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Host Influence on the Density of Chlamydiae in Renografin Gradients
Zbl. Bakt. Hyg., I.Abt. Orig. A 247,526-529 (1980) Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
Host Influence on the Density of Chlamydiae in Renografin Gradients 1 EinfluB des Wirtssystems auf die Chlamydiendichte im RenografinGradienten MICHAEL R. NEUMAN, NONNA KORDOvA, and JOHN C. WILT
Received December 9, 1979
Abstract Egg-grown and L cell-grown C. psittaci 6BC strains formed two bands when centrifuged through preformed Renografin gradients. No additional bands were observed by extending the time of centrifugation. Particles present in the bands which were collected from the Renografin gradients were examined by electron microscopy. Purified elementary bodies from both egg- and L cell-grown sources were located in a lower, minor band; egg-derived elementary bodies showed a higher density (p < 0.01) than elementary bodies derived from L cells. Particles present in the major, upper band of both egg- and L cell-derived strains had similar densities and contained mixed populations of elementary bodies and polymorphic reticulate bodies.
Zusammenfassung Auf Hiihnereiern und L-Zellen geziichtete Stamme von C. psittaci 6BC bildeten bei ins Zentrifugieren iiber vorher ausgebildete Renografin-Gradienten zwei Bander. Nach Verlangerung der Zentrifugierzeit zeigten sich keine weiteren Bander. In den Banden vorhandene Partikel, die als Proben aus den Renografin-Gradienten entnommen worden waren, wurden elektronenoptisch untersucht. Gereinigte Elementarkorperchen aus der Anzucht auf Eiern und L-Zellen waren in einem unteren, kleineren Band lokalisiert. Die aus Eiern gewonnenen Elementarkorperchen zeigten eine hohere Dichte (p < 0,01) als die
Host Influence on the Density of Chlamydiae in Renografin Gradients
527
Introduction In our previous studies (11) we observed that L cell-grown Chlamydia psittaci 6BC strains sedimented differently than egg-grown strains in sucrose gradients. The phenomenon of host influenced (L cells versus eggs) modification of physical properties of the 6BC variant strains was of particular interest because the strains derived from the 2 sources differed in their cytopathogenicity for L cells and macrophages in vitro and in vivo (9, 10). There were however, no differences found in the ultrastructure of the surface envelopes of the 6BC variant strains from the 2 sources (4). Allan and Pears in 1979 described (1, 2) a differential infectivity of Renografin (or KCI) purified egg-grown and McCoy cell-grown C. psittaci GP-1C strains for McCoy cell-monolayers. The present study was undertaken to examine the banding patterns of 6BC variant strains using Renografin gradients and to determine whether the host-related density differences of chlamydiae previously observed in sucrose gradients (11) could also be seen on isopycnic Renografin density gradients. Material and Methods The two Chlamydia psittaci 6BC strains used in this investigation were the same as used earlier for centrifugation through sucrose gradients: one strain was grown in yolk sacs of the chick embryo (CE), the other in L cells as described previously (3, 10). Both CE- and L cell-derived stock inocula were kept as crude preparations at - 70°C. Comparison of the morphology of 6BC strains derived from CE and from L cells showed no ultrastructural differences in the surface envelopes of the individual particles (4). Twelve samples of L cell-derived chlamydiae and 6 samples of egg-grown chlamydiae were prepared and their banding patterns in Renografin density gradients were examined. In the work reported here, a Renografin density gradient method previously described in detail by others (5, 7) was used. Both 6BC strains were partially freed from host-cell debris by differential centrifugation; 0.5 ml of this partially purified chlamydial suspension was layered on 4.5 ml of a preformed linear gradient of 20 to 45% Renografin (Squibb and Sons, Inc., Princeton, N. Y., U.S.A.), and centrifuged at 40,700 X g for 2 hours in a Beckman SW 65 rotor. The bands were then collected for density measurements and electron microscopy. Fixation, embedding and staining of chlamydial preparations were carried out as previously described (3, 4). The preparations were examined using a Philips 201 electron microscope.
Results and Discussion The results of centrifugation of the two 6BC strains through Renografin gradients are summarized in Table 1. The values shown are the averages of density determinations from 12 samples of L cell-derived and from 6 samples of egg-derived chlamydial preparations. The error is expressed as the standard error of these analyses from its respective mean. Chlamydiae derived from chick embryo and L cells each formed two visible bands; no additional bands were observed by extending the time of centrifugation from 2 to 18 hours. The major, wide upper band formed at the same location on the gradient and had similar densities (mean density of 1.18 g/cm3 ) for both strains. Electron microscopy showed that this band of both strains consisted of a mixed population of small and intermediate elementary bodies 35 Zbl. Bakt. Hyg., I. Abt. Orig. A 247
528
M. R. Neuman, N. Kordova, and J. C. Wilt
Table 1. Banding patterns of C. psittaci 6BC derived from infected yolk sac (CE) or from L cells by centrifugation through Renografin gradients No. of repeti tions CE-derived 6BC CE-derived 6BC L Cell-derived 6BC L Cell-derived 6BC
upper band lower band upper baEd lower band
6 6 12 12
Mean density g/cm 4 (± S.E.) 1.188 1.224 1.185 1.197
± 0.003 ± 0.002 ± 0.001 ± 0.001
The mean density values are the averages of density determinations of 18 independent samples. The error is expressed as the standard error (± S. E.) of each of these analyses. The analysis shows a statistically significant difference between the mean density of the lower bands of the two 6BC stains (p < 0.01). There is no significant difference between the correspor:ding upper bands.
and of polymorphic (by size and shape) reticulate bodies. Both egg- and L cellderived strains formed a minor, thinner lower band which by electron microscopy contained small, dense-centered elementary bodies free from contaminating material. However, egg-derived elementary bodies banded at a mean density of 1.20. The analysis showed a significant difference (p < 0.01) between the densities of the 6BC elementary bodies derived from the two different host systems. Centrifugation through Renografin gradients is currently the method of choice for purification of viable, antigenic ally unaltered Chlamydia from contaminating host material of infected cultured cells (5) or chick embryo (7). The present studies using Renografin gradients support our earlier studies using sucrose gradients (11) that the density of 6BC elementary bodies grown in chicken embryo is different than elementary bodies grown in L cells. In the present studies L cell-derived elementary bodies banded at a mean density of 1.20. Friis reported (5) that L cellderived elementary bodies of a meningopneumonitis agent (C. psittaci) banded in Renografin gradients at a density of 1.21. In the present studies, both egg-grown and L cell-grown chlamydiae formed major bands (consisting of mixed populations of elementary- and reticulate bodies) at similar densities of Renografin. Sucrose gradients in contrast showed the major bands of the two 6BC strains to be located at different densities (11). We have previously shown that chlamydial particles are damaged by the various steps commonly used in purification (including differential centrifugation) and that the envelopes of the reticulate bodies and the less condensed smaller forms (intermediate bodies) are the most susceptible to damage (3). Possibly, Renografin exerts different effects on the permeability of the envelopes of chlamydial particles than does sucrose. Although host related changes in virulence, antigenicity and immunogenicity of chlamydiae have been reported by several earlier authors (6, 13, 14), reports of host-controlled variations are rare in the more recent literature The reasons for host-related differences in physical properties of chlamydiae are not understood but it is most probable that they reflect changes in chemical composition of chlamydial surfaces that occur when grown in different host-systems (8, 12).
Host Influence on the Density of Chlamydiae in Renografin Gradients
529
References 1. Allan, I. and T. H. Pearce: Host Modification of Chlamydiae: Differential Infectivity for Cell Monolayers of Chlamydiae Grown in Eggs and Monolayers. J. gen. Microbial. 112 (1979) 53-59 2. Allan, I. and T. H. Pearce: Host Modification of Chlamydiae: Presence of an EggAntigen on the Surface of Chlamydiae grown in the Chick Embryo. J. gen. Microbial. 112 (1979) 61-66 3. Costerton, J. W., L.Poffenroth, J. C. Wilt, and N.Kordova: The effect of purification on the ultrastructure and infectivity of egg-attenuated Chlamydia psittaci (6BC). Canad. J. Microbial. 21 (1975) 1448-1463 4. Costerton, J. W., L.Poffenroth, J. C. Wilt, and N.Kordova: Ultrastructural studies of Chlamydia psittaci 6BC "in situ" in yolk sac explants and L cells: a comparison with gram-negative bacteria. Canad. J. Microbial. 21 (1975) 1433-1447 5. Friis, R. R.: Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J. Bact. 110 (1972) 706-721 6. Graham, D. M.: Growth and immunogenicity of TRIC agents in mice. Amer. J. Ophthalmol. 63 (1967) 1173/147-1190/164 7. Howard, L., N. S. Orenstein, and N. W. King: Purification on Renografin density gradients of Chlamydia trachomatis grown in the yolk sac of eggs. Appl. Microbial. 27 (1974) 102-106 8. Jenkin, H. M.: Comparative lipid composition of psittacosis and trachoma agents. Part II. Amer. J. Ophthalmol. 63 (1967) 1087-1098 9. Kordova, N., L. Poffenroth, and J. C. Wilt: Lysosomes and the "toxicity" of Rickettsias. III. Response of L cells infected with egg-attenuated C. psittaci 6BC strain. Canad. J. Microbial. 19 (1972) 1417-1423 10. Kordovd, N., J. C. Wilt, and C. Martin: Lysosomes and the "toxicity" of Rickettsias. VI. In vivo response of mouse peritoneal phagocytes to L cell grown Chlamydia psittaci 6BC strains. Canad. J. Microbial. 21 (1975) 323-331 11. Kordovd, N., C.Martin, J.C. Wilt, and M.Neuman: Sucrose density differences of Chlamydia psittaci 6BC in relation to its host. Canad. J. Microbial. 23 (1977) 649-652 12. Makino, S., H.M.Jenkin, H.M. Yu, and D. Townsend: Lipid composition of Chlamydia psittaci grown in monkey kidney cells in defined medium. J. Bact. 103 (1970) 62-70 13. Meyer, K. F.: Demonstration of host-species antigens in Chlamydia psittaci by the plaque reduction test in L-cell tissue cultures. Arch. ges. Virusforsch. 31 (1970) 1-10 14. Officer, T.E. and A.Brown: Growth of psittacosis virus in tissue culture. J. infect. Dis. 107 (1960) 283-299 Pre doctoral graduate student M.R.Neuman, Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
Host Influence on the Density of Chlamydiae in Renografin Gradients 1 EinfluB des Wirtssystems auf die Chlamydiendichte im RenografinGradienten MICHAEL R. NEUMAN, NONNA KORDOvA, and JOHN C. WILT
Received December 9, 1979
Abstract Egg-grown and L cell-grown C. psittaci 6BC strains formed two bands when centrifuged through preformed Renografin gradients. No additional bands were observed by extending the time of centrifugation. Particles present in the bands which were collected from the Renografin gradients were examined by electron microscopy. Purified elementary bodies from both egg- and L cell-grown sources were located in a lower, minor band; egg-derived elementary bodies showed a higher density (p < 0.01) than elementary bodies derived from L cells. Particles present in the major, upper band of both egg- and L cell-derived strains had similar densities and contained mixed populations of elementary bodies and polymorphic reticulate bodies.
Zusammenfassung Auf Hiihnereiern und L-Zellen geziichtete Stamme von C. psittaci 6BC bildeten bei ins Zentrifugieren iiber vorher ausgebildete Renografin-Gradienten zwei Bander. Nach Verlangerung der Zentrifugierzeit zeigten sich keine weiteren Bander. In den Banden vorhandene Partikel, die als Proben aus den Renografin-Gradienten entnommen worden waren, wurden elektronenoptisch untersucht. Gereinigte Elementarkorperchen aus der Anzucht auf Eiern und L-Zellen waren in einem unteren, kleineren Band lokalisiert. Die aus Eiern gewonnenen Elementarkorperchen zeigten eine hohere Dichte (p < 0,01) als die
Host Influence on the Density of Chlamydiae in Renografin Gradients
527
Introduction In our previous studies (11) we observed that L cell-grown Chlamydia psittaci 6BC strains sedimented differently than egg-grown strains in sucrose gradients. The phenomenon of host influenced (L cells versus eggs) modification of physical properties of the 6BC variant strains was of particular interest because the strains derived from the 2 sources differed in their cytopathogenicity for L cells and macrophages in vitro and in vivo (9, 10). There were however, no differences found in the ultrastructure of the surface envelopes of the 6BC variant strains from the 2 sources (4). Allan and Pears in 1979 described (1, 2) a differential infectivity of Renografin (or KCI) purified egg-grown and McCoy cell-grown C. psittaci GP-1C strains for McCoy cell-monolayers. The present study was undertaken to examine the banding patterns of 6BC variant strains using Renografin gradients and to determine whether the host-related density differences of chlamydiae previously observed in sucrose gradients (11) could also be seen on isopycnic Renografin density gradients. Material and Methods The two Chlamydia psittaci 6BC strains used in this investigation were the same as used earlier for centrifugation through sucrose gradients: one strain was grown in yolk sacs of the chick embryo (CE), the other in L cells as described previously (3, 10). Both CE- and L cell-derived stock inocula were kept as crude preparations at - 70°C. Comparison of the morphology of 6BC strains derived from CE and from L cells showed no ultrastructural differences in the surface envelopes of the individual particles (4). Twelve samples of L cell-derived chlamydiae and 6 samples of egg-grown chlamydiae were prepared and their banding patterns in Renografin density gradients were examined. In the work reported here, a Renografin density gradient method previously described in detail by others (5, 7) was used. Both 6BC strains were partially freed from host-cell debris by differential centrifugation; 0.5 ml of this partially purified chlamydial suspension was layered on 4.5 ml of a preformed linear gradient of 20 to 45% Renografin (Squibb and Sons, Inc., Princeton, N. Y., U.S.A.), and centrifuged at 40,700 X g for 2 hours in a Beckman SW 65 rotor. The bands were then collected for density measurements and electron microscopy. Fixation, embedding and staining of chlamydial preparations were carried out as previously described (3, 4). The preparations were examined using a Philips 201 electron microscope.
Results and Discussion The results of centrifugation of the two 6BC strains through Renografin gradients are summarized in Table 1. The values shown are the averages of density determinations from 12 samples of L cell-derived and from 6 samples of egg-derived chlamydial preparations. The error is expressed as the standard error of these analyses from its respective mean. Chlamydiae derived from chick embryo and L cells each formed two visible bands; no additional bands were observed by extending the time of centrifugation from 2 to 18 hours. The major, wide upper band formed at the same location on the gradient and had similar densities (mean density of 1.18 g/cm3 ) for both strains. Electron microscopy showed that this band of both strains consisted of a mixed population of small and intermediate elementary bodies 35 Zbl. Bakt. Hyg., I. Abt. Orig. A 247
528
M. R. Neuman, N. Kordova, and J. C. Wilt
Table 1. Banding patterns of C. psittaci 6BC derived from infected yolk sac (CE) or from L cells by centrifugation through Renografin gradients No. of repeti tions CE-derived 6BC CE-derived 6BC L Cell-derived 6BC L Cell-derived 6BC
upper band lower band upper baEd lower band
6 6 12 12
Mean density g/cm 4 (± S.E.) 1.188 1.224 1.185 1.197
± 0.003 ± 0.002 ± 0.001 ± 0.001
The mean density values are the averages of density determinations of 18 independent samples. The error is expressed as the standard error (± S. E.) of each of these analyses. The analysis shows a statistically significant difference between the mean density of the lower bands of the two 6BC stains (p < 0.01). There is no significant difference between the correspor:ding upper bands.
and of polymorphic (by size and shape) reticulate bodies. Both egg- and L cellderived strains formed a minor, thinner lower band which by electron microscopy contained small, dense-centered elementary bodies free from contaminating material. However, egg-derived elementary bodies banded at a mean density of 1.20. The analysis showed a significant difference (p < 0.01) between the densities of the 6BC elementary bodies derived from the two different host systems. Centrifugation through Renografin gradients is currently the method of choice for purification of viable, antigenic ally unaltered Chlamydia from contaminating host material of infected cultured cells (5) or chick embryo (7). The present studies using Renografin gradients support our earlier studies using sucrose gradients (11) that the density of 6BC elementary bodies grown in chicken embryo is different than elementary bodies grown in L cells. In the present studies L cell-derived elementary bodies banded at a mean density of 1.20. Friis reported (5) that L cellderived elementary bodies of a meningopneumonitis agent (C. psittaci) banded in Renografin gradients at a density of 1.21. In the present studies, both egg-grown and L cell-grown chlamydiae formed major bands (consisting of mixed populations of elementary- and reticulate bodies) at similar densities of Renografin. Sucrose gradients in contrast showed the major bands of the two 6BC strains to be located at different densities (11). We have previously shown that chlamydial particles are damaged by the various steps commonly used in purification (including differential centrifugation) and that the envelopes of the reticulate bodies and the less condensed smaller forms (intermediate bodies) are the most susceptible to damage (3). Possibly, Renografin exerts different effects on the permeability of the envelopes of chlamydial particles than does sucrose. Although host related changes in virulence, antigenicity and immunogenicity of chlamydiae have been reported by several earlier authors (6, 13, 14), reports of host-controlled variations are rare in the more recent literature The reasons for host-related differences in physical properties of chlamydiae are not understood but it is most probable that they reflect changes in chemical composition of chlamydial surfaces that occur when grown in different host-systems (8, 12).
Host Influence on the Density of Chlamydiae in Renografin Gradients
529
References 1. Allan, I. and T. H. Pearce: Host Modification of Chlamydiae: Differential Infectivity for Cell Monolayers of Chlamydiae Grown in Eggs and Monolayers. J. gen. Microbial. 112 (1979) 53-59 2. Allan, I. and T. H. Pearce: Host Modification of Chlamydiae: Presence of an EggAntigen on the Surface of Chlamydiae grown in the Chick Embryo. J. gen. Microbial. 112 (1979) 61-66 3. Costerton, J. W., L.Poffenroth, J. C. Wilt, and N.Kordova: The effect of purification on the ultrastructure and infectivity of egg-attenuated Chlamydia psittaci (6BC). Canad. J. Microbial. 21 (1975) 1448-1463 4. Costerton, J. W., L.Poffenroth, J. C. Wilt, and N.Kordova: Ultrastructural studies of Chlamydia psittaci 6BC "in situ" in yolk sac explants and L cells: a comparison with gram-negative bacteria. Canad. J. Microbial. 21 (1975) 1433-1447 5. Friis, R. R.: Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J. Bact. 110 (1972) 706-721 6. Graham, D. M.: Growth and immunogenicity of TRIC agents in mice. Amer. J. Ophthalmol. 63 (1967) 1173/147-1190/164 7. Howard, L., N. S. Orenstein, and N. W. King: Purification on Renografin density gradients of Chlamydia trachomatis grown in the yolk sac of eggs. Appl. Microbial. 27 (1974) 102-106 8. Jenkin, H. M.: Comparative lipid composition of psittacosis and trachoma agents. Part II. Amer. J. Ophthalmol. 63 (1967) 1087-1098 9. Kordova, N., L. Poffenroth, and J. C. Wilt: Lysosomes and the "toxicity" of Rickettsias. III. Response of L cells infected with egg-attenuated C. psittaci 6BC strain. Canad. J. Microbial. 19 (1972) 1417-1423 10. Kordovd, N., J. C. Wilt, and C. Martin: Lysosomes and the "toxicity" of Rickettsias. VI. In vivo response of mouse peritoneal phagocytes to L cell grown Chlamydia psittaci 6BC strains. Canad. J. Microbial. 21 (1975) 323-331 11. Kordovd, N., C.Martin, J.C. Wilt, and M.Neuman: Sucrose density differences of Chlamydia psittaci 6BC in relation to its host. Canad. J. Microbial. 23 (1977) 649-652 12. Makino, S., H.M.Jenkin, H.M. Yu, and D. Townsend: Lipid composition of Chlamydia psittaci grown in monkey kidney cells in defined medium. J. Bact. 103 (1970) 62-70 13. Meyer, K. F.: Demonstration of host-species antigens in Chlamydia psittaci by the plaque reduction test in L-cell tissue cultures. Arch. ges. Virusforsch. 31 (1970) 1-10 14. Officer, T.E. and A.Brown: Growth of psittacosis virus in tissue culture. J. infect. Dis. 107 (1960) 283-299 Pre doctoral graduate student M.R.Neuman, Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3