JOURNAL OF ULTRASTRUCTURE RESEARCH 67, 152-160 (1979)
Changes in the Ultrastructure of Chlamydia psittaci Produced by Treatment of the Host Cell with DEAE-Dextran and Cycloheximide P. SPEARS AND J. STORZ Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 Received July 31, 1978, and in revised form, January 15, 1979 The ultrastructure of two Chlamydia psittaci serotypes was investigated in infected L-cell monolayers fixed and embedded in situ. This preparation preserved the relationship of the parasite to the internal structure of the host cells. Chlamydial inclusions in cells treated with diethylaminoethyl-dextran (DEAE-D) and cycloheximide were compared to inclusions in untreated cells. C. psittaci serotype 1 had the same or larger yields of infectious progeny per infected cell in treated cells as in untreated cells. This serotype developed normal inclusions following both treatments. C. psittaci serotype 2 had decreased infectivity yields per infected cell and developed aberrant chlamydial forms in treated cell monolayers. Serotype 2 inclusions contained pycnotic chlamydial forms and numerous miniature reticulate bodies in cycloheximide-treated cells. In DEAE-Dtreated cells, some serotype 2 reticulate bodies were abnormally pleomorphic and contained amorphous material of intermediate electron density. Other reticulate bodies had lost electron density, degenerated, and contained only fragments of the reticulum. Maturation of reticulate bodies into infectious elementary bodies was greatly decreased in serotype 2 inclusions that contained aberrant forms.
The chlamydiae are prokaryotic pathogens that are obligately parasitic upon eukaryotic cells. A distinguishing feature of chlamydiae is the developmental cycle which involves a defined sequence of morphological and functional changes. The infectious extracellular form, the elementary body, and the shape of the intracellular inclusion can be observed by light microscopy, but ultrastructural studies are required for the morphological analysis of the normal developmental cycle and changes produced by antibiotics (Storz and Spears, 1977). Isolates of Chlamydia psittaci from cattle and sheep have been divided into serotypes 1 and 2 (Schachter et al., 1974, 1975). Serotype 1 strains are etiological agents of abortion, genital tract infection, and enteritis; serotype 2 strains cause polyarthritis, conjunctivitis, and sporadic bovine encephalomyelitis. The growth characteristics of C. psittaci serotypes 1 and 2 in mouse L cells that were treated with diethylaminoethyl-dextran (DEAE-D) and cycloheximide were previously studied (Spears, 152 0022-5320/79/050152-09502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
1978). These treatments are reported to increase chlamydial infectivity (Harrison, 1970; Hobson, et al., 1977). The polycation DEAE-D is thought to cause increased uptake of some types of chlamydiae by the host cells (Kuo and Grayston, 1976), which results in the development of more inclusions in DEAE-D-treated cells. DEAE-D treatment caused little change either in the number of inclusion-containing cells or in the yield of infectious progeny produced by a serotype 1 strain. In contrast, a serotype 2 strain infected nine times the number of cells in a DEAE-D-treated monolayer, but the yield per infected cell was much lower than in untreated cells. This indicates that although more of the serotype 2 elementary bodies began the developmental cycle, many of the resulting inclusions did not mature normally in the treated cells. A defect in the development of a serotype 2 strain was also suspected in cycloheximide-treated cells. Cycloheximide inhibits eukaryotic protein synthesis and presumably increases the soluble amino acid pool available to the prokaryotic parasite
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(Hatch, 1975). A serotype 1 strain formed threefold the number of inclusions and produced 30 times the infectious progeny in cycloheximide-treated cells. Under the same conditions, the serotype 2 strain formed about twice the number of inclusions, but only one-fifth the infectious progeny of untreated cells. In the present study, we examined the ultrastructure of C. psittaci serotypes I and 2 during their development in L cells treated with DEAE-D and cycloheximide. Infected monolayers were fixed and embedded in situ to preserve the native arrangement of chlamydial forms within the host cell's cytoplasm. The inclusions of the serotype 1 strain were ultrastructurally normal in the treated cells, but several types of abhormal-chlamydial forms were observed in serotype 2 inclusions. MATERIALS AND METHODS Mouse L cells were planted in 35-mm plastic petri dishes and infected with an inoculum of chlamydiae which had been prepared from freshly harvested, infected chicken embryo yolk sacs. The infected yolk sacs were homogenized in a Ten Broeck grinder with 1 ml of buffer per yolk sac. The suspension was centrifuged at 1000 rpm for 10 min to remove large debris and then diluted 1:100 in cell culture medium. Chlamydiae used were strain B577 from serotype 1 and stain LW-613 from serotype 2. Treatment of the cells with DEAE-D and cycloheximide was performed as described previously (Spears, 1978). Briefly, monolayers were rinsed three times with Dulbecco's phosphate-buffered saline containing 20 ~zg DEAE-D/ml prior to addition of the inoculum. DEAE-D was omitted from rinses for control monolayers. A 0.1-ml in0culum was adsorbed onto each monolayer for 2 hr at 37°C. The monolayers were washed once after adsorption and the culture medium, Eagle's minimal essential
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medium containing 5% fetal calf serum, 75 ttg vancomycin/ml, and 500 ttg streptomycin/ml, was added. Cycloheximide-treated monolayers received 2 /~g of the inhibitor per milliliter of culture medium. Monolayers were prepared for electron microscopy using a modification of the method of Brinkley and co-workers (1967). At intervals after infection, the monolayers were washed with Dulbecco's phosphate-buffered saline and fixed for 1 hr at 4°C with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.1 M sucrose at pH 7.4. After three rinses with 0.1 M sodium cacodylate buffer containing 0.25 M sucrose, the cells were fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer, rinsed in water, stained with 2% aqueous uranyl acetate, dehydrated with ethanol followed by hydroxypropyl methacrylate, and embedded in Epon. After polymerization of the epoxy, the monolayers were examined at 200x using an inverted phase microscope and areas of interest were marked. Squares of about 4 mm 2 were cut from the embedded monolayer and glued to Epon blocks with the cell side up. The blocks were trimmed and thin sections were cut approximately parallel to the monolayer with a Porter-Blum MT2-B ultramicrotome and stained with uranyl acetate and lead citrate (Reynolds, 1963). Sections were examined with a Hitachi HU-12 electron microscope at 75 kV. RESULTS
Effect of Cell Treatments on the Ultrastructure of C. psittaci Serotype 1 The inclusions of C. psittaci strain B577 developed with an entire margin and were closely packed with chlamydial forms (Fig. 1). In DEAE-D-treated, cycloheximidetreated, and untreated cells, mature inclusions that contained reticulate bodies, condensing forms, and elementary bodies were observed. There were few apparent differences between the chlamydial forms found in treated or untreated cells (Figs. 1, 2). Miniature reticulate bodies (MRB) were
FIG. 1. Chlamydial inclusion of strain B577 (serotype 1) in an L cell. This inclusion contains reticulate bodies (RB), a condensing form (CF), and an elementary body (EB). Miniature reticulate bodies (MRB) are present within the cell wall of the parent reticulate body. × 25 000. FIG. 2. An inclusion of strain B577 in an L cell treated with cycloheximide. Chlamydial forms are ultrastrucrurally normal. Elongated miniature reticulate bodies (MRB) are present within the cell wall of a reticulate body. × 31 000. Fro. 3. A diffuse portion of an inclusion formed by strain LW-613 in an L cell. This serotype 2 strain retained the inclusion membrane (arrows) as it spread through the host cell cytoplasm. × 37 000. Fro. 4. A chlamydial inclusion of strain LW-613 in an L cell. Reticulate bodies (RB), condensing forms (CF), and elementary bodies (EB) are ultrastructurally normal. Small, membrane vesicles (arrow) are abundant. A mitochondrion (M) is closely associated with the inclusion. × 25 000.
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frequently seen in serotype 1 inclusions. Some of the MRB were small, membranebound areas of chlamydial cytoplasm that clearly lay within the outer membrane of the parent reticulate body (Fig. 1). Other MRB were surrounded by both inner and outer membranes and were not seen to be associated with a parent reticulate body. The MRB were spherical or short cylindrical rods with rounded ends in untreated cells, but elongated forms were seen in some of the inclusions in cells treated with DEAE-D or cycloheximide (Fig. 2). Budding of the MRB from the parent reticulate body was not seen in any of the preparations. Empty membrane vesicles of varying sizes up to about one-quarter of the diameter of the elementary bodies were present between the chlamydial forms.
Effect of Cell Treatments on the Ultrastructure of C. psittaci Serotype 2 Strain LW-613 exhibited a different pattern of inclusion formation in untreated cells than did strain B577. Instead of forming an inclusion with an entire margin, the reticulate bodies of the serotype 2 strain often indented individually into the surrounding host cell cytoplasm, which gave the inclusion a highly irregular shape. The inclusion membrane was retained, however, so that the chlamydial forms were not free in the cytoplasm (Fig. 3). Serotype 2 inclusions often included a more compact area and a spreading, irregular area. Empty membrane vesicles were common within the inclusions (Fig. 4). There were few MRB formed, and those present had cell walls of their own, instead of lying within the cell wall of the parent reticulate body. In cycloheximide-treated cells, many strain LW-613 chlamydial forms were highly pycnotic. The chlamydial forms stained darkly and numerous MRB appeared within the cell walls of the altered reticulate bodies (Fig. 5). The reticulate bodies had amorphous areas and areas packed with dark-staining granules the size
of ribosomes. Segregated lighter patches containing filamentous material were seen in some aberrant reticulate bodies (Fig. 6). The rough endoplasmic reticulum of the host cell was dilated and the cytoplasm was more electron dense than that of neighboring uninfected cells. The mitochondria had condensed matrices and the membranes of the cristae were irregular and indistinct (Figs. 4, 6). Chlamydial condensing forms were occasionally seen but few or no elementary bodies were present. The pycnotic chlamydial forms were seen most often in the later time periods of the growth cycle. Earlier, when inclusions were small and contained only reticulate bodies, fewer abnormalities were noted. The serotype 2 strain exhibited several additional changes in DEAE-D-treated cells. The reticulate bodies were often enlarged and more electron lucent than normal (Fig. 7). The chlamydial forms were abnormally pleomorphic and the boundaries between reticulate bodies were less distinct (Fig. 8). Amorphous areas of intermediate electron density appeared within chlamydial forms. The cytoplasm of some chlamydial forms appeared degenerated and consisted of membranes and cell walls that enclosed a few strands of the previous reticulum (Fig. 9). Although the rough endoplasmic reticulum was dilated in host cells that contained these abnormal inclusions, the mitochondria were not as severely affected as were those in cells that contained the pycnotic inclusions. A few abnormal chlamydial forms could be seen in untreated cells, but the aberrant forms predominated in treated cells. In L cells that received both DEAE-D and cycloheximide treatments, a large proportion of the inclusions was affected and the defective chlamydial forms appeared earlier. Control monolayers that received uninfected yolk sac and that were treated with cycloheximide did not contain cells with the denser cytoplasm and altered mitochondria that were seen in cycloheximidetreated cells infected with strain LW-613.
ABERRANT FORMS OF Chlamydia psittaci DISCUSSION The species Chlamydiapsittaci has been described as forming inclusions whose limiting m e m b r a n e ruptures and releases the microorganisms into the cyoplasm of the host cell (Cutlip, 1970). This description probably came a b o u t from photomicrographs of inclusions t h a t had been sectioned tangentially to the inclusion membrane. In our study, fixation and embedding of the m o n o l a y e r in situ preserved the relationship between the chlamydial forms and the internal structures of the host cell. Although C. psittaci serotype 2 could indeed develop with interspersed areas of cytoplasm, the chlamydial forms were always surrounded by an inclusion membrane. T h e inclusions of the serotype 1 strain were more compact and h a d well-defined, entire margins. Individual serotype 1 reticulate bodies were not seen to spread t h r o u g h o u t the cytoplasm surrounding the inclusion as did serotype 2. Differences in inclusion m o r p h o l o g y between the two serotypes have also been seen in Giemsa-stained monolayers using light microscopy (Spears, 1978). The miniature reticulate b o d y (MRB) form of Chlamydia was described by Tanami and Y a m a d a (1973) from ultrastructural studies of a goat pneumonitis chlamydial strain. T h e budding of M R B which they described was not seen in our study. A distinction should be made between M R B that are b o u n d e d only by inner (cytoplas-
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mic) m e m b r a n e and those also b o u n d e d by outer m e m b r a n e (cell wall). T h e mechanism for the formation of these two types of M R B is probably different. T h e reason for the formation of M R B or the e m p t y m e m b r a n e vesicles is not known, although uncoordinated synthesis of chlamydial cytoplasmic m e m b r a n e or cell wall m a y be part of the mechanism. Previous studies have d e m o n s t r a t e d antigenic and pathogenic differences between C. psittaci serotypes 1 and 2 (Storz, 1966). T h e present investigation further elucidated biological differences between the two serotypes in their morphological response to D E A E - D and cycloheximide. Serotype 2 strains appeared to require more host-specific biochemical activities t h a n serotype 1 strains in order to complete the normal developmental cycle. T h e inclusions of C. psittaci serotype 1 were not changed in host cells t h a t h a d been treated with D E A E - D or cycloheximide. T h e complete normal developmental cycle was present in b o t h treated and untreated cells. T h e r e were no qualitative ultrastructural differences to explain the tenfold higher yield of infectious p r o g e n y from inclusions in cycloheximide-treated monolayers. Light microscopy has revealed t h a t the cells treated with cycloheximide contain larger inclusions t h a n do u n t r e a t e d cells (Hobson et al., 1977). T h e higher yield of serotype 1 from cycloheximide-treated cells m a y result from a larger volume of inclusion r a t h e r t h a n any qualitative differ-
FIG. 5. A chlamydial inclusion of strain LW-613 in a cycloheximide-treated cell. Chlamydial forms are pycnotic (PC) and there are numerous miniature reticulate bodies (MRB). The rough endoplasmic reticulum (RER) is dilated. An extensive array of membranes (ME) surrounds the area of the inclusion. The cytoplasm ofthe infected cell is more electron dense than that of the neighboring uninfected cell (arrow). × 13 000. Fro. 6. Pycnotic chlamydial forms (PC) in an inclusion formed by strain LW-613in a cycloheximide-treated cell. Amorphous areas (A) and areas containing fibrils (F) are present within the pycnotic reticulate bodies. Mitochondria (M) have condensed matrices and the membranes of the cristae are altered. × 29 000. FIG. 7. Enlarged, pleomorphic reticulate bodies (PRB) within an inclusion of strain LW-613in a DEAE-Dtreated L cell. The granulofibrillar structure is less dense and areas (arrow) are devoid of the granular components (ribosomes). × 21 000. FIG. 8. An inclusion of strain LW-613 in an L cell treated with DEAE-D. The abnormally pleomorphic reticulate bodies contain amorphous material (A). × 48 000. Fro. 9. An inclusion of strain LW-613 in an L cell treated with DEAE-D. Degenerated reticulate bodies contain a few strands of the previous reticulum (S). x 60 000.
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ence. The ability of the serotype I strain to use the nutritional environment provided by inhibition of host cell protein synthesis is important in development of such large inclusions. The decreased yields of infectious progeny from C. psittaci serotype 2 grown in cells treated with DEAE-D or cycloheximide can be explained by the aberrant chlamydial forms observed in the present study. The pycnotic forms in cycloheximide-treated monolayers were not caused by a specific effect of the cycloheximide, since this type of inclusionwas also seen in DEAE-D treated cells and infrequently in untreated cells. The configuration of the mitochondria in host cells that contained pycnotic inclusions suggested that the eukaryotic cells had received an injury and perhaps were no longer able to supply the parasite with the energy it required. The chlamydiae probably contributed to the injury of the host cells since control cell monolayers which received uninfected yolk sac suspensions did not show the same changes, even though degenerating cells were present. While chlamydiae multiply in balance with a normal host cell (Moulder, 1974), the serotype 2 strains may overtax a stressed host, since this serotype appears to require more host-specific synthesis than serotype 1. The effect of DEAE-D treatment on serotype 2 inclusions was more specific than the effect of cycloheximide since the type of change produced was not seen unless cells were treated with DEAED. The increased polymorphism of the chlamydial forms in DEAE-D-treated cells implies defects in the cell wall of the parR-
site. The abnormalities, like those produced by cycloheximide, may arise from a combination of damage to the host cell's ability to produce substances required by serotype 2 strains and the extra demands placed on the cell by the chlamydiae. We thank Mary L. Hegedus for her excellent assistance in specimen preparation and ultramicrotomy. This study was supported by Grant AI-08420 from the National Institute of Allergy and Infectious Diseases, by the Colorado Agricultural Experiment Station through Western Research Project W-112, and by the Dr. Tracy Rhodes Research Fund. This work was part of a dissertation submitted by P. Spears in partial fulfillment of the requirements for the Ph.D. degree in microbiology from Colorado State University. Journal Article 2380 of the Colorado Agricultural Experiment Station. REFERENCES BRINKLEY, B. R., MURPHY, M., AND RICHARDSON,L. C. {1967) J. Cell Biol. 35, 279-283. CUTLIP, R. C. (1970) Infec. Immunity 1, 499-502. HARRISON, M. J. (1970) AuNt. J. Exp. Biol. Med. Sci. 48, 207-213. HATCH, T. P. {1975) Infec. Immunity 12, 211-220. HOBSON, D., JOHNSON, F. W. A., AND BYNG, R. E. (1977) J. Comp. Pathol. 87, 155-159. I~uo, C. C., AND GRAYSTON, J. T. (1976) Infec. Immunity 13, 1103-1109. MOULDER, J. W. (1974) J. Infec. Dis. 130, 300-306. REYNOLDS, E. S. {1963) J. Cell Biol. 17, 208-213. SCHACHTER,J., BANKS,J., SUGG,N., SUNG, M., STORZ, J., AND MEYER, K. F. (1974) Infec. Immunity 9, 9294. SCHACHTER,J., BANKS,J., SUGG,N., SUNG,M., STORZ, J., AND MEYER, K. F. (1975) Infec. Immunity 11, 904-907. SPEARS, P. (1978) Ph.D. thesis, Colorado State University, Fort Collins. STORZ,J. (1966) J. Comp. Pathol. 76, 351-362. STORZ, J., AND SPEARS, P. (1977) Curr. Top. Microbiol. Immunol. 76, 167-214. TANAMI, Y., AND YAMADA,Y. (1973) J. Bacteriol. 114, 408-412.
Changes in the Ultrastructure of Chlamydia psittaci Produced by Treatment of the Host Cell with DEAE-Dextran and Cycloheximide P. SPEARS AND J. STORZ Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 Received July 31, 1978, and in revised form, January 15, 1979 The ultrastructure of two Chlamydia psittaci serotypes was investigated in infected L-cell monolayers fixed and embedded in situ. This preparation preserved the relationship of the parasite to the internal structure of the host cells. Chlamydial inclusions in cells treated with diethylaminoethyl-dextran (DEAE-D) and cycloheximide were compared to inclusions in untreated cells. C. psittaci serotype 1 had the same or larger yields of infectious progeny per infected cell in treated cells as in untreated cells. This serotype developed normal inclusions following both treatments. C. psittaci serotype 2 had decreased infectivity yields per infected cell and developed aberrant chlamydial forms in treated cell monolayers. Serotype 2 inclusions contained pycnotic chlamydial forms and numerous miniature reticulate bodies in cycloheximide-treated cells. In DEAE-Dtreated cells, some serotype 2 reticulate bodies were abnormally pleomorphic and contained amorphous material of intermediate electron density. Other reticulate bodies had lost electron density, degenerated, and contained only fragments of the reticulum. Maturation of reticulate bodies into infectious elementary bodies was greatly decreased in serotype 2 inclusions that contained aberrant forms.
The chlamydiae are prokaryotic pathogens that are obligately parasitic upon eukaryotic cells. A distinguishing feature of chlamydiae is the developmental cycle which involves a defined sequence of morphological and functional changes. The infectious extracellular form, the elementary body, and the shape of the intracellular inclusion can be observed by light microscopy, but ultrastructural studies are required for the morphological analysis of the normal developmental cycle and changes produced by antibiotics (Storz and Spears, 1977). Isolates of Chlamydia psittaci from cattle and sheep have been divided into serotypes 1 and 2 (Schachter et al., 1974, 1975). Serotype 1 strains are etiological agents of abortion, genital tract infection, and enteritis; serotype 2 strains cause polyarthritis, conjunctivitis, and sporadic bovine encephalomyelitis. The growth characteristics of C. psittaci serotypes 1 and 2 in mouse L cells that were treated with diethylaminoethyl-dextran (DEAE-D) and cycloheximide were previously studied (Spears, 152 0022-5320/79/050152-09502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
1978). These treatments are reported to increase chlamydial infectivity (Harrison, 1970; Hobson, et al., 1977). The polycation DEAE-D is thought to cause increased uptake of some types of chlamydiae by the host cells (Kuo and Grayston, 1976), which results in the development of more inclusions in DEAE-D-treated cells. DEAE-D treatment caused little change either in the number of inclusion-containing cells or in the yield of infectious progeny produced by a serotype 1 strain. In contrast, a serotype 2 strain infected nine times the number of cells in a DEAE-D-treated monolayer, but the yield per infected cell was much lower than in untreated cells. This indicates that although more of the serotype 2 elementary bodies began the developmental cycle, many of the resulting inclusions did not mature normally in the treated cells. A defect in the development of a serotype 2 strain was also suspected in cycloheximide-treated cells. Cycloheximide inhibits eukaryotic protein synthesis and presumably increases the soluble amino acid pool available to the prokaryotic parasite
ABERRANT FORMS OF
(Hatch, 1975). A serotype 1 strain formed threefold the number of inclusions and produced 30 times the infectious progeny in cycloheximide-treated cells. Under the same conditions, the serotype 2 strain formed about twice the number of inclusions, but only one-fifth the infectious progeny of untreated cells. In the present study, we examined the ultrastructure of C. psittaci serotypes I and 2 during their development in L cells treated with DEAE-D and cycloheximide. Infected monolayers were fixed and embedded in situ to preserve the native arrangement of chlamydial forms within the host cell's cytoplasm. The inclusions of the serotype 1 strain were ultrastructurally normal in the treated cells, but several types of abhormal-chlamydial forms were observed in serotype 2 inclusions. MATERIALS AND METHODS Mouse L cells were planted in 35-mm plastic petri dishes and infected with an inoculum of chlamydiae which had been prepared from freshly harvested, infected chicken embryo yolk sacs. The infected yolk sacs were homogenized in a Ten Broeck grinder with 1 ml of buffer per yolk sac. The suspension was centrifuged at 1000 rpm for 10 min to remove large debris and then diluted 1:100 in cell culture medium. Chlamydiae used were strain B577 from serotype 1 and stain LW-613 from serotype 2. Treatment of the cells with DEAE-D and cycloheximide was performed as described previously (Spears, 1978). Briefly, monolayers were rinsed three times with Dulbecco's phosphate-buffered saline containing 20 ~zg DEAE-D/ml prior to addition of the inoculum. DEAE-D was omitted from rinses for control monolayers. A 0.1-ml in0culum was adsorbed onto each monolayer for 2 hr at 37°C. The monolayers were washed once after adsorption and the culture medium, Eagle's minimal essential
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medium containing 5% fetal calf serum, 75 ttg vancomycin/ml, and 500 ttg streptomycin/ml, was added. Cycloheximide-treated monolayers received 2 /~g of the inhibitor per milliliter of culture medium. Monolayers were prepared for electron microscopy using a modification of the method of Brinkley and co-workers (1967). At intervals after infection, the monolayers were washed with Dulbecco's phosphate-buffered saline and fixed for 1 hr at 4°C with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.1 M sucrose at pH 7.4. After three rinses with 0.1 M sodium cacodylate buffer containing 0.25 M sucrose, the cells were fixed with 1% OsO4 in 0.1 M sodium cacodylate buffer, rinsed in water, stained with 2% aqueous uranyl acetate, dehydrated with ethanol followed by hydroxypropyl methacrylate, and embedded in Epon. After polymerization of the epoxy, the monolayers were examined at 200x using an inverted phase microscope and areas of interest were marked. Squares of about 4 mm 2 were cut from the embedded monolayer and glued to Epon blocks with the cell side up. The blocks were trimmed and thin sections were cut approximately parallel to the monolayer with a Porter-Blum MT2-B ultramicrotome and stained with uranyl acetate and lead citrate (Reynolds, 1963). Sections were examined with a Hitachi HU-12 electron microscope at 75 kV. RESULTS
Effect of Cell Treatments on the Ultrastructure of C. psittaci Serotype 1 The inclusions of C. psittaci strain B577 developed with an entire margin and were closely packed with chlamydial forms (Fig. 1). In DEAE-D-treated, cycloheximidetreated, and untreated cells, mature inclusions that contained reticulate bodies, condensing forms, and elementary bodies were observed. There were few apparent differences between the chlamydial forms found in treated or untreated cells (Figs. 1, 2). Miniature reticulate bodies (MRB) were
FIG. 1. Chlamydial inclusion of strain B577 (serotype 1) in an L cell. This inclusion contains reticulate bodies (RB), a condensing form (CF), and an elementary body (EB). Miniature reticulate bodies (MRB) are present within the cell wall of the parent reticulate body. × 25 000. FIG. 2. An inclusion of strain B577 in an L cell treated with cycloheximide. Chlamydial forms are ultrastrucrurally normal. Elongated miniature reticulate bodies (MRB) are present within the cell wall of a reticulate body. × 31 000. Fro. 3. A diffuse portion of an inclusion formed by strain LW-613 in an L cell. This serotype 2 strain retained the inclusion membrane (arrows) as it spread through the host cell cytoplasm. × 37 000. Fro. 4. A chlamydial inclusion of strain LW-613 in an L cell. Reticulate bodies (RB), condensing forms (CF), and elementary bodies (EB) are ultrastructurally normal. Small, membrane vesicles (arrow) are abundant. A mitochondrion (M) is closely associated with the inclusion. × 25 000.
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frequently seen in serotype 1 inclusions. Some of the MRB were small, membranebound areas of chlamydial cytoplasm that clearly lay within the outer membrane of the parent reticulate body (Fig. 1). Other MRB were surrounded by both inner and outer membranes and were not seen to be associated with a parent reticulate body. The MRB were spherical or short cylindrical rods with rounded ends in untreated cells, but elongated forms were seen in some of the inclusions in cells treated with DEAE-D or cycloheximide (Fig. 2). Budding of the MRB from the parent reticulate body was not seen in any of the preparations. Empty membrane vesicles of varying sizes up to about one-quarter of the diameter of the elementary bodies were present between the chlamydial forms.
Effect of Cell Treatments on the Ultrastructure of C. psittaci Serotype 2 Strain LW-613 exhibited a different pattern of inclusion formation in untreated cells than did strain B577. Instead of forming an inclusion with an entire margin, the reticulate bodies of the serotype 2 strain often indented individually into the surrounding host cell cytoplasm, which gave the inclusion a highly irregular shape. The inclusion membrane was retained, however, so that the chlamydial forms were not free in the cytoplasm (Fig. 3). Serotype 2 inclusions often included a more compact area and a spreading, irregular area. Empty membrane vesicles were common within the inclusions (Fig. 4). There were few MRB formed, and those present had cell walls of their own, instead of lying within the cell wall of the parent reticulate body. In cycloheximide-treated cells, many strain LW-613 chlamydial forms were highly pycnotic. The chlamydial forms stained darkly and numerous MRB appeared within the cell walls of the altered reticulate bodies (Fig. 5). The reticulate bodies had amorphous areas and areas packed with dark-staining granules the size
of ribosomes. Segregated lighter patches containing filamentous material were seen in some aberrant reticulate bodies (Fig. 6). The rough endoplasmic reticulum of the host cell was dilated and the cytoplasm was more electron dense than that of neighboring uninfected cells. The mitochondria had condensed matrices and the membranes of the cristae were irregular and indistinct (Figs. 4, 6). Chlamydial condensing forms were occasionally seen but few or no elementary bodies were present. The pycnotic chlamydial forms were seen most often in the later time periods of the growth cycle. Earlier, when inclusions were small and contained only reticulate bodies, fewer abnormalities were noted. The serotype 2 strain exhibited several additional changes in DEAE-D-treated cells. The reticulate bodies were often enlarged and more electron lucent than normal (Fig. 7). The chlamydial forms were abnormally pleomorphic and the boundaries between reticulate bodies were less distinct (Fig. 8). Amorphous areas of intermediate electron density appeared within chlamydial forms. The cytoplasm of some chlamydial forms appeared degenerated and consisted of membranes and cell walls that enclosed a few strands of the previous reticulum (Fig. 9). Although the rough endoplasmic reticulum was dilated in host cells that contained these abnormal inclusions, the mitochondria were not as severely affected as were those in cells that contained the pycnotic inclusions. A few abnormal chlamydial forms could be seen in untreated cells, but the aberrant forms predominated in treated cells. In L cells that received both DEAE-D and cycloheximide treatments, a large proportion of the inclusions was affected and the defective chlamydial forms appeared earlier. Control monolayers that received uninfected yolk sac and that were treated with cycloheximide did not contain cells with the denser cytoplasm and altered mitochondria that were seen in cycloheximidetreated cells infected with strain LW-613.
ABERRANT FORMS OF Chlamydia psittaci DISCUSSION The species Chlamydiapsittaci has been described as forming inclusions whose limiting m e m b r a n e ruptures and releases the microorganisms into the cyoplasm of the host cell (Cutlip, 1970). This description probably came a b o u t from photomicrographs of inclusions t h a t had been sectioned tangentially to the inclusion membrane. In our study, fixation and embedding of the m o n o l a y e r in situ preserved the relationship between the chlamydial forms and the internal structures of the host cell. Although C. psittaci serotype 2 could indeed develop with interspersed areas of cytoplasm, the chlamydial forms were always surrounded by an inclusion membrane. T h e inclusions of the serotype 1 strain were more compact and h a d well-defined, entire margins. Individual serotype 1 reticulate bodies were not seen to spread t h r o u g h o u t the cytoplasm surrounding the inclusion as did serotype 2. Differences in inclusion m o r p h o l o g y between the two serotypes have also been seen in Giemsa-stained monolayers using light microscopy (Spears, 1978). The miniature reticulate b o d y (MRB) form of Chlamydia was described by Tanami and Y a m a d a (1973) from ultrastructural studies of a goat pneumonitis chlamydial strain. T h e budding of M R B which they described was not seen in our study. A distinction should be made between M R B that are b o u n d e d only by inner (cytoplas-
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mic) m e m b r a n e and those also b o u n d e d by outer m e m b r a n e (cell wall). T h e mechanism for the formation of these two types of M R B is probably different. T h e reason for the formation of M R B or the e m p t y m e m b r a n e vesicles is not known, although uncoordinated synthesis of chlamydial cytoplasmic m e m b r a n e or cell wall m a y be part of the mechanism. Previous studies have d e m o n s t r a t e d antigenic and pathogenic differences between C. psittaci serotypes 1 and 2 (Storz, 1966). T h e present investigation further elucidated biological differences between the two serotypes in their morphological response to D E A E - D and cycloheximide. Serotype 2 strains appeared to require more host-specific biochemical activities t h a n serotype 1 strains in order to complete the normal developmental cycle. T h e inclusions of C. psittaci serotype 1 were not changed in host cells t h a t h a d been treated with D E A E - D or cycloheximide. T h e complete normal developmental cycle was present in b o t h treated and untreated cells. T h e r e were no qualitative ultrastructural differences to explain the tenfold higher yield of infectious p r o g e n y from inclusions in cycloheximide-treated monolayers. Light microscopy has revealed t h a t the cells treated with cycloheximide contain larger inclusions t h a n do u n t r e a t e d cells (Hobson et al., 1977). T h e higher yield of serotype 1 from cycloheximide-treated cells m a y result from a larger volume of inclusion r a t h e r t h a n any qualitative differ-
FIG. 5. A chlamydial inclusion of strain LW-613 in a cycloheximide-treated cell. Chlamydial forms are pycnotic (PC) and there are numerous miniature reticulate bodies (MRB). The rough endoplasmic reticulum (RER) is dilated. An extensive array of membranes (ME) surrounds the area of the inclusion. The cytoplasm ofthe infected cell is more electron dense than that of the neighboring uninfected cell (arrow). × 13 000. Fro. 6. Pycnotic chlamydial forms (PC) in an inclusion formed by strain LW-613in a cycloheximide-treated cell. Amorphous areas (A) and areas containing fibrils (F) are present within the pycnotic reticulate bodies. Mitochondria (M) have condensed matrices and the membranes of the cristae are altered. × 29 000. FIG. 7. Enlarged, pleomorphic reticulate bodies (PRB) within an inclusion of strain LW-613in a DEAE-Dtreated L cell. The granulofibrillar structure is less dense and areas (arrow) are devoid of the granular components (ribosomes). × 21 000. FIG. 8. An inclusion of strain LW-613 in an L cell treated with DEAE-D. The abnormally pleomorphic reticulate bodies contain amorphous material (A). × 48 000. Fro. 9. An inclusion of strain LW-613 in an L cell treated with DEAE-D. Degenerated reticulate bodies contain a few strands of the previous reticulum (S). x 60 000.
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ence. The ability of the serotype I strain to use the nutritional environment provided by inhibition of host cell protein synthesis is important in development of such large inclusions. The decreased yields of infectious progeny from C. psittaci serotype 2 grown in cells treated with DEAE-D or cycloheximide can be explained by the aberrant chlamydial forms observed in the present study. The pycnotic forms in cycloheximide-treated monolayers were not caused by a specific effect of the cycloheximide, since this type of inclusionwas also seen in DEAE-D treated cells and infrequently in untreated cells. The configuration of the mitochondria in host cells that contained pycnotic inclusions suggested that the eukaryotic cells had received an injury and perhaps were no longer able to supply the parasite with the energy it required. The chlamydiae probably contributed to the injury of the host cells since control cell monolayers which received uninfected yolk sac suspensions did not show the same changes, even though degenerating cells were present. While chlamydiae multiply in balance with a normal host cell (Moulder, 1974), the serotype 2 strains may overtax a stressed host, since this serotype appears to require more host-specific synthesis than serotype 1. The effect of DEAE-D treatment on serotype 2 inclusions was more specific than the effect of cycloheximide since the type of change produced was not seen unless cells were treated with DEAED. The increased polymorphism of the chlamydial forms in DEAE-D-treated cells implies defects in the cell wall of the parR-
site. The abnormalities, like those produced by cycloheximide, may arise from a combination of damage to the host cell's ability to produce substances required by serotype 2 strains and the extra demands placed on the cell by the chlamydiae. We thank Mary L. Hegedus for her excellent assistance in specimen preparation and ultramicrotomy. This study was supported by Grant AI-08420 from the National Institute of Allergy and Infectious Diseases, by the Colorado Agricultural Experiment Station through Western Research Project W-112, and by the Dr. Tracy Rhodes Research Fund. This work was part of a dissertation submitted by P. Spears in partial fulfillment of the requirements for the Ph.D. degree in microbiology from Colorado State University. Journal Article 2380 of the Colorado Agricultural Experiment Station. REFERENCES BRINKLEY, B. R., MURPHY, M., AND RICHARDSON,L. C. {1967) J. Cell Biol. 35, 279-283. CUTLIP, R. C. (1970) Infec. Immunity 1, 499-502. HARRISON, M. J. (1970) AuNt. J. Exp. Biol. Med. Sci. 48, 207-213. HATCH, T. P. {1975) Infec. Immunity 12, 211-220. HOBSON, D., JOHNSON, F. W. A., AND BYNG, R. E. (1977) J. Comp. Pathol. 87, 155-159. I~uo, C. C., AND GRAYSTON, J. T. (1976) Infec. Immunity 13, 1103-1109. MOULDER, J. W. (1974) J. Infec. Dis. 130, 300-306. REYNOLDS, E. S. {1963) J. Cell Biol. 17, 208-213. SCHACHTER,J., BANKS,J., SUGG,N., SUNG, M., STORZ, J., AND MEYER, K. F. (1974) Infec. Immunity 9, 9294. SCHACHTER,J., BANKS,J., SUGG,N., SUNG,M., STORZ, J., AND MEYER, K. F. (1975) Infec. Immunity 11, 904-907. SPEARS, P. (1978) Ph.D. thesis, Colorado State University, Fort Collins. STORZ,J. (1966) J. Comp. Pathol. 76, 351-362. STORZ, J., AND SPEARS, P. (1977) Curr. Top. Microbiol. Immunol. 76, 167-214. TANAMI, Y., AND YAMADA,Y. (1973) J. Bacteriol. 114, 408-412.
