Deficient Mutants of Streptococcus salivarius Reveal that the

Vol. 165, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1986, p. 746-755 0021-9193/86/030746-10$02.00/0 Copyright C 1986, American Society for Microbiology

Negative Staining and Immunoelectron Microscopy of AdhesionDeficient Mutants of Streptococcus salivarius Reveal that the Adhesive Protein Antigens Are Separate Classes of Cell Surface Fibril ANTON H.

WEERKAMP,1* PAULINE S. HANDLEY,2 ADA BAARS,3 AND JAN W. SLOT3

Department of Oral Biology, Dental School, University of Groningen, 9713 AV Groningen, The Netherlands'; Department of Bacteriology and Virology, The Medical School, Manchester M13 9PT, England2; and Center for Electron Microscopy, Medical School, University of Utrecht, 3511 HG Utrecht, The Netherlands3 Received 19 June 1985/Accepted 5 November 1985

The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease-sensitive fibrils 91 and 72 nm in length, respectively. A third class of only very sparsely distributed short fibrils (63 nm) was observed on mutant HB-V51, which lacks both wall-associated AgB and AgC antigens. The identity of these fibrils and whether they are present on the wild type are not clear. The function of long, protease-resistant fibrils of 178 nm, which are also present on the wild-type strain, remains unknown.

antiphagocytic molecule (18) which also may be involved in the adhesion of the bacteria to human tracheal epithelial cells (2). The fuzzy coat has furthermore been reported to contain the R and T proteins (8). M protein-like structures with receptor activities for various serum proteins have been observed in these bacteria, as well as in group B, C, and G streptococci (10) among others. S. salivarius K+ cells carry an elaborate fuzzy coat (5) consisting of two morphologically distinct types of fibrils (6) and at least two categories of specific functional, proteinaceous compounds, or adhesins, mediating attachment to host surfaces and to various gram-negative oral bacteria, respectively (21, 23, 24). Two adhesins, the host attachment factor (antigen C; AgC) and the Veillonella-binding protein (AgB), respectively, were previously isolated from cell walls of S. salivarius HB and purified to apparent homogeneity, in addition to a third cell wall protein antigen (AgD) whose function is not known (22). It would therefore be of interest to attempt the correlation of morphological and functional studies of the cell surface. S. salivarius is a successful inhabitant of the human oral cavity which preferentially attaches to keratinized oral epithelial cells, reflecting its preference for the tonque dorsum (16). In addition, S. salivarius readily forms aggregates with

The involvement of surface appendages in the adhesion of a wide variety of indigenous and pathogenic bacteria to host

surfaces is well documented. Surface appendages which mediate the attachment of streptococci to host surfaces are often associated with a fibrillar layer outside the cell wall, historically termed the "fuzzy coat" on the basis of early observations of thin sections of cells. This morphologically indistinct layer is present in such organisms as Streptococcus pyogenes (2), Streptococcus salivarius (5), Streptococcus mitior (11), and Streptococcus mutans (13). The presence of proteinaceous compounds in this layer is indicated by the observation that protease treatment removes the fuzzy coat (2). More recent studies on the morphology of the fuzzy coat of S. salivarius Lancefield group K cells (6) showed that this layer consists of densely packed fibrils with a loose, amorphous appearance. Similar observations were subsequently made on strains of Streptococcus sanguis (7). However, little detailed knowledge is available on the fine structure and the chemical and functional components of these layers. The fuzzy coat of S. pyogenes contains the M protein, an *

Corresponding author. 746

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LOCALIZATION OF CELL SURFACE FIBRILLAR ANTIGENS

several oral anaerobic, gram-negative bacteria, which is suggested to enhance intergeneric ecological relationships in multicellular microbial conglomerates (21). In this paper we describe the localization of the S. salivarius adhesins by immunoelectron microscopy (IEM). We also present studies of the surface morphology of mutant strains specifically lacking these adhesins, using the negative-stain technique. A schematic picture of the cell surface based on these observations, and involving at least two functionally different fibril types, has been constructed. MATERIALS AND METHODS Bacterial strains and growth conditions. S. salivarius HB and mutant strains HB-7 and HB-V5 have been described previously (23, 24). Mutant strain HB-V51 was prepared from strain HB-V5 by nitrosoguanidine mutagenesis according to previously described procedures (23) and was unable to attach to buccal epithelial cells or to aggregate with saliva; in addition it was unable to coaggregate with veillonellae. Unless otherwise stated, the strains were grown in batch culture in Todd-Hewitt broth for 16 h at 37°C in air with 5%

CO2. Preparation of antisera. Monospecific polyclonal antisera against AgB, AgC, and AgD were prepared by immunoaffinity chromatography of whole antiserum against S. salivarius HB cell walls (24) on columns of the purified antigens immobilized on Affigel-10 (Bio-Rad Laboratories). The antigens were isolated from lysates of cell wall from the wild-type strain and purified by a series of procedures including ion-exchange and gel permeation chromatography, preparative isoelectric focusing, and affinity chromatography on concanavalin-Sepharose, as described in the accompanying paper (27). The immunoaffinity columns (6 by 1.0 cm) were preequilibrated with 0.1 M phosphate buffer (pH 7.3) containing 0.5 M NaCl. After application of the serum sample (4 ml), the columns were eluted with the same buffer until no UV-absorbing material was detected in the eluate. Bound antibodies were subsequently released by eluting the column with 0.1 M glycine hydrochloride buffer (pH 2.5). The pH of the eluate was quickly neutralized by the addition of solid Tris. The eluate was then either dialyzed and freeze dried or concentrated by ultrafiltration using an Amicon cell equipped with a PM30 filter (Amicon Benelux, Oosterhout, The Netherlands) and stored at -20°C in the presence of 1% bovine serum albumin. The antisera were tested for purity by crossed immunoelectrophoresis with crude cell wall digests of strain HB. Standard immunoelectrophoretic techniques in agarose gels using a Tris-Veronal buffer system (pH 8.6) were described previously (22). Bromophenol blue was used as a marker for electrophoresis in the first dimension. IEM. The subcellular location of antigens was tested using IEM on thin sections (~~ ~ ~ ~ ~ ~ t~ ~ ~ ~ ~.~ ~ ~ ~.~S~A.~ ~.~ ~ ~ ~ ~ ~ ~ ~ . 0

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FIG. 3. S. salivarius mutant strains deficient in host-associated adhesive functions, indirectly immunolabeled with anti-AgC serum and protein A-colloidal gold probe. (A) Strain HB-7; (B) strain HB-V51. Bar, 0.5 ,um.

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antiserum. No cross-reactivity was observed between any of the antisera. Comparison of the electrophoretic mobilities with those obtained with purified antigens moreover confirmed that the purified antisera reacted with AgB, AgC, and AgD (not shown). Although highly purified antigens were used to prepare the specific antisera, this procedure leaves a theoretical chance that the antisera contain antibody against other cell wall antigens which do not show up in crossed immunoelectrophoresis. Preliminary experiments using spot blotting as well as IEM with antiserum against lipoteichoic acid indicated that lipoteichoic acid was not associated with the purified antigens (not shown). Subcellular localization of protein antigens. Preliminary experiments, in which whole cells were immunolabeled with ferritin-coupled immunoglobulins before conventional embedding and sectioning, showed label only at the outer edge of the fibrillar coat, presumably because diffusion of the antibodies and label was hindered by this dense coat. Therefore immunolabeling of ultrathin cryosections of mildly fixed cells with colloidal gold particles complexed with Staphylococcus aureus coat protein A, a technique which has been applied to tissue (4) and recently to other bacteria (15, 19), was attempted to localize the antigens more precisely. Immunolabeling of thin sections of S. salivarius HB cells with monospecific immunoglobulins against AgC showed that this antigen is located almost exclusively in a region distal to the solid cell wall (Fig. 2). This layer would therefore correspond to the fibrillar layer or fuzzy coat observed in thin sections of cells conventionally stained with uranyl and lead salts (5). As a consequence of the staining

procedure applied in IEM, no structures could be seen associated with the colloidal gold particles. However, in most preparations the fibrillar layer was easily recognized as a clear zone surrounding the cell, which results from the exclusion of the gelatin in which the cells are embedded and which shows up as a grayish background. The colloidal gold particles sometimes appeared as bundles or tufts and were oriented in a radial fashion on the cell surface. Essentially no gold particles were found in the solid cell wall and cytoplasmic membrane regions. However, small clusters of label were visible randomly throughout the cytoplasmic area, suggesting a macromolecular association of the antigen. The amount of intracellular label of AgC varied somewhat between different batches of cells (Fig. 2) and may reflect a dependency on the growth phase. In contrast to the parent strain HB, little label was observed in the region distal to the solid cell wall of mutant HB-7 (Fig. 3A), which lacks AgC in solubilized cell wall preparations (22). In this mutant a remarkably heavy labeling of the cytoplasmic area was observed. The intracellular label was found in all cells and in different batches of cells. Although the label was sometimes distributed over the entire cytoplasmic region, the gold particles appeared preferentially associated with electrondense areas of the cytoplasm. Similar observations were made with mutant HB-V51, which lacks both AgB and AgC from the cell wall (Fig. 3B). Immunolabeling of the wild-type strain HB using antibodies against AgB resulted in the deposition of gold particles mainly in the same fibrillar layer distal to the solid cell wall (Fig. 4A). The density of the label appeared lower than that

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\A'4.KL14 FIG. 7. Diagrammatic representation of the surface structures observed by negative staining on the wild-type strain HB and its mutants. (A) Strain HB carries long (178 + 11 nm) and short (91 ± 5 nm) fibrils. (B) Strain HB-VS carries long (166 ± 11 nm) and short (72 ± 3 nm) fibrils. (C) Strain HB-7 carries only 90 ± 4-nm fibrils which are less dense than those of either strain HB or HB-V5. (D) Strain HB-V51 carries very sparse, short (63 ± 7 nm) fibrils only.

observed with AgC, more irregularly distributed, and preferentially located at the peripheral fringe of the fibrillar coat. Little label was observed in the solid cell wall, membrane, and cytoplasmic area. In contrast to AgC, the intracellular AgB label showed up as discrete gold particles and not as clusters. Mutant HB-V5, which specifically lacks wallassociated AgB (24), did not show label in the fibrillar layer upon treatment with antibodies against AgB (Fig. 4B). Strain HB-V5 reacted similarly to the wild-type strain when antiserum against AgC was applied. Only a weak labeling, similar to that found in the wild-type strain, was found in the cytoplasmic area of the mutant. S. salivarius HB was also treated with antibodies against AgD (Fig. 5). AgD appeared to reside mainly in the fibrillar layer, but label was also found in the cytoplasmic area, similar to AgC, and presumably also in the solid cell walL A negative control could not be carried out with AgD because no mutants were available which specifically lack this antigen. Negative staining. Whole cells of the wild-type strain HB and mutant strains were observed by negative staining in the electron microscope to compare the structural composition of the cell surfaces with the presence of particular protein antigens (Fig. 6). As shown previously (6), negatively stained cells of strain HB apparently carry two types of fibrils on the cell surface (Fig. 6A). These can be distin-

178

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guished from each other on the basis of their length, density, and protease sensitivity (6). The long, slender fibrils have a mean length of 178 ± 11 nm, are protease resistant, and are more sparsely distributed than the short fibrils. The shorter, more globular, densily packed fibrils (91 ± 5 nm) have an uneven outside edge (Fig. 6A) and are protease sensitive (6). Surface structure analysis of the adhesion-deficient mutants u'sing negative staining has revealed that the shorter fibrils consist of two or three separate fibril classes that can be distinguished from each other on the basis of length (Fig. 7). Mutant HB-V5 appeared to be very similar to strain HB, as it carries both long and short fibrils (Fig. 6B). The long fibrils (166 ± 11 nm) are not significantly shorter than those on strain HB (178 ± 11 nm). However, the short fibrils are only 72 ± 3 nm long and are significantly shorter by 19 nm (P < 0.05) than the 91 + 5-nm fibrils on strain HB. This difference in length suggests either that the 91-nm, fibrils have lost length or that the 72-nm fibrils represent a distinct class of fibrils obscured in the parent strain. These shorter fibrils have no measurable width when on the cell surface and have globular ends (Fig. 6B). Mutant HB-7 carries only short fibrils with a length of 90 ± 4 nm and has completely lost the longer, more sparsely distributed fibrils. These 90-nm fibrils are not significantly shorter than the 91-nm fibrils on the parent strain HB. Mutant HB-V51 carries very sparsely distributed fibrils which are 63 ± 7 nm long and have globular ends. The 178-, 91-, and 72-nm-long fibril classes have been lost (Fig. 6D). These 63-nm fibrils are significantly shorter (P < 0.05) than the 72-nm class of short fibrils and could represent a third structural and functional class of short fibrils, obscured in both strains HB-7 and HB-V5. Figure 8 is a composite diagram of all the classes of fibrils probably found on the wild-type strain HB.

DISCUSSION Cell walls of S. salivarius HB and most other Lancefield group K strains of this species contain three major proteinaceous components (22), at least two of which now appear to be associated with different structural classes of fibril on the surface of the cells. These fibrils form a densely packed, amorphous layer with an almost confluent outer edge (6), representing the characteristic fuzzy coat of S. salivarius K+ cells. A similar layer is also found in a variety of other streptococci (2, 4, 11, 13). A remarkable finding of this and our previous work (6, 22, 26) is that the fuzzy coat

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FIG. 8. Composite diagram to show the lengths and possible functions of some of the fibril classes on S. salivarius HB. Long, 178-nm fibrils: protease resistant; function(s) unknown. Short, 91-nm fibrils: protease sensitive; coaggregation adhesit AgB. Short, 72-nm fibrils: protease sensitive; -host-associated adhesin AgC. Short, 63-nm fibrils- protease sensitive; identity and function(s) unknown; actual presence on strain HB not established.

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appears to be composed of biochemically, antigenically, and structurally different fibrils which carry distinct adhesive functions. Each fibril class could represent single molecules of the respective protein antigen. The fibrillar nature of one of the antigens, AgC, was corroborated by the recent demonstration that this compound contains a high level of a-helix configuration (26). In an accompanying paper (27) we furthermore demonstrate that purified AgB and AgC have fibrillar configurations when observed in the electron microscope. AgC, which is a glycoprotein containing about 42% by weight of hexose and hexosamine, was shown to be involved in adhesion of bacteria to host surfaces and aggregation with saliva (22). AgB, which contains little carbohydrate, mediates coaggregation of the bacteria with oral Veillonella strains and various other gram-negative bacteria (21, 24). The biological functions of the third protein antigen, AgD, are so far not known. The presence of antigenically and functionally different surface appendages on the same cell was already demonstrated for oral strains of Actinomyces viscosus (1), but these structures appear to be very different from the fibrils studied here and would be classified as fimbriae according to the nomenclature applied by Handley and co-workers (6). In gram-negative bacteria, different piliassociated adhesins may also be present simultaneously (9). Obviously such a situation will broaden the opportunities of a bacterium to colonize a host. A comparison of the surface structures on negatively stained cells of parent and mutant strains and the localization of adhesins by IEM allow us to draw a schematic picture of the cell surface of S. salivarius K+ cells (Fig. 8). For a variety of reasons, it is postulated that AgC is represented by the 72-nm class of fibrils present on the cells and not by the long 178-nm fibrils. First, protease-treated cells still carry the long fibrils, have lost the short fibrils (6), and have simultaneously lost the host-associated adhesive function (23). Second, mutant HB-V5, which has normal amounts of wall-associated AgC, carries a 72-nm fibrillar fringe that is significantly shorter than that observed on both the wild-type strain HB and strain HB-7 (91 nm). Third, IEM showed that AgC label did not extend beyond the outside edge formed by the short fibrils. In addition, we show in an acconmpanying paper (27) that free, isolated AgC is only 87 nm long and is protease sensitive (22). It is therefore probable that in addition to losing the long fibrils, mutant HB-7 has also lost the shorter 72-nm class of fibrils. This loss is masked by the continued presence of the longer 90-nm fibrils responsible for coaggregation. This hypothesis is supported by ruthenium red-stained sections of strain HB-7 organisms which show a marked reduction in density of the short fibrils (unpublished data) consistent with the loss both of long fibrils and of the short 72-nm class of fibrils. The nature and possible function of the protease-resistant long (178-nm) fibrils and the reason for their simultaneous absence in mutants HB-7 and HB-V51 remain unknown. AgB is apparently represented by the longer type of short fibrils (91 nm), since these are still present in mutant HB-7 but not in strain HB-V5. This conclusion is supported by the IEM data, which suggest that AgB locates slightly more peripherally than AgC. It was noted previously that coaggregation reactions associated with AgB were less sensitive to protease treatment than reactions associated with AgC (22). Although most fibrils are finally removed from the cells by protease treatment, as judged by negative-stain electron microscopy, the short (72-nm) fibrils were lost more easily than the 91-nm fibrils (unpublished data). Thus, even upon

J. BACTERIOL.

prolonged protease treatment an amount of functionally active adhesin sufficient to cause the coaggregation reaction is still present on the cells. The observation that the sparsely distributed fibrils on mutant HB-V51 are significantly shorter than those on parent strain HB-V5 could, in a similar way, indicate the presence of yet another structural and functional class of protease-sensitive fibrils, obscured in both strains HB and HB-V5. A possible candidate for the 63-nm fibril class would be AgD, since cell walls of mutant HB-V51 contain amounts of this antigen comparable to those found in wild-type strain HB (unpublished data). Alternatively, the 63-ntn fibrils may represent structurally incomplete and functionally inactive remnants of one of the other fibril classes. The isolation of mutants specifically lacking AgD should permit the identification of this compound. Fibrils morphologically similar to those found in S. salivarius K+ have been observed on a variety of other oral streptococci (3, 7). Cell wall-associated protein antigens, some apparently involved in adhesion, are also present in these bacteria (12, 14). It therefore seems not unlikely that a similar mechanismn of carrying separate functions on specialized fibrils has evolved in the other species as well. Such a mechanism mnay facilitate the differential expression of adhesive fuhctions such as observed with S. salivarius HB under different growth conditions (25). However, the recent report by Fives-Taylor and Thompson (3) on a collection of mutants of S. sanguis FW213 which were defective in cell surface fibrils is in favor of a model in which activities mediated by different gene products may be associated with the same fibrils. A second important finding is that the genetic changes leading to the selective disappearance from the cell surface of either AgB or AgC seem to be expressed at different biochemical levels. Mutants defective in wall-associated AgC accutnulated AgC reactivity intracellularly, whereas no such accumulation was observed with AgB. Mutants defective in AgB, on the other hand, did not accumulate antigens in the cytoplasm. Unfortunately, very little is known about the genetics and the mechanisms of synthesis, assembly, and excretion of fibrillar proteins in gram-positive bacteria that co'uld help to explain the observations. The mutation in strain HB-7 may have led to the blocking or absence of a putative membrane receptor--mediated secretory apparatus, as was recently suggested for the secretion of alkaline phosphatase in a strain of Bacillus licheniformis (19). The accumulation of intracellular alkaline phosphatase in a nonsecretory mutant of this strain was demonstrated by colloidal gold immnunolabeling, analogous to our observations on S. salivarius. Mutants of this type could be very helpful in elucidating the complex mechanisms of fibril synthesis and assembly. ACKNOWLEDGMENTS We thank Alan Gibbs of the Department of Community Health, The Medical School, Manchester University, for the statistical analysis and Henny van der Mei, Department of Oral Biology, Groningen University, for assistance in preparing the specific antisera.

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