Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril

Abstract

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.

Description of Two Morphotypes of Peptostreptococcus micros

“…Streptococcus sanguis, for example, has adhesins (5, 13), fibrillar glycoproteins (20), and cell surface components related to aggregation, adherence, and hydrophobicity (19). Streptococcus salivanus expresses three different classes of fibrils, which have distinct adhesive functions (34,35). It has been suggested that subunits of the fibrils of these oral streptococci range in size from 70 to 90 kDa (19) to more than 300 kDa (20).…”

The electrophoretic softness of the surface of Staphylococcus epidermidis cells grown in a liquid medium and on a solid agar

“…Recently, we studied the softness of two oral streptococcal strains, Streptococcus salivarius HB and HBC12, by particulate microelectrophoresis in KCl solutions of varying ionic strengths (Bos et al, 1998). Electron microscopy of negatively stained organisms, and X-ray photoelectron spectroscopy, confirmed that strain HB had several classes of proteinaceous fibrils with lengths up to 178 nm on its outermost surface (Weerkamp et al, 1986), while variant HBC12 had a bald, peptidoglycanrich outer surface. The fibrillated strain HB appeared as relatively soft (1\λ l 1n4 nm) from analysis of its electrophoretic mobility according to Ohshima, while the bald variant HBC12 was hard (1\λ l 0n7 nm) because of its comparatively rigid, peptidoglycan-rich outer surface, characteristic of Gram-positive bacteria.…”

Molecular Strategies for Fimbrial Expression and Assembly

“…These fimbriae are documented in S. parasanguis (Fives- Taylor, 1982;Fachon-Kalweit et al, 1985), S. salivarius and S. songuis 12 (Handley et al, 1984(Handley et al, , 1985 Intermediate fimbriae are less than 150 nm in length, but their diameter has not been determined because of their ability to aggregate with each other. These fimbriae have been described for the sanguis streptococci (FivesTaylor, 1982); similar fimbriae are also present in the staphylococci (Weerkamp et al, 1986).…”

“…sanguis to SHA (Morris et al, 1987 (Weerkamp et al, 1986). A 200-kDa high-molecular-weight protein, Fapl, previously named Antigen I (Ag!…”