal M protein, p. 74-76. In M. T. Parker(ed.), Pathogenic streptococci. Reedbooks Ltd., Chertsey, En-gland.

21. Kronvall, G., C. Schonbeck, and E. Myhre. 1979. Fibrinogenbinding structures in f-hemolytic streptococci group A, C, andG. Acta Pathol. Microbiol. Scand. Sect. B 87:303-310.

VOL. 57, 1989

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2404 RYC ET AL.

22. Kronvall, G., A. Simmons, E. B. Myhre, and S. Jonsson. 1979.Specific absorption of human serum albumin, immunoglobulinA and immunoglobulin G with selected strains of group A and Gstreptococci. Infect. Immun. 25:1-10.

23. Kuhnemund, O., J. Havlicek, H. Knoll, J. Sjoquist, and W.Kohler. 1985. Interaction of group A type 1 streptococcal Mprotein with fibrinogen. Acta Pathol. Microbiol. Scand. Sect. B93:201-209.

24. Lammler, C., G. S. Chhatwal, and H. Blobel. 1983. Variations inthe binding of mammalian fibrinogens to streptococci of dif-ferent animal origin. Med. Microbiol. Immunol. 172:191-196.

25. Lancefield, R. C. 1962. Current knowledge of type-specific Mantigens of group A streptococci. J. Immunol. 89:307-313.

26. Marder, V. J., C. W. Francis, and R. F. Doolittle. 1982.Fibrinogen structure and physiology, p. 145-163. In R. W.Colman, J. Hirsch, V. J. Marder, and E. W. Salzman (ed.).Hemostasis and thrombosis: basic principles and clinical prac-tice. J. B. Lippincott Co., Philadelphia.

27. Mouw, A. R., E. H. Beachey, and V. Burdett. 1988. Molecularevolution of streptococcal M protein: cloning and nucleotidesequence of the type 24 M protein gene and relation to othergenes of Streptococcus pyogenes. J. Bacteriol. 170:676-684.

28. Pancholi, V., and V. A. Fischetti. 1988. Isolation and character-ization of the cell-associated region of group A streptococcal M6protein. Infect. Immun. 170:2618-2624.

29. Phillips, G. N., Jr., P. F. Flicker, C. Cohen, B. N. Manjula, andV. A. Fischetti. 1981. Streptococcal M protein: alpha-helicalcoiled-coil structure and arrangement on the cell surface. Proc.Natl. Acad. Sci. USA 78:4689-4693.

30. Reutersward, A., H. Miorner, M. Wagner, and G. Kronvall.1985. Variations in binding of mammalian fibrinogens to strep-tococci groups A, B, C, E, G and to Staphylococcius aurieuis.Acta Pathol. Microbiol. Scand. Sect. B 93:77-82.

31. Reynolds, E. S. 1963. The use of lead citrate at high pH as anelectron-opaque stain in electron microscopy. J. Cell Biol.19:208-212.

32. Runehagen, A., C. Schonbeck, U. Hedner, B. Hessel, and G.Kronvall. 1981. Binding of fibrinogen degradation products to S.aureus and to ,3-hemolytic streptococci group A, C and G. Acta

Pathol. Microbiol. Scand. Sect. B 89:49-55.33. Ryc, M., B. Wagner, M. Wagner, P. Petras, and J. Havlicek.

1985. Binding of horse-spleen ferritin to group A streptococci.Microbios 44:261-270.

34. Scott, J. R., W. M. Pulliam, S. K. Hollingshead, and V. A.Fischetti. 1985. Relationship of M protein genes in group Astreptococci. Proc. Natl. Acad. Sci. USA 82:1822-1826.

35. Swanson, J., K. C. Hsu, and E. C. Gotschlich. 1969. Electronmicroscopic studies on streptococci: I. M antigen. J. Exp. Med.130:1063-1091.

36. Switalski, L. M., A. Ljungh, C. Ryden, K. Rubin, M. Hook, andT. Wadstrom. 1982. Binding of fibronectin to the surface ofgroup A, C, and G streptococci isolated from human infections.Eur. J. Clin. Microbiol. 1:383-387.

37. Tillet, W. S., and R. L. Garner. 1934. The agglutination ofhemolytic streptococci by plasma and fibrinogen. Bull. JohnsHopkins Hosp. 54:145-156.

38. Wagner, B., K. H. Schmidt, M. Wagner, and W. Kohler. 1986.Albumin bound to the surface of M protein-positive streptococciincreased their phagocytosis by human polymorphonuclear leu-kocytes in the absence of complement and bactericidal antibod-ies. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1Orig. Reihe A 261:432-446.

39. Whitnack, E., and E. H. Beachey. 1982. Antiopsonic activity offibrinogen bound to M protein on the surface of group Astreptococci. J. Clin. Invest. 69:1042-1045.

40. Whitnack, E., and E. H. Beachey. 1985. Biochemical andbiological properties of the binding of human fibrinogen to Mprotein in group A streptococci. J. Bacteriol. 164:350-358.

41. Whitnack, E., and E. H. Beachey. 1985. Inhibition of comple-ment-mediated opsonization and phagocytosis of Streptococcuspyogenes by D fragments of fibrinogen and fibrin bound to cellsurface M protein. J. Exp. Med. 162:1983-1997.

42. Whitnack, E., J. B. Dale, and E. H. Beachey. 1983. Streptococ-cal defences against host immune attack: the M protein-fi-brinogen interaction. Trans. Assoc. Am. Physicians 96:197-202.

43. Whitnack, E., J. B. Dale, and E. H. Beachey. 1984. Commonprotective antigens of group A streptococcal M proteins maskedby fibrinogen. J. Exp. Med. 159:1201-1212.

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