Location of Polysaccharide on Chlamydia Psittaci by Silver-Methenamine Staining and Electron Microscopy SANITTAR P

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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 98195 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 rep- resents, or is a component of, the group antigen of these organisms.

Recently, we reported the characterization of is dependent upon the production of aldehyde a serologically active polysaccharide fraction groups after periodate oxidation of the poly- from the complement-fixing lipid group an- saccharide. The exposed aldehyde groups react tigen of Chlamydia trachomatis (2). The sero- with silver-methenamine causing a deposition logical activity of the polysaccharide was of silver at specific sites. The deposition is found to be sensitive to low concentrations of proportional to the numbers of reactive groups periodate (0.005 M sodium metaperiodate). The present and can be observed by the electron data also indicated that this polysaccharide microscope as discrete electron-dense spots antigen contained a 2-keto-3-deoxyoctanoic (granules). acid which appeared critical for the serological activity of the polysaccharide (Dhir et al., J. MATERIALS AND METHODS Immunol., 1972, in press). This acidic com- Organisms. Meningopneumonitis Cal-10 strain of pound was also readily oxidized by periodate psittacosis (3) was propagated in HeLa-M cell sus- with the production of aldehyde groups. The pension culture as described elsewhere (Dhir et al., J. present study was undertaken to determine Immunol., 1972, in press). Organisms were liberated the location of exposed polysaccharide of a by brief sonic treatment (Biosonik, Bronwill Scien- chlamydial (meningopneumonitis) organism tific Co., Rochester, N.Y., 120 w for 30 sec) from 7 x after ultrathin sectioning and staining for alde- 107 HeLa cells (70-80% infected). Cell debris was hyde groups by the silver-methenamine tech- removed by centrifugation at 500 x g for 10 min. nique. Purification of organisms. Organisms from the crude sonic extract were concentrated by high-speed Walker and Short (8) utilized a silver-meth- centrifugation (30,000 x g for 30 min at 4 C). The enamine technique to stain polysaccharide- deposits were suspended in 0.001 M phosphate containing material at the surface of bacteria buffer, pH 7.6, and the cycle of differential (500 and embedded and sectioned for electron micros- 30,000 x g) centrifugation was repeated. The final copy. The specificity of this staining procedure pellet was suspended in 2.0 ml of phosphate buffer 267 268 DHIR AND BOATMAN J. BACTERIOL. and treated with 1.0 ml of trichlorotrifluoroethane membrane was about 6 nm. Adjacent to the (Genetron) for 10 min in a mechanical shaker. The internal surface of the membrane there was in organisms were recovered in the upper phase by cen- some cells evidence of membrane-like trifugation. The organisms were further purified by frag- filtration on a Bio-Gel A-150m (0.5 by 20 cm col- ments of dimensions similar to the outer umn, Bio-Rad Laboratories, Richmond, Calif.). The membrane (Fig. 1, inset). Within the cyto- column was eluted with phosphate buffer. Fractions plasm of both small and large forms, less (1 ml) were collected and examined for chlamydial dense areas of fibrillar material were found particles by the electron microscope. Fractions con- and in certain planes of sectioned cells containing organisms were pooled and centrifuged to densations of electron-dense material (Fig. 1C). obtain a pellet. Treatment of sections with sodium deoxy- Fixation. The pellets were fixed either with 1.5% cholate before staining with uranyl acetate glutaraldehyde in 0.001 M phosphate buffer, pH 7.6, failed to alter the appearance of the sectioned overnight at 4 C and postfixed with 0.8% osmium organisms. tetroxide in phosphate buffer for 1 hr at 22 C, or fixed with buffered osmium tetroxide only. The pel- Silver-methenamine stain. Only a low lets were washed three times with phosphate buffer, level of silver staining was achieved when sec- dehydrated in ascending concentrations of alcohol, tions were stained with silver-methenamine and embedded in Epon 812 by the method of Luft either after treatment with sodium deoxycho- (6). Sections were cut with a Porter-Blum MT-2 ul- late or without prior periodate oxidation (Fig. tramicrotome and a diamond knife. Sections were 2). However, sections stained with silver- placed on nickle, 200-mesh parlodion-coated grids, methenamine after periodate oxidation treat- and in groups of five (i.e., one grid for each proce- ment showed a significant increase in the dure) were treated as follows: (i) stained with 3% overall staining of the sectioned uranyl acetate; (ii) extracted with 1% sodium deoxy- chlamydial cholate in phosphate buffer, pH 7.4, at 45 C for 4 hr, particles (Fig. 3). This enhancement of washed three times with distilled water, and stained staining was particularly evident at the pe- with uranyl acetate; (iii) stained with silver-methen- riphery of the sectioned organisms where a amine stain without prior periodate oxidation; (iv) double layer of small, regularly spaced silver stained with silver-methenamine with prior per- grains was observed. Although the majority of iodate oxidation; (v) treated with sodium deoxycho- silver grains observed by electron microscopy late, oxidized with periodate, and stained with were fairly uniform in size, the inner layer of silver-methenamine. this membrane profile often appeared denser Silver-methenamine staining procedure. Grids than the outer layer (Fig. 3, arrows). Sections were immersed in 0.005 M periodic acid for 20 min at 22 C, rinsed twice in distilled water, and stained for extracted with deoxycholate, oxidized with 70 min at 50 C in freshly made silver-methenamine periodate, and stained with silver-methena- solution of the following composition: 0.25% silver mine appeared similar to sections stained with nitrate, 10.0 ml; hexamethylenetetramine, 0.3 g; 5% silver-methenamine without periodate oxida- sodium borate, 8.0 ml; and distilled water, 12.0 ml. tion. After staining, grids were rinsed four times in dis- DISCUSSION tilled water, treated with 0.5% sodium thiosulfate for 2 min at 22 C, washed twice in distilled water, and The demonstration by silver-methenamine dried on filter paper. staining of periodate-sensitive polysaccharide Electron microscopy. The grids were observed in at the cell surface of sectioned chlamydial or- an RCA 3G electron microscope at 100 kv. ganisms is pertinent to the data obtained from chemical analysis of these organisms. Chlamy- RESULTS diae in general are known to have small Sectioned material: uranyl acetate amounts of carbohydrate, and the agent of staining. The profiles of the sectioned orga- meningopneumonitis in particular has about nisms (Fig. 1) varied according to the plane of 2% total carbohydrate, a third of which is hex- section, but measurements of cells cut in true osamine (4). A rather diffuse staining is ob- cross section (i.e., when the limiting mem- served by use of the silver-methenamine proce- brane of a cell was well defined) indicated that dure on bacteria which contain large amounts the population consisted of essentially two of carbohydrates (8). The discrete staining ob- types: a large form (reticulate body) about 500 served in the present study is probably due to nm in diameter and a smaller form (elementary the low content of carbohydrate in these orga- body) about 300 nm in diameter. There were nisms. four to five small forms for every large form. The observation that periodate-sensitive Cells of both types were mostly circular in polysaccharide is removed by extraction with cross section and displayed a distinct "unit" sodium deoxycholate suggests that this poly- type limiting membrane. The width of this saccharide is bound to lipids. Recent studies VOL. 111, 1972 LOCATION OF C. PSITTACI POLYSACCHARIDE 269

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FIG. 2. Electron micrograph of sectioned Chlamydia psittaci. Section stained with silver-methenamine without prior treatment with periodic acid. Silver stainingo, is generally low, compared to Fig. 3, particularly at the surface membrane.

FIG. 3. Electron micrograph of sectioned Chlamydia psittaci. Section treated with 0.005 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 VOL. 111, 1972 LOCATION OF C. PSITTACI POLYSACCHARIDE 271 have indicated that the group antigen of these will be necessary to study the permeability of organisms is a glycolipid, composed of an intact cell walls to radioactively labeled mole- acidic polysaccharide and long-chain fatty cules both before, and after, detergent treat- acids (2). The acidic component is sensitive to ment of the organisms. low concentrations of periodate. Furthermore, this group antigen can be extracted from the ACKNOWLEDGMENTS as organisms with sodium deoxycholate (5), This investigation was supported by Public Health well as with sodium lauryl sulfate (1). In view Service research grant 2-R01-EY00219 from the National of the fact that treatment of sections with so- Eye Institute and the training grant AI-206 from the Na- dium deoxycholate before staining greatly re- tional Institute of Allergy and Infectious Diseases. Acknowledgments are due to G. E. Kenny, Chairman, duced the development of silver deposits at Department of Pathobiology, School of Public Health and the cell surface layers, it is probable that the Community Medicine, Univ. of Washington, for his helpful polysaccharide-silver complex observed by suggestions and criticism. electron microscopy is the group antigen itself. The extent of carbohydrate removal by LITERATURE CITED deoxycholate is only apparent when followed 1. Benedict, A. A., and E. O'Brien. 1956. Antigenic studies by silver staining. If deoxycholate treatment is on the psittacosis-lymphogranuloma venereum group of followed by staining with uranyl acetate the viruses. II. Characterisation of complement fixing anti- appearances of these sectioned cells are iden- gens extracted from sodium lauryl sulfate. J. Immunol. tical with that of untreated cells. Jenkin et al. 76:293-300. 2. Dhir, S. P., G. E. Kenny, and J. T. Grayston. 1971. Char- (5) found that prior treatment with deoxycho- acterization of the group antigen of Chlamydia tra- late was most helpful in digesting cell cyto- chomatis. Infect. Immunity 4:725-730. plasmic material with trypsin during proce- 3. Francis, T., Jr., and T. P. Magill. 1938. An unidentified dures for the preparation of cell walls. This virus producing acute meningitis and pneumonitis in experimental animals. J. Exp. Med. 68:147-160. immediate change from trypsin resistance to 4. Jenkin, H. M. 1960. Preparation and properties of cell trypsin sensitivity suggests a chemical altera- walls of the agent of meningopneumonitis. J. Bacteriol. tion of the cell wall by deoxycholate. Tamura 80:639-647. et al. (7) observed that treatment with 1% so- 5. Jenkin, H. M., M. R. Ross, and J. W. Moulder. 1961. Species-specific antigens from cell walls of the agent of dium dodecyl sulfate dissolved the inner layer meningopneumonitis and feline pneumonitis. J. Bac- of isolated envelopes (cell walls) of these orga- teriol. 86:123-127. nisms. If the group glycolipid antigen is lo- 6. Luft, J. H. 1961. Improvements in epoxy resin embedding cated on the surface of these organisms in a methods. J. Biophys. Biochem Cytol. 9:409-414. 7. Tamura, A., A. Matsumoto, G. P. Manire, and N. Hi- double layer or as a structural component of gashi. 1971. Electron microscopic observations on the the membrane itself, as the findings of the structure of the envelopes of mature elementary bodies present study suggest, then a possible function and developmental reticulate forms of Chlamydia psit- for this glycolipid layer could be participation taci. J. Bacteriol. 105:355-360. 8. Walker, P. D., and J. Short. 1969. Location of bacterial in the control of cell membrane permeability. polysaccharide during various phases of growth. J. Bac- To obtain further insight into this problem it teriol. 98:1342-1354.