psittaci by Silver-Methenamine Staining and

Transcription

1 JOURNAL OF BACTERIOLOGY, July 1972, p Copyright 1972 American Society for Microbiology Vol. 111, No. 1 Printed in U.S.A. Location of Polysaccharide on Chlamydia psittaci by Silver-Methenamine Staining and Electron Microscopy SANITTAR P. DHIR AND EDWIN S. BOATMAN Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington Received for publication 21 March 1972 Previous serological studies have indicated that the group antigen of chlamydial organisms is composed of an acidic polysaccharide and a lipid component. The present study was undertaken in an effort to locate this polysaccharide complex by use of electron microscopy and a silver-methenamine marker. The meningopneumonitis strain of Chlamydia psittaci was propagated in HeLa-M cell culture. Organisms were purified by differential centrifugation, treatment with Genetron, and by gel filtration. After fixation and embedding, sections were obtained for electron microscopy. Sections were stained for carbohydrates with silver-methenamine. A double layer of regularly spaced silver grains of uniform size was observed at the periphery of the sectioned organisms tracing the contours of the surface membrane (cell wall). This intensity of staining was observed only when sections were oxidized with periodate prior to silver-methenamine staining. Prior treatment with 1% sodium deoxycholate resulted in a significant reduction in staining. It is considered probable that the periodate-sensitive polysaccharide found at the periphery of the organisms represents, or is a component of, the group antigen of these organisms. Recently, we reported the characterization of a serologically active polysaccharide fraction from the complement-fixing lipid group antigen of Chlamydia trachomatis (2). The serological activity of the polysaccharide was found to be sensitive to low concentrations of periodate (0.005 M sodium metaperiodate). The data also indicated that this polysaccharide antigen contained a 2-keto-3-deoxyoctanoic acid which appeared critical for the serological activity of the polysaccharide (Dhir et al., J. Immunol., 1972, in press). This acidic compound was also readily oxidized by periodate with the production of aldehyde groups. The present study was undertaken to determine the location of exposed polysaccharide of a chlamydial (meningopneumonitis) organism after ultrathin sectioning and staining for aldehyde groups by the silver-methenamine technique. Walker and Short (8) utilized a silver-methenamine technique to stain polysaccharidecontaining material at the surface of bacteria embedded and sectioned for electron microscopy. The specificity of this staining procedure 267 is dependent upon the production of aldehyde groups after periodate oxidation of the polysaccharide. The exposed aldehyde groups react with silver-methenamine causing a deposition of silver at specific sites. The deposition is proportional to the numbers of reactive groups present and can be observed by the electron microscope as discrete electron-dense spots (granules). MATERIALS AND METHODS Organisms. Meningopneumonitis Cal-10 strain of psittacosis (3) was propagated in HeLa-M cell suspension culture as described elsewhere (Dhir et al., J. Immunol., 1972, in press). Organisms were liberated by brief sonic treatment (Biosonik, Bronwill Scientific Co., Rochester, N.Y., 120 w for 30 sec) from 7 x 107 HeLa cells (70-80% infected). Cell debris was removed by centrifugation at 500 x g for 10 min. Purification of organisms. Organisms from the crude sonic extract were concentrated by high-speed centrifugation (30,000 x g for 30 min at 4 C). The deposits were suspended in M phosphate buffer, ph 7.6, and the cycle of differential (500 and 30,000 x g) centrifugation was repeated. The final pellet was suspended in 2.0 ml of phosphate buffer

2 268 DHIR AND BOATMAN J. BACTERIOL. and treated with 1.0 ml of trichlorotrifluoroethane (Genetron) for 10 min in a mechanical shaker. The organisms were recovered in the upper phase by centrifugation. The organisms were further purified by filtration on a Bio-Gel A-150m (0.5 by 20 cm column, Bio-Rad Laboratories, Richmond, Calif.). The column was eluted with phosphate buffer. Fractions (1 ml) were collected and examined for chlamydial particles by the electron microscope. Fractions containing organisms were pooled and centrifuged to obtain a pellet. Fixation. The pellets were fixed either with 1.5% glutaraldehyde in M phosphate buffer, ph 7.6, overnight at 4 C and postfixed with 0.8% osmium tetroxide in phosphate buffer for 1 hr at 22 C, or fixed with buffered osmium tetroxide only. The pellets were washed three times with phosphate buffer, dehydrated in ascending concentrations of alcohol, and embedded in Epon 812 by the method of Luft (6). Sections were cut with a Porter-Blum MT-2 ultramicrotome and a diamond knife. Sections were placed on nickle, 200-mesh parlodion-coated grids, and in groups of five (i.e., one grid for each procedure) were treated as follows: (i) stained with 3% uranyl acetate; (ii) extracted with 1% sodium deoxycholate in phosphate buffer, ph 7.4, at 45 C for 4 hr, washed three times with distilled water, and stained with uranyl acetate; (iii) stained with silver-methenamine stain without prior periodate oxidation; (iv) stained with silver-methenamine with prior periodate oxidation; (v) treated with sodium deoxycholate, oxidized with periodate, and stained with silver-methenamine. Silver-methenamine staining procedure. Grids were immersed in M periodic acid for 20 min at 22 C, rinsed twice in distilled water, and stained for 70 min at 50 C in freshly made silver-methenamine solution of the following composition: 0.25% silver nitrate, 10.0 ml; hexamethylenetetramine, 0.3 g; 5% sodium borate, 8.0 ml; and distilled water, 12.0 ml. After staining, grids were rinsed four times in distilled water, treated with 0.5% sodium thiosulfate for 2 min at 22 C, washed twice in distilled water, and dried on filter paper. Electron microscopy. The grids were observed in an RCA 3G electron microscope at 100 kv. RESULTS Sectioned material: uranyl acetate staining. The profiles of the sectioned organisms (Fig. 1) varied according to the plane of section, but measurements of cells cut in true cross section (i.e., when the limiting membrane of a cell was well defined) indicated that the population consisted of essentially two types: a large form (reticulate body) about 500 nm in diameter and a smaller form (elementary body) about 300 nm in diameter. There were four to five small forms for every large form. Cells of both types were mostly circular in cross section and displayed a distinct "unit" type limiting membrane. The width of this membrane was about 6 nm. Adjacent to the internal surface of the membrane there was in some cells evidence of membrane-like fragments of dimensions similar to the outer membrane (Fig. 1, inset). Within the cytoplasm of both small and large forms, less dense areas of fibrillar material were found and in certain planes of sectioned cells condensations of electron-dense material (Fig. 1C). Treatment of sections with sodium deoxycholate before staining with uranyl acetate failed to alter the appearance of the sectioned organisms. Silver-methenamine stain. Only a low level of silver staining was achieved when sections were stained with silver-methenamine either after treatment with sodium deoxycholate or without prior periodate oxidation (Fig. 2). However, sections stained with silvermethenamine after periodate oxidation treatment showed a significant increase in the overall staining of the sectioned chlamydial particles (Fig. 3). This enhancement of staining was particularly evident at the periphery of the sectioned organisms where a double layer of small, regularly spaced silver grains was observed. Although the majority of silver grains observed by electron microscopy were fairly uniform in size, the inner layer of this membrane profile often appeared denser than the outer layer (Fig. 3, arrows). Sections extracted with deoxycholate, oxidized with periodate, and stained with silver-methenamine appeared similar to sections stained with silver-methenamine without periodate oxidation. DISCUSSION The demonstration by silver-methenamine staining of periodate-sensitive polysaccharide at the cell surface of sectioned chlamydial organisms is pertinent to the data obtained from chemical analysis of these organisms. Chlamydiae in general are known to have small amounts of carbohydrate, and the agent of meningopneumonitis in particular has about 2% total carbohydrate, a third of which is hexosamine (4). A rather diffuse staining is observed by use of the silver-methenamine procedure on bacteria which contain large amounts of carbohydrates (8). The discrete staining observed in the present study is probably due to the low content of carbohydrate in these organisms. The observation that periodate-sensitive polysaccharide is removed by extraction with sodium deoxycholate suggests that this polysaccharide is bound to lipids. Recent studies

3 VOL. 111, * ji;27 *',' LOCATION OF C. PSITTACI POLYSACCHARIDE 269 fstf,l s Ut-F., S.*.$s Y + z FE.. B s'sf w r \va. }z 7 s,!l l2-,;..k -,A. 6;! i". -, ir il;vv.;*-... :-,.. :A.,- -4:. Z, N. ;.. g.+s 4-w: *' t J'*,e'M,\,2 io Downloaded from 'r a.; '. f ^ 0 5/Am I 0 I I -... ;i. FIG. 1. Electron micrograph of sectioned Chlamydia psittaci stained with uranyl acetate. Profiles of elementary bodies are shown (E) and one reticulate body (R) and condensations of electron-dense material (C). Inset: Membrane-like fragments (arrows) close to the well defined limiting membrane. on August 26, 2018 by guest

4 * 1.-'*.,* *34p* * I. v v t D * o, * - s e 5 4 w - b 0 6t ; * * * w%x*es%w e * t-- _, ;<'_ F. Z b.,: ; ; b ;* J'-t * itd t v t ', $ ';s ' -' 9 t '-.^. J S o s..*.!', *. ;; ;.*.^ w.;, ^ *''',',-X.'.,",'"''*-'"i z ' * e t;.; v'-b - s Zo \ * b - '' o.t>ent FIG. 2. Electron micrograph of sectioned Chlamydia psittaci. Section stained with silver-methenamine without prior treatment with periodic acid. Silver staining is generally low, compared to Fig. 3, particularly at the surface membrane. Downloaded from on August 26, 2018 by guest FIG. 3. Electron micrograph of sectioned Chlamydia psittaci. Section treated with M periodic acid and stained with silver-methenamine. Note well defined limiting membrane with double layer of silver grains (arrows indicate denser inner layer). 270

5 VOL. 111, 1972 LOCATION OF C. PSITTACI POLYSACCHARIDE 271 have indicated that the group antigen of these organisms is a glycolipid, composed of an acidic polysaccharide and long-chain fatty acids (2). The acidic component is sensitive to low concentrations of periodate. Furthermore, this group antigen can be extracted from the organisms with sodium deoxycholate (5), as well as with sodium lauryl sulfate (1). In view of the fact that treatment of sections with sodium deoxycholate before staining greatly reduced the development of silver deposits at the cell surface layers, it is probable that the polysaccharide-silver complex observed by electron microscopy is the group antigen itself. The extent of carbohydrate removal by deoxycholate is only apparent when followed by silver staining. If deoxycholate treatment is followed by staining with uranyl acetate the appearances of these sectioned cells are identical with that of untreated cells. Jenkin et al. (5) found that prior treatment with deoxycholate was most helpful in digesting cell cytoplasmic material with trypsin during procedures for the preparation of cell walls. This immediate change from trypsin resistance to trypsin sensitivity suggests a chemical alteration of the cell wall by deoxycholate. Tamura et al. (7) observed that treatment with 1% sodium dodecyl sulfate dissolved the inner layer of isolated envelopes (cell walls) of these organisms. If the group glycolipid antigen is located on the surface of these organisms in a double layer or as a structural component of the membrane itself, as the findings of the present study suggest, then a possible function for this glycolipid layer could be participation in the control of cell membrane permeability. To obtain further insight into this problem it will be necessary to study the permeability of intact cell walls to radioactively labeled molecules both before, and after, detergent treatment of the organisms. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant 2-R01-EY00219 from the National Eye Institute and the training grant AI-206 from the National Institute of Allergy and Infectious Diseases. Acknowledgments are due to G. E. Kenny, Chairman, Department of Pathobiology, School of Public Health and Community Medicine, Univ. of Washington, for his helpful suggestions and criticism. LITERATURE CITED 1. Benedict, A. A., and E. O'Brien Antigenic studies on the psittacosis-lymphogranuloma venereum group of viruses. II. Characterisation of complement fixing antigens extracted from sodium lauryl sulfate. J. Immunol. 76: Dhir, S. P., G. E. Kenny, and J. T. Grayston Characterization of the group antigen of Chlamydia trachomatis. Infect. Immunity 4: Francis, T., Jr., and T. P. Magill An unidentified virus producing acute meningitis and pneumonitis in experimental animals. J. Exp. Med. 68: Jenkin, H. M Preparation and properties of cell walls of the agent of meningopneumonitis. J. Bacteriol. 80: Jenkin, H. M., M. R. Ross, and J. W. Moulder Species-specific antigens from cell walls of the agent of meningopneumonitis and feline pneumonitis. J. Bacteriol. 86: Luft, J. H Improvements in epoxy resin embedding methods. J. Biophys. Biochem Cytol. 9: Tamura, A., A. Matsumoto, G. P. Manire, and N. Higashi Electron microscopic observations on the structure of the envelopes of mature elementary bodies and developmental reticulate forms of Chlamydia psittaci. J. Bacteriol. 105: Walker, P. D., and J. Short Location of bacterial polysaccharide during various phases of growth. J. Bacteriol. 98: