An Electron Microscopic Study of the Location of Peptidoglycan in Group A and C Streptococcal Cell Walls

Abstract

The morphological appearance of deproteinized Group A and C streptococcal walls after treatment by different procedures extracting teichoic acids and polysaccharides (formamide, hydrochloric acid, nitrous acid, trichloroacetic acid, sulphuric acid, sodium hydroxide and sodium deoxycholate) was compared with the content of teichoic acids and polysaccharides remaining in the treated walls. All procedures extracted teichoic acids almost completely, but polysaccharides were extracted to various degrees. The ultrastructural appearance of walls after these extractions still exhibited the triple-layered wall profile; only a reduction of thickness of the wall and of electron density of the layers occurred. Therz was no direct correlation between the reduction of rhamnose content and thickness of walls.The ultrastructural localization of peptidoglycan in the streptococcal walls was explored by means of the indirect immunoferritin technique using anti-peptidoglycan antibodies isolated from anti-Group A-variant antisera. Ferritin particles were bound predominantly to filamentous structures which protruded from both surfaces of peptidoglycan fragments and isolated walls. Peptidoglycan was also detected on the filamentous protrusions of whole cocci. These results contradict models of the streptococcal wall in which peptidoglycan forms the innermost layer and support a mosaic structure in which peptidoglycan forms a network of the peptidoglycan-polysaccharide complex. INTRODUCTIONThe chemical and morphological structures of the walls of Group A and C streptococci are very similar (Krause & McCarty, 1962;Cole, 1968). Group A streptococcal wall consists of four chemically defined components -proteins (M, T and R), polysaccharide, peptidoglycan and teichoic acid (Swanson et al., 1969). In ultrathin sections three morphologically distinct layers can usually be distinguished (Cole, 1968;Swanson et al., 1969; Wagner & Wagner, 1972~). The wall is composed of an electron-dense inner layer, a middle layer of medium electron density and an outer layer which in most strains is covered by filamentous protrusions. The morphological appearance of the wall, and the fact that proteins can easily be removed by proteolytic enzymes without affecting the viability of the streptococci (Lancefield, 1943) For the extraction studies, the bacteria were grown with shaking in Todd-Hewitt broth (Difco) for 4 h at 37 "C and then treated twice with trypsin (Difco; 0.1 %, w/v, in 0.1 M-phosphate buffer, pH 7.8) at 37 "C for 2 h. After treatment, the organisms were repeatedly washed in 0.1 M-phosphate buffer before being subjected to the different extraction procedures. For the immunoelectron microscopic studies, the bacteria were grown with shaking in Proteose broth at 37 "C overnight, then centrifuged and washed with R-K buffer (Ryter & Kellenberger, 1958).The presence of the Fc-binding factor in these strains was determined by the method of Christensen et al. (1976).Preparation of isolated walls and peptidoglycan. Bacterial suspensions were shaken with ...

Teichoic and teichuronic acids: biosynthesis, assembly, and location.

“…Under these conditions, the walls did not show a trilamellar structure, whereas this structure was regained together with wall teichoic acid on transfer to the permissive temperature. On the other hand, extraction of the walls of group A and group C streptococci with various reagents which removed from 20 to 95% of the rhamnose present did not alter the trilamellar appearance, although there were considerable reductions in thickness (169). Similar results were obtained with walls of S. faecalis (68,165) and L. arabinosus (13), from which the secondary polymers were extracted with trichloroacetic acid.…”

Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci

“…Identical results were obtained with choline-containing and choline-free cell walls examined with rabbit anti-C-Ps antiserum. Because this antiserum is known to react with peptidoglycan fragments (17,25), the experiments were repeated with monoclonal antibody specific for the phosphoryl-choline part of the C-Ps (16). In agreement with the previous results the monoclonal antibody bound to both sides of choline-containing cell walls but, as expected, not to the choline-free cell walls.…”

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

“…The RhaCWP is a major component in streptococcal species comprising about 40%–60% of the cell wall by weight (McCarty 1952 ; Krause and McCarty 1962a ; Krause 1963 ; Doran and Mattingly 1982 ). Correspondingly, cell wall thickness is visually reduced by 40%–50% after chemical extraction of the Group-specific carbohydrate (Swanson and Gotschlich 1973 ; Wagner and Wagner 1978 ). Recent mutagenesis studies confirmed the structural importance of streptococcal group antigens; complete loss of RhaCWP expression resulted in severe growth and cell division abnormalities (Fig.…”