Vol. 12, No. 3 Printed in U.S.A.
INFECTION AND IMMUNrrY, Sept. 1975, p. 638-646 Copyright 0 1975 American Society for Microbiology
Ultrastructural Cytochemical Evidence for the Activation of Lysosomes in the Cytocidal Effect of Chlamydia psittaci W. J. TODD* AND J. STORZ Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 Received for publication 14 March 1975
The cytopathic effect of the polyarthritis strain of Chlamydia psittaci was studied in cultured bovine fetal spleen cells and found to be mediated by the release of lysosomal enzymes into the host cytoplasm during the late stages of chlamydial development. Ultrastructural cytochemical analysis and cell fractionation studies of infected cells revealed a close relationship between the stage of chlamydial development, fine structural features of the host, and localization of lysosomal enzyme activities. After adsorption, chlamydiae entered the host cells by endocytosis. The endocytic vacuoles containing individual chlamydiae and later the inclusion vacuoles containing the different chlamydial developmental forms were always free from lysosomal enzyme activity. Even after extensive multiplication of chlamydiae, lysosomal enzymes remained localized within lysosomes or their precursors in the host cell. Coincident with the process of chlamydial maturation, lysosomal enzymes were released into the host cytoplasm and were always associated with disintegration of host cell constituents and lysis. The chlamydiae appeared to be protected from this lysosomal enzyme activity by the inclusion membrane. After release from the inclusion, elementary bodies maintained their fine structural features, whereas all other chlamydial developmental forms lost their ultrastructural integrity.
Chlamydiae are obligate intracellular para- Malmquist et al. (25). After three passages, minimal sites widely disseminated in nature and respon- essential growth medium containing 10% lamb serum sible for diverse disease syndromes in animals and 500 jtg of streptomycin and 500 U of penicillin per with lactalbumin vitamin medium and humans. These organisms are distinguished ml was replaced 10% heat-inactivated fetal calf serum and from other biological entities by a unique devel- containing Ag of streptomycin per ml. Cells were used in the opmental cycle. Death and lysis of the host cell, 500 4th to 10th subpassage. Mouse L-929 fibroblast cells which occurs upon completion of this develop- for plaque assay were grown in minimal essential mental cycle, is closely linked with the release medium with 10% heat-inactivated fetal calf serum of mature and infectious chlamydial forms. and 500 Ag of streptomycin per ml. The polyarthritis strain LW-613 representing speAlthough many investigations provide detailed information about the developmental cycle, cies C. psittaci was used throughout these investigaultrastructural features, chemical composition, tions (39). This strain was propagated by infecting and biochemical functions of chlamydiae, few monolayers of BFS cells with approximately 10 units per cell. At 36 to 48 h after ultrastructural studies have been directed to- plaque-forming were harvested as described by infection, chlamydiae wards the chlamydia-induced lysis of the host Schechter (35) and stored Bovernick's buffer (9) at cell. To elucidate the cytocidal mechanisms -57 C. Chlamydiae were inpassed 10 to 15 times in functioning in cell lysis, cultured bovine fetal BFS cells prior to use in these experiments. Chlamydspleen cells (BFS) were infected with a polyar- iae were titered by plaque assay according to Banks et thritis strain of Chlamydia psittaci and exam- al. (4). ined by ultrastructural cytochemistry and cell Fractionation of BFS cells and assay of lysosomal enzymes. For study of lysosomal enzymes in fractionation. (This investigation was presented by W.J.T. cytoplasmic fractions, chlamydia-infected BFS cells in partial fulfillment of the requirements for the grown in lactalbumin vitamin medium without phered were harvested and resuspended in 0.25 M Ph.D. degree at Colorado State University, Fort nol cold sucrose containing 0.001 M disodium Collins, Colo.) ethylenediaminetetraacetate. Cells were disrupted MATERIALS AND METHODS Propagation of cells and C. psittaci. Primary cultures of BFS cells were prepared according to 638
with a Dounce homogenizer (12) by the method of Wolff and Bubel (41) and fractionated according to Flanagan (14). Lysosomal enzymes were freed from cell debris by sound treatment with a Branson sonifier
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CYTOCIDAL EFFECT OF C. PSITTACI
for 20 s at 100 W, followed by addition of Triton X-100 to 0.1% and centrifugation at 45,000 x g for 30 min. Enzymes in the supernatant and pellet fractions were assayed by spectrophotometric procedures for acid phosphatase (EC 3.1.3.2), arylsulfatase (EC 3.1.6.1), and ,@-acetylglucosaminidase (EC 3.2.1.30) as described by Barrett (6). Ultrastructural cytochemistry. Samples of chlamydia-infected BFS cells were processed according to Brunk and Ericsson (10) by fixation for 10 to 16 h at 0 C in 0.1 M sodium cacodylate-hydrochloride buffer, pH 7.3, containing 0.1 M sucrose and 2% glutaraldehyde. Fixed cells were washed for 2 h in two changes of 0.05 M tris(hydroxymethyl)aminomethane-maleate buffer, pH 7.2, containing 0.22 M sucrose and 10% dimethyl sulfoxide. The specimens were incubated for 90 min at 37 C in a Barka and Anderson (5) modified Gomori (16) medium at pH 5.0, with the addition of sucrose to 0.22 M and 10% dimethyl sulfoxide. Fixation was completed at 4 C with 1% osmium tetroxide in 0.2 M collidine buffer at pH 7.4. Dehydration and embedding in Epon 812 were by standard procedures. Thin sections were cut with a diamond knife (E. I. DuPont de Nemours Co., Inc.) on a Porter-Blum MT2-B ultramicrotome and stained with uranyl acetate (40) and lead citrate (32). Grids were examined with a Hitachi HU-12 electron microscope. Control samples of uninfected cells and infected cells processed without the acid phosphatase substrate, ,Bglycerophosphate, were studied in parallel. Samples without osmium tetroxide, uranyl acetate, and lead citrate were also examined.
RESULTS Growth curve of chlamydial strain LW-613 and host cell death. A one-step growth curve of chlamydiae in BFS cells is illustrated in Fig. 1. After adsorption, an initial decrease in the number of plaque-forming units continuing for 16 h postinfection was observed. The decrease in titer reflects reorganization of infectious elementary bodies to noninfectious reticulate forms (27). Between 16 and 24 h the number of
plaque-forming units began to increase, indicating the formation of infectious progeny. The process of secondary reorganization to form infectious elementary bodies continued until about 48 h postinoculation. Host cell death was associated with this second reorganization from noninfectious reticulate forms to infectious forms. Most cells died between 24 and 36 h postinoculation, as indicated by loss of their ability to exclude trypan blue dye (Fig. 1) and by cell lysis observed by light and electron microscopy (24). Cell fractionation studies of lysosomal enzymes in chlamydia-infected BFS cells. Infected and uninfected BFS cells were periodically harvested, disrupted by the Dounce method, and separated by differential centrifugation into sedimentable and supernatant fractions. Activities of the lysosomal enzymes acid phosphatase, arylsulfatase, and #-acetylglucosaminidase were measured. The percentage of activity of each of the three enzymes present in the supernatant fraction was calculated and plotted against the duration of infection (Fig. 2). By 24 h postinfection most of the lysosomal enzyme activity was located within the supernatant fraction. These results indicate lysosomal enzymes were released from sedimentable lysosomal structures during the later stage of chlamydial development. Release of lysosomal enzymes preceded cell lysis. Ultrastructural analysis of acid phosphatase activity in normal and infected BFS cells. Acid phosphatase activity within uninfected BFS cells was found only within the cisternae system of the cell. The electron-opaque reaction products of acid phosphatase were localized within segments of endoplasmic reticulum, certain stacks of the Golgi apparatus lOG
o ACID PHOSPHATASE o - ACETYLGLUCOSAMINIDASE 80 a ARYLSULFASE
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ag
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FIG. 1. One-step growth curve of C. psittaci in cultured BFS cells (0) and host-cell viability measured by the trypan blue dye exclusion test (0).
8
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40 24 32 16 HOURS AFTER INFECTION
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FIG. 2. Percentage of lysosomal enzyme activity in the cytoplasmic fraction of chlamydia-infected BFS cells plotted as a function of the duration of infection.
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FIG. 3. Electron micrograph of a Golgi complex (G) containing acid phosphatase reaction products within one element of the Golgi. Other elements are free from this activity. x 112,000.
FIG. 4. Electron micrograph of acid phosphatase activity localized within an unifected cultured BFS cell. The electron-opaque reaction products are within membrane-bound vacuoles defined as lysosomes (L). The cytoplasmic matrix and other host cell organelles are free from acid phosphatase activity. x 14,400. 640
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CYTOCIDAL EFFECT OF C. PSITTACI
641
(Fig. 3), and within lysosomes (Fig. 4). The cyto- illustrated in Fig. 6 to 9. In the beginning stage plasmic matrix was free from electron-opaque of chlamydial maturation (Fig. 6) a few secondreaction products. The electron-opaque reaction ary reorganized forms and electron-dense maproducts were not observed in control cells ture forms are found among the reticulate processed in the same manner without ,B-glycer- bodies. At this stage numerous intact lysosomes ophosphate substrate for acid phosphatase. The are located at the periphery of the infected cells. fine structure of cells processed for acid phos- Acid phosphatase activity was found localized phatase activity was well preserved. within lysosomes. The first degenerative alteraThe early events of infection, adsorption and tion observed during the late stage of chlamyentry by endocytosis proceeded without in- dial development was focal disintegration of the volvement of lysosomes. Endocytic vacuoles host cell (Fig. 7). In those areas acid phosphacontaining chlamydiae were always free from tase activity was observed outside lysosomes acid phosphatase activity. Primary reorganiza- within the host cytoplasm. Continued release of tion and subsequent multiplication of reticulate lysosomal enzymes resulted in acid phosphatase forms occurred within an inclusion surrounded activity throughout the host cytoplasm (Fig. 8). by a membrane derived from the endocytic Associated with this activity was digestion of vacuole (27, 37). Release of acid phosphatase host cell organelles. Of the different cellular activity into the chlamydial inclusions was organelles, mitochondria retained their ultranever observed (Fig. 5). Even after extensive structural features longest. multiplication of reticulate forms, the fine A chlamydia-infected cell with total cytoplasstructural features of the host remained intact mic disintegration and containing an ultraand the acid phosphatase activity remained structurally intact inclusion is shown in Fig. 9. localized. The physical limit of this cell is marked by the The process of chlamydial maturation to thin ballooning plasmalemma. The cell is deinfectious dense forms and host cell lysis is void of all organelles; only a pyknotic nucleus
FIG. 5. Electron micrograph of an early stage of chlamydial development in a BFS cell processed for acid phosphatase activity. The early inclusion containing reticulated forms (RF) is free from the electron-opaque reaction products. x33,600.
642
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INFECT. IMMUN.
TODD AND STORZ
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FIG. 6. Electron micrograph of a chlamydia-infected BFS cell processed cytochemically for acid phosphatase activity. The lysosomes, organelles containing the electron-opaque reaction products of acid phosphatase, are located predominately in the extreme peripheral areas of the cell. x8,650.
e~~~~4 X, !!tt&~X FIG. 7. Electron micrograph of acid phosphatase activity (arrows) outside a lysosome (1) in an area of focal cytoplasmic disintegration; an adjacent lysosome (2) remains intact. The cytoplasm around this lysosome is ultrastructurally normal as are the chlamydial forms within the inclusion. x25,350..
8
I
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FIG. 8. Photograph of a montage constructed from six overlapping electron micrographs showing electronopaque reaction products of acid phosphatase scattered throughout the digested cytoplasm of a chlamydiainfected BFS cell. The chlamydial forms remain free of this activity and are ultrastructurally intact.
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3 FIG. 9. Electron micrograph of the late stage of disintegration of a chlamydia-infected BFS cell processed cytochemically for acid phosphatase activity (arrows). Only the inclusion remains ultrastructurally intact and free of acid phosphatase reaction products. Plasma membrane (PM); pyknotic nucleus (N); mitochondrion (M). x8,450. 643
644
TODD AND STORZ
and fragmented rough endoplasmic reticulum remain. The pyknotic nucleus consists of clumped chromatin-like material and is surrounded by fragments of the nuclear membrane. The cytoplasmic matrix is as electron translucent as the epoxy embedding medium. Despite this extreme degeneration of all host cell constituents, the fine structural details of the chlamydial inclusion remained intact. Within the inclusion is a large vacuole containing a degenerated mitochondrion. Acid phosphatase reaction products are found within this vacuole and the cell cytoplasm, but not within the area of the inclusion containing chlamydiae. The chlamydiae are separated from this vacuole and the cell cytoplasm by the inclusion membrane. The inclusion membrane may serve to protect the maturing chlamydiae from the mechanism of host cell lysis which occurs through release of lysosomal enzymes.
DISCUSSION Lysosomal involvement in the cytopathic effect of viruses was initially suggested by Defendi in 1962 (11). Subsequently, investigations by Allison and Sandelin (3) and others (2, 8, 14, 17, 23, 29, 34, 41) on lytic cycles of viruses and by Kordova and co-workers (19-22) on lytic cycles of chlamydiae have provided evidence supporting Defendi's hypothesis. This evidence is based primarily on cell fractionation studies and cytochemistry at the level of light microscopy. This present study refines and extends these initial observations to the level of ultrastructural cytochemical analysis. Ultrastructural cytochemical analysis of chlamydia-infected cells during all stages of parasite development revealed a consistent relationship between localization and containment of lysosomal enzymes, ultrastructural integrity of the host cell, and the different stages of chlamydial development. After adsorption of chlamydiae and uptake by endocytosis, the usual host response to foreign bodies consisting of fusion of lysosomes with endocytic vacuoles and subsequent digestion of endocytized material was not observed for vacuoles containing chlamydiae. The mechanism by which chlamydiae avoid this fate is unknown; however, an interesting study of Friis (15) showed chlamydiae are digested by lysosomal enzymes if the chlamydiae are heat inactivated or treated with specific antiserum prior to infection. Apparently an intrinsic property of viable or antibody-free chlamydiae is essential to avoid the initial lysosomal response. The promotion of intracellular fusion between lysosomes and endosomes containing parasite-
INFECT. IMMUN.
antibody complexes is also reported for toxoplasma, tubercle bacteria, rickettsiae, and vaccinia virus (33). This initial fusion of lysosomes and endosomes induced by antibody-coated parasites may be an important mechanism in neutralizing the infectivity of the infectious agent. The parasites listed possess the natural ability to avoid initiation of the lysosomal response in the absence of specific antibodies. After uptake, primary reorganization and replication of reticulate forms proceeded within an inclusion surrounded by a membrane initially derived from the endocytic vacuole (37). As chlamydiae multiplied, the inclusion membrane increased in size. According to Stokes (37), the inclusion membrane is made primarily from membranes or membrane precursors present in the host cell prior to infection. Chlamydia-directed modification of the inclusion membrane through glycosylation (38) or by addition of fatty acids not found in uninfected cells is likely (13). Chlamydia-mediated modification of the inclusion membrane may be important to further chlamydial development by regulating the flow of molecules between the parasite and the host cell and by limiting the lysosomal response. Lysosomal enzymes were not released into the inclusion. The chlamydial forms remained ultrastructurally intact and free from reaction products of acid phosphatase during the entire developmental cycle. Throughout the course of extensive multiplication of reticulate forms, the fine structural features of the host cell remained intact. Consequently, host cell lysis is not simply due to increased chlamydial biomass. A viable host cell is essential because chlamydiae are dependent on host metabolic activity for the generation of energy and some precursors essential to their development (27, 28). Extensive ultrastructural and cytochemical changes occurred within the host cell as the chlamydial developmental cycle progressed through secondary reorganization to reform infectious elementary bodies. The dislocation of lysosomes towards the peripheries of infected cells was probably due to the displacement of lysosomes and other cell organelles caused by growth of the large centrally located chlamydial inclusions. This relocation of lysosomes observed in the late stages of chlamydial development is of strategic interest. Release of hydrolytic enzymes from lysosomes in the peripheral areas of infected cells leads to digestion of primarily host cell constituents. The released lysosomal enzymes contribute extensively to cell lysis. The different developmental forms of chlamydiae appear to be protected from this
VOL. 13, 1975
CYTOCIDAL EFFECT OF C. PSITTACI
activity by the inclusion membrane. The action of lysosomal enzymes is followed by rupture of the host cell plasmalemma and the inclusion membrane, resulting in release of chlamydiae. After release, the mature elementary bodies which are known to be highly resistant to enzyme action and adverse environmental factors remain viable (27). The earlier developmental forms appear degenerated. The versatility of the lysosome response to chlamydiae and other intracellular parasites is remarkable. During the early stage of infection, failure of lysosomes to fuse with parasite-containing endosomes, as occurs with chlamydiae, enables the infectious process to' proceed (15). In the unique case of reovirus infection, fusion of lysosomes with the reovirus-containing endosomes is essential to the infectious process by providing the mechanism for uncoating the viral genome (36). In contrast, many organisms (and other foreign bodies) are digested and eliminated by the fusion of lysosomes with parasite-containing endosomes as originally described by Metchnikoff (26). In the later stages of parasite development, extensive release of lysosomal enzymes contributed to cell death as shown for chlamydiae (19-22) and many cytolytic viruses (1-3, 8, 10, 11, 14, 17, 23, 29, 34, 36, 41). A second and limited type of lysosomal enzyme release is reported to occur in polykaryon formation induced by viruses (30, 31). Poste suggested that limited and focal release of lysosomal enzymes at the surface of the host cell plasmalemma would be necessary to destabilize membranes for virus-induced cell fusion (31). Finally, in certain productive and nonproductive viral infections lysosomal enzymes are not released and the host cells are not killed. The failure of lysosomes to release enzymes may be an essential step in the formation of steady-state infections and virus-induced cell transformations. The mechanisms which regulate the diverse spectrum of lysosomal responses in infections of cells are completely unknown. Most of the evidence for these responses is based on mechanical disruption of cells (10, 14, 17, 29, 34, 36, 41), which in itself can cause release of lysosomal enzymes (18), and on cytochemical studies at the level of light microscopy (1-3, 19-23, 30). Unfortunately, light microscopy cannot resolve whether lysosomal enzyme activity occurs inside or outside of membrane-bound structures. The technique of ultrastructural cytochemistry as employed here to study the activation of lysosomes in the cytopathic effect of chlamydiae has the potential to resolve some of these controversies (7, 18, 23) concerning the
interaction between intracellular parasites and lysosomes. This method should be more widely used to provide clearer insight into cytocidal mechanisms of viruses and other obligate intracellular parasites.
645
ACKNOW'LEDGMENTS This investigation was supported by Public Health Service research grant AI-08420 from the National Institute of Allergy and Infectious Diseases, Bethesda, Md. LITERATURE CITED 1. Allison, A. C! 1967. Lysosomes in virus-infected cells, p. 29-55. In M. Pollard (ed.), Prospects in virology, vol. 5. Academic Press Inc., New York. 2. Allison, A. C., and L. Mallucci. 1965. Histochemical studies of lysosomes and lysosomal enzymes in virusinfected cell cultures. J. Exp. Med. 121:463-476. 3. Allison, A. C., and K. Sandelin. 1963. Activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effect. J. Exp. Med. 117:879-887. 4. Banks, J., B. Eddie, J. Schachter, and K. F. Meyer. 1970. Plaque formation by Chiamydia in L cells. Infect. Immun. 1:259-262. 5. Barka, T., and P. J. Anderson. 1962. Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10:741-753. 6. Barrett, A. J. 1972. Lysosomal enzymes, p. 46-71. In J. Dingle (ed.), Lysosomes, a laboratory handbook. North-Holland Publishing Co., Amsterdam. 7. Bienz, K., D. Egger, and D. A. Wolff. 1973. Virus replication, cytopathology, and lysosomal enzyme response of mitotic and interphase HEp-2 cells infected with poliovirus. J. Virol. 11:565-574. 8. Blackman, K. E., and H. C. Bubel. 1969. Poliovirusinduced cellular injury. J. Virol. 4:203-208. 9. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1960. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol.
59:509-522. 10. Brunk, U. T., and J. L. E. Ericsson. 1972. The demonstration of acid phosphatase in in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability. Histochem. J. 4:349-363. 11. Defendi, V. 1962. Cytopathology of virus infection. Fed. Proc. 21:1113-1117. 12. Dounce, A. L. 1963. The isolation of nuclei from tumor cells. Exp. Cell Res. 9(Suppl.):126-143. 13. Fan, V. S. C., and H. M. Jenkin. 1974. Lipid metabolism of monkey kidney cells (LLC-MK-2) infected with Chiamydia trachomatis strain lymphogranuloma venereum. Infect. Immun. 10:464-470. 14. Flanagan, J. F. 1966. Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus. J. Bacteriol. 91:789-797. 15. Friis, R. R. 1972. Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J. Bacteriol. 110:706-721. 16. Gomori, G. 1952. Microsopic histochemistry principles and practices. University of Chicago Press, Chicago. 17. Guskey, L. E., P. C. Smith, and D. A. Wolff. 1970. Patterns of cytopathology and lysosomal enzyme release in poliovirus-infected HEp-2 cells treated with either 2-(a-hydroxybenzyl)-benzimidazole or guanidine HCl. J. Gen. Virol. 6:151-161. 18. Killington, R. A., D. Lee, E. J. Scott, and J. A. Osborne. 1974. Studies on the lysosomes of L 132 cells infected
646
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20.
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27. 28.
29.
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with either rhinovirus type 2 or poliovirus type 1. J. Gen. Virol. 22:303-307. Kordova, N., L. Poffenroth, and J. C. Wilt. 1972. Lysosomes and the "toxicity" of Rickettsiales. II. Non-cytocidal interactions of egg-grown C. psittaci 6BC and in vitro macrophages. Can J. Microbiol. 18:869-873. Kordova, N., L. Poffenroth, and J. C. Wilt. 1972. Lysosomes and the "toxicity" of Rickettsiales. III. Response of L cells infected with egg-attenuated C. psittaci 6BC strain. Can. J. Microbiol. 18:1343-1348. Kordova, N., J. C. Wilt, and L. Poffenroth. 1973. Lysosomes and the "toxicity" of Rickettsiales. V. In vivo relationship of peritoneal phagocytes and eggattenuated C. psittaci 6BC. Can. J. Microbiol. 19:1417-1423. Kordova, N., J. C. Wilt, and M. Sadiq. 1971. Lysosomes in L cells infected with Chlamydia psittaci 6BC strain. Can. J. Microbiol. 17:955-959. Koschel, K., H. M. Aus, and V. Ter Meulen. 1974. Lysosomal enzyme activity in poliovirus-infected HeLa cells and vesicular stomatitis virus-infected L cells: biochemical and histochemical comparative analysis with computer-aided techniques. J. Gen. Virol. 25:359-369. McLimans, W. F., E. V. Davis, F. L. Glover, and G. W. Rake. 1957. The submerged culture of mammalian cells: the spinner culture. J. Immunol. 79:428-433. Malmquist, W. A., M. J. Van Der Maaten, and A. D. Boothe. 1969. Isolation, immunodiffusion, immunofluorescence, and electron microscopy of a syncytical virus of lymphosarcomatous and apparently normal cattle. Cancer Res. 29:118-200. Metchnikoff, E. 1893. Lectures on the comparative pathology of inflammation delivered at the Pasteur Institute, 1891 (English translation). K. Paul, Trench, Triibner and Co., Ltd., London. Moulder, J. W. 1964. The psittacosis group as bacteria. CIBA lectures in microbial biochemistry. John Wiley & Sons, Inc., New York. Moulder, J. W. 1974. Intracellular parasitism: life in an extreme environment. J. Infect. Dis. 130:300-306. Ogier, G., Y. Chardonnet, and L. Gozzolo. 1974. Role of
INFECT. IMMUN. lysosomes during infection with Shope fibroma virus of primary rabbit kidney tissue culture cells. J. Gen. Virol. 22:249-253. 30. Poste, G. 1971. The role of lysosomes in virus-induced cell fusion. II. Modification of cell surface. Microbios 3:105-112. 31. Poste, G. 1972. Mechanisms of virus-induced cell fusion. Int. Rev. Cytol. 33:157-237. 32. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-212. 33. Rezai, H. R. 1974. Immunity to mycobacteria and other nonviral intracellular parasites, p. 337-340. In L. Brent and J. Holborow (ed.), Progress in immunology II, vol. 4, Clinical aspects II. North-Holland Publishing Co., Amsterdam. 34. Sato, K., F. Righthand, and D. T. Karzon. 1971. Effect of host cell on distribution of a lysosomal enzyme during virus infection. J. Virol. 7:467-472. 35. Schechter, E. M. 1966. Synthesis of nucleic acid and protein in L cells infected with the agent of meningopneumonitis. J. Bacteriol. 91:2069-2080. 36. Silverstein, S. C., and S. Dales. 1968. The penetration of reovirus RNA and initiation of its genetic function in L-strain fibroblasts (mouse). J. Cell Biol. 36:197-221. 37. Stokes, G. V. 1973. Formation and destruction of internal membranes in L cells infected with Chlamydia psittaci. Infect. Immun. 3:173-177. 38. Stokes, G. V. 1974. Cycloheximide-resistant glycosylation in L cells infected with Chlamydia psittaci. Infect. Immun. 9:497-499. 39. Storz, J., R. A. Smart, M. E. Marriott, and R. V. Davis. 1966. Polyarthritis of calves. Isolation of psittacosis agents from affected joints. Am. J. Vet. Res. 27:633-641. 40. Watson, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475-478. 41. Wolff, D. A., and H. C. Bubel. 1964. The deposition of lysosomal enzymes as related to specific viral cytopathic effects. Virology 24:502-505.
INFECTION AND IMMUNrrY, Sept. 1975, p. 638-646 Copyright 0 1975 American Society for Microbiology
Ultrastructural Cytochemical Evidence for the Activation of Lysosomes in the Cytocidal Effect of Chlamydia psittaci W. J. TODD* AND J. STORZ Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523 Received for publication 14 March 1975
The cytopathic effect of the polyarthritis strain of Chlamydia psittaci was studied in cultured bovine fetal spleen cells and found to be mediated by the release of lysosomal enzymes into the host cytoplasm during the late stages of chlamydial development. Ultrastructural cytochemical analysis and cell fractionation studies of infected cells revealed a close relationship between the stage of chlamydial development, fine structural features of the host, and localization of lysosomal enzyme activities. After adsorption, chlamydiae entered the host cells by endocytosis. The endocytic vacuoles containing individual chlamydiae and later the inclusion vacuoles containing the different chlamydial developmental forms were always free from lysosomal enzyme activity. Even after extensive multiplication of chlamydiae, lysosomal enzymes remained localized within lysosomes or their precursors in the host cell. Coincident with the process of chlamydial maturation, lysosomal enzymes were released into the host cytoplasm and were always associated with disintegration of host cell constituents and lysis. The chlamydiae appeared to be protected from this lysosomal enzyme activity by the inclusion membrane. After release from the inclusion, elementary bodies maintained their fine structural features, whereas all other chlamydial developmental forms lost their ultrastructural integrity.
Chlamydiae are obligate intracellular para- Malmquist et al. (25). After three passages, minimal sites widely disseminated in nature and respon- essential growth medium containing 10% lamb serum sible for diverse disease syndromes in animals and 500 jtg of streptomycin and 500 U of penicillin per with lactalbumin vitamin medium and humans. These organisms are distinguished ml was replaced 10% heat-inactivated fetal calf serum and from other biological entities by a unique devel- containing Ag of streptomycin per ml. Cells were used in the opmental cycle. Death and lysis of the host cell, 500 4th to 10th subpassage. Mouse L-929 fibroblast cells which occurs upon completion of this develop- for plaque assay were grown in minimal essential mental cycle, is closely linked with the release medium with 10% heat-inactivated fetal calf serum of mature and infectious chlamydial forms. and 500 Ag of streptomycin per ml. The polyarthritis strain LW-613 representing speAlthough many investigations provide detailed information about the developmental cycle, cies C. psittaci was used throughout these investigaultrastructural features, chemical composition, tions (39). This strain was propagated by infecting and biochemical functions of chlamydiae, few monolayers of BFS cells with approximately 10 units per cell. At 36 to 48 h after ultrastructural studies have been directed to- plaque-forming were harvested as described by infection, chlamydiae wards the chlamydia-induced lysis of the host Schechter (35) and stored Bovernick's buffer (9) at cell. To elucidate the cytocidal mechanisms -57 C. Chlamydiae were inpassed 10 to 15 times in functioning in cell lysis, cultured bovine fetal BFS cells prior to use in these experiments. Chlamydspleen cells (BFS) were infected with a polyar- iae were titered by plaque assay according to Banks et thritis strain of Chlamydia psittaci and exam- al. (4). ined by ultrastructural cytochemistry and cell Fractionation of BFS cells and assay of lysosomal enzymes. For study of lysosomal enzymes in fractionation. (This investigation was presented by W.J.T. cytoplasmic fractions, chlamydia-infected BFS cells in partial fulfillment of the requirements for the grown in lactalbumin vitamin medium without phered were harvested and resuspended in 0.25 M Ph.D. degree at Colorado State University, Fort nol cold sucrose containing 0.001 M disodium Collins, Colo.) ethylenediaminetetraacetate. Cells were disrupted MATERIALS AND METHODS Propagation of cells and C. psittaci. Primary cultures of BFS cells were prepared according to 638
with a Dounce homogenizer (12) by the method of Wolff and Bubel (41) and fractionated according to Flanagan (14). Lysosomal enzymes were freed from cell debris by sound treatment with a Branson sonifier
VOL. 13, 1975
639
CYTOCIDAL EFFECT OF C. PSITTACI
for 20 s at 100 W, followed by addition of Triton X-100 to 0.1% and centrifugation at 45,000 x g for 30 min. Enzymes in the supernatant and pellet fractions were assayed by spectrophotometric procedures for acid phosphatase (EC 3.1.3.2), arylsulfatase (EC 3.1.6.1), and ,@-acetylglucosaminidase (EC 3.2.1.30) as described by Barrett (6). Ultrastructural cytochemistry. Samples of chlamydia-infected BFS cells were processed according to Brunk and Ericsson (10) by fixation for 10 to 16 h at 0 C in 0.1 M sodium cacodylate-hydrochloride buffer, pH 7.3, containing 0.1 M sucrose and 2% glutaraldehyde. Fixed cells were washed for 2 h in two changes of 0.05 M tris(hydroxymethyl)aminomethane-maleate buffer, pH 7.2, containing 0.22 M sucrose and 10% dimethyl sulfoxide. The specimens were incubated for 90 min at 37 C in a Barka and Anderson (5) modified Gomori (16) medium at pH 5.0, with the addition of sucrose to 0.22 M and 10% dimethyl sulfoxide. Fixation was completed at 4 C with 1% osmium tetroxide in 0.2 M collidine buffer at pH 7.4. Dehydration and embedding in Epon 812 were by standard procedures. Thin sections were cut with a diamond knife (E. I. DuPont de Nemours Co., Inc.) on a Porter-Blum MT2-B ultramicrotome and stained with uranyl acetate (40) and lead citrate (32). Grids were examined with a Hitachi HU-12 electron microscope. Control samples of uninfected cells and infected cells processed without the acid phosphatase substrate, ,Bglycerophosphate, were studied in parallel. Samples without osmium tetroxide, uranyl acetate, and lead citrate were also examined.
RESULTS Growth curve of chlamydial strain LW-613 and host cell death. A one-step growth curve of chlamydiae in BFS cells is illustrated in Fig. 1. After adsorption, an initial decrease in the number of plaque-forming units continuing for 16 h postinfection was observed. The decrease in titer reflects reorganization of infectious elementary bodies to noninfectious reticulate forms (27). Between 16 and 24 h the number of
plaque-forming units began to increase, indicating the formation of infectious progeny. The process of secondary reorganization to form infectious elementary bodies continued until about 48 h postinoculation. Host cell death was associated with this second reorganization from noninfectious reticulate forms to infectious forms. Most cells died between 24 and 36 h postinoculation, as indicated by loss of their ability to exclude trypan blue dye (Fig. 1) and by cell lysis observed by light and electron microscopy (24). Cell fractionation studies of lysosomal enzymes in chlamydia-infected BFS cells. Infected and uninfected BFS cells were periodically harvested, disrupted by the Dounce method, and separated by differential centrifugation into sedimentable and supernatant fractions. Activities of the lysosomal enzymes acid phosphatase, arylsulfatase, and #-acetylglucosaminidase were measured. The percentage of activity of each of the three enzymes present in the supernatant fraction was calculated and plotted against the duration of infection (Fig. 2). By 24 h postinfection most of the lysosomal enzyme activity was located within the supernatant fraction. These results indicate lysosomal enzymes were released from sedimentable lysosomal structures during the later stage of chlamydial development. Release of lysosomal enzymes preceded cell lysis. Ultrastructural analysis of acid phosphatase activity in normal and infected BFS cells. Acid phosphatase activity within uninfected BFS cells was found only within the cisternae system of the cell. The electron-opaque reaction products of acid phosphatase were localized within segments of endoplasmic reticulum, certain stacks of the Golgi apparatus lOG
o ACID PHOSPHATASE o - ACETYLGLUCOSAMINIDASE 80 a ARYLSULFASE
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FIG. 1. One-step growth curve of C. psittaci in cultured BFS cells (0) and host-cell viability measured by the trypan blue dye exclusion test (0).
8
8
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2
3
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40 24 32 16 HOURS AFTER INFECTION
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FIG. 2. Percentage of lysosomal enzyme activity in the cytoplasmic fraction of chlamydia-infected BFS cells plotted as a function of the duration of infection.
A~~~~~~~~~~~~~~~~~~~~~~~~~t
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FIG. 3. Electron micrograph of a Golgi complex (G) containing acid phosphatase reaction products within one element of the Golgi. Other elements are free from this activity. x 112,000.
FIG. 4. Electron micrograph of acid phosphatase activity localized within an unifected cultured BFS cell. The electron-opaque reaction products are within membrane-bound vacuoles defined as lysosomes (L). The cytoplasmic matrix and other host cell organelles are free from acid phosphatase activity. x 14,400. 640
VOL. 13, 1975
CYTOCIDAL EFFECT OF C. PSITTACI
641
(Fig. 3), and within lysosomes (Fig. 4). The cyto- illustrated in Fig. 6 to 9. In the beginning stage plasmic matrix was free from electron-opaque of chlamydial maturation (Fig. 6) a few secondreaction products. The electron-opaque reaction ary reorganized forms and electron-dense maproducts were not observed in control cells ture forms are found among the reticulate processed in the same manner without ,B-glycer- bodies. At this stage numerous intact lysosomes ophosphate substrate for acid phosphatase. The are located at the periphery of the infected cells. fine structure of cells processed for acid phos- Acid phosphatase activity was found localized phatase activity was well preserved. within lysosomes. The first degenerative alteraThe early events of infection, adsorption and tion observed during the late stage of chlamyentry by endocytosis proceeded without in- dial development was focal disintegration of the volvement of lysosomes. Endocytic vacuoles host cell (Fig. 7). In those areas acid phosphacontaining chlamydiae were always free from tase activity was observed outside lysosomes acid phosphatase activity. Primary reorganiza- within the host cytoplasm. Continued release of tion and subsequent multiplication of reticulate lysosomal enzymes resulted in acid phosphatase forms occurred within an inclusion surrounded activity throughout the host cytoplasm (Fig. 8). by a membrane derived from the endocytic Associated with this activity was digestion of vacuole (27, 37). Release of acid phosphatase host cell organelles. Of the different cellular activity into the chlamydial inclusions was organelles, mitochondria retained their ultranever observed (Fig. 5). Even after extensive structural features longest. multiplication of reticulate forms, the fine A chlamydia-infected cell with total cytoplasstructural features of the host remained intact mic disintegration and containing an ultraand the acid phosphatase activity remained structurally intact inclusion is shown in Fig. 9. localized. The physical limit of this cell is marked by the The process of chlamydial maturation to thin ballooning plasmalemma. The cell is deinfectious dense forms and host cell lysis is void of all organelles; only a pyknotic nucleus
FIG. 5. Electron micrograph of an early stage of chlamydial development in a BFS cell processed for acid phosphatase activity. The early inclusion containing reticulated forms (RF) is free from the electron-opaque reaction products. x33,600.
642
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INFECT. IMMUN.
TODD AND STORZ
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FIG. 6. Electron micrograph of a chlamydia-infected BFS cell processed cytochemically for acid phosphatase activity. The lysosomes, organelles containing the electron-opaque reaction products of acid phosphatase, are located predominately in the extreme peripheral areas of the cell. x8,650.
e~~~~4 X, !!tt&~X FIG. 7. Electron micrograph of acid phosphatase activity (arrows) outside a lysosome (1) in an area of focal cytoplasmic disintegration; an adjacent lysosome (2) remains intact. The cytoplasm around this lysosome is ultrastructurally normal as are the chlamydial forms within the inclusion. x25,350..
8
I
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FIG. 8. Photograph of a montage constructed from six overlapping electron micrographs showing electronopaque reaction products of acid phosphatase scattered throughout the digested cytoplasm of a chlamydiainfected BFS cell. The chlamydial forms remain free of this activity and are ultrastructurally intact.
9
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3 FIG. 9. Electron micrograph of the late stage of disintegration of a chlamydia-infected BFS cell processed cytochemically for acid phosphatase activity (arrows). Only the inclusion remains ultrastructurally intact and free of acid phosphatase reaction products. Plasma membrane (PM); pyknotic nucleus (N); mitochondrion (M). x8,450. 643
644
TODD AND STORZ
and fragmented rough endoplasmic reticulum remain. The pyknotic nucleus consists of clumped chromatin-like material and is surrounded by fragments of the nuclear membrane. The cytoplasmic matrix is as electron translucent as the epoxy embedding medium. Despite this extreme degeneration of all host cell constituents, the fine structural details of the chlamydial inclusion remained intact. Within the inclusion is a large vacuole containing a degenerated mitochondrion. Acid phosphatase reaction products are found within this vacuole and the cell cytoplasm, but not within the area of the inclusion containing chlamydiae. The chlamydiae are separated from this vacuole and the cell cytoplasm by the inclusion membrane. The inclusion membrane may serve to protect the maturing chlamydiae from the mechanism of host cell lysis which occurs through release of lysosomal enzymes.
DISCUSSION Lysosomal involvement in the cytopathic effect of viruses was initially suggested by Defendi in 1962 (11). Subsequently, investigations by Allison and Sandelin (3) and others (2, 8, 14, 17, 23, 29, 34, 41) on lytic cycles of viruses and by Kordova and co-workers (19-22) on lytic cycles of chlamydiae have provided evidence supporting Defendi's hypothesis. This evidence is based primarily on cell fractionation studies and cytochemistry at the level of light microscopy. This present study refines and extends these initial observations to the level of ultrastructural cytochemical analysis. Ultrastructural cytochemical analysis of chlamydia-infected cells during all stages of parasite development revealed a consistent relationship between localization and containment of lysosomal enzymes, ultrastructural integrity of the host cell, and the different stages of chlamydial development. After adsorption of chlamydiae and uptake by endocytosis, the usual host response to foreign bodies consisting of fusion of lysosomes with endocytic vacuoles and subsequent digestion of endocytized material was not observed for vacuoles containing chlamydiae. The mechanism by which chlamydiae avoid this fate is unknown; however, an interesting study of Friis (15) showed chlamydiae are digested by lysosomal enzymes if the chlamydiae are heat inactivated or treated with specific antiserum prior to infection. Apparently an intrinsic property of viable or antibody-free chlamydiae is essential to avoid the initial lysosomal response. The promotion of intracellular fusion between lysosomes and endosomes containing parasite-
INFECT. IMMUN.
antibody complexes is also reported for toxoplasma, tubercle bacteria, rickettsiae, and vaccinia virus (33). This initial fusion of lysosomes and endosomes induced by antibody-coated parasites may be an important mechanism in neutralizing the infectivity of the infectious agent. The parasites listed possess the natural ability to avoid initiation of the lysosomal response in the absence of specific antibodies. After uptake, primary reorganization and replication of reticulate forms proceeded within an inclusion surrounded by a membrane initially derived from the endocytic vacuole (37). As chlamydiae multiplied, the inclusion membrane increased in size. According to Stokes (37), the inclusion membrane is made primarily from membranes or membrane precursors present in the host cell prior to infection. Chlamydia-directed modification of the inclusion membrane through glycosylation (38) or by addition of fatty acids not found in uninfected cells is likely (13). Chlamydia-mediated modification of the inclusion membrane may be important to further chlamydial development by regulating the flow of molecules between the parasite and the host cell and by limiting the lysosomal response. Lysosomal enzymes were not released into the inclusion. The chlamydial forms remained ultrastructurally intact and free from reaction products of acid phosphatase during the entire developmental cycle. Throughout the course of extensive multiplication of reticulate forms, the fine structural features of the host cell remained intact. Consequently, host cell lysis is not simply due to increased chlamydial biomass. A viable host cell is essential because chlamydiae are dependent on host metabolic activity for the generation of energy and some precursors essential to their development (27, 28). Extensive ultrastructural and cytochemical changes occurred within the host cell as the chlamydial developmental cycle progressed through secondary reorganization to reform infectious elementary bodies. The dislocation of lysosomes towards the peripheries of infected cells was probably due to the displacement of lysosomes and other cell organelles caused by growth of the large centrally located chlamydial inclusions. This relocation of lysosomes observed in the late stages of chlamydial development is of strategic interest. Release of hydrolytic enzymes from lysosomes in the peripheral areas of infected cells leads to digestion of primarily host cell constituents. The released lysosomal enzymes contribute extensively to cell lysis. The different developmental forms of chlamydiae appear to be protected from this
VOL. 13, 1975
CYTOCIDAL EFFECT OF C. PSITTACI
activity by the inclusion membrane. The action of lysosomal enzymes is followed by rupture of the host cell plasmalemma and the inclusion membrane, resulting in release of chlamydiae. After release, the mature elementary bodies which are known to be highly resistant to enzyme action and adverse environmental factors remain viable (27). The earlier developmental forms appear degenerated. The versatility of the lysosome response to chlamydiae and other intracellular parasites is remarkable. During the early stage of infection, failure of lysosomes to fuse with parasite-containing endosomes, as occurs with chlamydiae, enables the infectious process to' proceed (15). In the unique case of reovirus infection, fusion of lysosomes with the reovirus-containing endosomes is essential to the infectious process by providing the mechanism for uncoating the viral genome (36). In contrast, many organisms (and other foreign bodies) are digested and eliminated by the fusion of lysosomes with parasite-containing endosomes as originally described by Metchnikoff (26). In the later stages of parasite development, extensive release of lysosomal enzymes contributed to cell death as shown for chlamydiae (19-22) and many cytolytic viruses (1-3, 8, 10, 11, 14, 17, 23, 29, 34, 36, 41). A second and limited type of lysosomal enzyme release is reported to occur in polykaryon formation induced by viruses (30, 31). Poste suggested that limited and focal release of lysosomal enzymes at the surface of the host cell plasmalemma would be necessary to destabilize membranes for virus-induced cell fusion (31). Finally, in certain productive and nonproductive viral infections lysosomal enzymes are not released and the host cells are not killed. The failure of lysosomes to release enzymes may be an essential step in the formation of steady-state infections and virus-induced cell transformations. The mechanisms which regulate the diverse spectrum of lysosomal responses in infections of cells are completely unknown. Most of the evidence for these responses is based on mechanical disruption of cells (10, 14, 17, 29, 34, 36, 41), which in itself can cause release of lysosomal enzymes (18), and on cytochemical studies at the level of light microscopy (1-3, 19-23, 30). Unfortunately, light microscopy cannot resolve whether lysosomal enzyme activity occurs inside or outside of membrane-bound structures. The technique of ultrastructural cytochemistry as employed here to study the activation of lysosomes in the cytopathic effect of chlamydiae has the potential to resolve some of these controversies (7, 18, 23) concerning the
interaction between intracellular parasites and lysosomes. This method should be more widely used to provide clearer insight into cytocidal mechanisms of viruses and other obligate intracellular parasites.
645
ACKNOW'LEDGMENTS This investigation was supported by Public Health Service research grant AI-08420 from the National Institute of Allergy and Infectious Diseases, Bethesda, Md. LITERATURE CITED 1. Allison, A. C! 1967. Lysosomes in virus-infected cells, p. 29-55. In M. Pollard (ed.), Prospects in virology, vol. 5. Academic Press Inc., New York. 2. Allison, A. C., and L. Mallucci. 1965. Histochemical studies of lysosomes and lysosomal enzymes in virusinfected cell cultures. J. Exp. Med. 121:463-476. 3. Allison, A. C., and K. Sandelin. 1963. Activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effect. J. Exp. Med. 117:879-887. 4. Banks, J., B. Eddie, J. Schachter, and K. F. Meyer. 1970. Plaque formation by Chiamydia in L cells. Infect. Immun. 1:259-262. 5. Barka, T., and P. J. Anderson. 1962. Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10:741-753. 6. Barrett, A. J. 1972. Lysosomal enzymes, p. 46-71. In J. Dingle (ed.), Lysosomes, a laboratory handbook. North-Holland Publishing Co., Amsterdam. 7. Bienz, K., D. Egger, and D. A. Wolff. 1973. Virus replication, cytopathology, and lysosomal enzyme response of mitotic and interphase HEp-2 cells infected with poliovirus. J. Virol. 11:565-574. 8. Blackman, K. E., and H. C. Bubel. 1969. Poliovirusinduced cellular injury. J. Virol. 4:203-208. 9. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1960. The influence of certain salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol.
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INFECT. IMMUN. lysosomes during infection with Shope fibroma virus of primary rabbit kidney tissue culture cells. J. Gen. Virol. 22:249-253. 30. Poste, G. 1971. The role of lysosomes in virus-induced cell fusion. II. Modification of cell surface. Microbios 3:105-112. 31. Poste, G. 1972. Mechanisms of virus-induced cell fusion. Int. Rev. Cytol. 33:157-237. 32. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-212. 33. Rezai, H. R. 1974. Immunity to mycobacteria and other nonviral intracellular parasites, p. 337-340. In L. Brent and J. Holborow (ed.), Progress in immunology II, vol. 4, Clinical aspects II. North-Holland Publishing Co., Amsterdam. 34. Sato, K., F. Righthand, and D. T. Karzon. 1971. Effect of host cell on distribution of a lysosomal enzyme during virus infection. J. Virol. 7:467-472. 35. Schechter, E. M. 1966. Synthesis of nucleic acid and protein in L cells infected with the agent of meningopneumonitis. J. Bacteriol. 91:2069-2080. 36. Silverstein, S. C., and S. Dales. 1968. The penetration of reovirus RNA and initiation of its genetic function in L-strain fibroblasts (mouse). J. Cell Biol. 36:197-221. 37. Stokes, G. V. 1973. Formation and destruction of internal membranes in L cells infected with Chlamydia psittaci. Infect. Immun. 3:173-177. 38. Stokes, G. V. 1974. Cycloheximide-resistant glycosylation in L cells infected with Chlamydia psittaci. Infect. Immun. 9:497-499. 39. Storz, J., R. A. Smart, M. E. Marriott, and R. V. Davis. 1966. Polyarthritis of calves. Isolation of psittacosis agents from affected joints. Am. J. Vet. Res. 27:633-641. 40. Watson, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475-478. 41. Wolff, D. A., and H. C. Bubel. 1964. The deposition of lysosomal enzymes as related to specific viral cytopathic effects. Virology 24:502-505.
