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Giant Cell Formation in Acholeplasma laidlawii, Strain JA1
Zbl. Bakt. Hyg., LAbt. Orig. A 248, 120-128 (1980) lInstitut fur Medizinische Mikrobiologie und Virologie der Universitat Dusseldorf, 2Institut fur Medizinische Mikrobiologie der Universitat Gielsen, 3Institut fur Hygiene und Infektionskrankheiten der Tiere der Universitat Giegen, Federal Republic of Germany
Giant Cell Formation in Acholeplasma laidlawii, Strain JA 1 Riesenzellbildung von Acholeplasma laidlawii, Stamm JAl HELMUT BRUNNER!, HERBERT SCHAAR 2, and HARTMUT KRAUSS3
With 7 Figures· Received March 18, 1980
Abstract Striking alterations, which looked like blebs, were observed in colonies of Acholeplasma laidlawii, strain JA 1. "Craters" were seen on the surface of these colonies by scanning electron microscopy. In addition, giant celIs of acholeplasma, up to 14,um in diameter, i.e. approximately 20 times the size of a normal A. laidlawii cell, were visible in these colonies. The large forms were surrounded by a unit membrane. After infection with group 1 and group 2 mycoplasma-viruses, the proportion of altered colonies and the number of large celIs within these colonies increased. A strain of A. laidlau/ii, which was not susceptible to infection with the three known acholeplasma-viruses did not exhibit comparable morphological changes. The development of the giant cells can be explained either by cell fusion or by a lag of cell division behind genome replication. Blebs and "craters" may result from the destruction of mycoplasma organisms within the complex structure of the colony. There is also suggestive evidence that strain JA 1 carries a virus in some cryptic form.
Zusammenfassung Auffallige Veranderungen, die wie Blaschen imponierten, wurden in den Kolonien von Acholeplasma laidlawii, Stamm JA 1, beobachtet. Im Rasrerelektronenmikroskop wurcn "Krater" in der Oberflache dieser Kolonien zu sehen. Aufserdem zeigten sich in den veranderten Kolonien Riesenzellen mit einem Durchmesser bis zu 14,um, d. h. der ungefahr 20fachen Grage einer normalen Acholeplasma-Zelle. Die grofsen Formen waren vollstandig von einer doppelschichtigen Membran umgeben. Nach Infektion mit Mycoplasma-Viren der Gruppen 1 und 2 nahm der AnteiI veranderrer Kolonien und die Zahl von Riesenzellen in diesen Kolonien zu. Ein Stamm von A. laidlawii, der nicht empfanglich war fur eine Infektion mit den drei bekannten Acholeplasma-Viren, zeigte keine vergleichbaren morphologischen Veranderungen. Die Entwicklung der Riesenzellen kann man entweder durch eine Zellfusion oder durch eine Verzogerung der Zellteilung gegenuber der Genomrepli-
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kation erklaren. Blaschen und "Krater" konnten das Resultat einer Zerstorung von Mycoplasmen innerhalb der komplexen Struktur einer Kolonie sein. Es gibt Hinweise dafiir, dag der Stamm JA 1 latent mit einem Virus infiziert ist.
Introduction Recently, Congdon and Kenny, observed strikingly altered and distinct colonies of Acholeplasma laidlawii, infected with Mycoplasmatales virus-laidlawii MV-Ll and MV-L3 (6). This is of special interest, since Mycoplasmatales are important pathogens for man, animals, plants and insects. Our knowledge about the pathogenic mechanisms of mycoplasmas is scarce. It should therefore be kept in mind, that the virulence of some pathogenic mycoplasma species may be determined by the presence of a phage, as has already been demonstrated for Corynebacterium diphtheriae and for group A Streptococci (3, 4, 12, 26). In addition, mycoplasmas are of special importance for basic research in microbiology, because they are the smallest free living organisms known. They lack a rigid cell wall and therefore expose their cytoplasmic membrane directly to the environment. In this publication, we provide evidence that A. laidlawii, strain JA 1, not artificially infected with a virus, forms colonies with bleb-like alterations. In scanning electron microscopy these blebs appear as "craters". In addition, we have seen giant cells of A.laidlawii, up to 14.um in diameter, in blebbed colonies. After infection with group 1 or group 2 acholeplasma-viruses the number of these giant cells increased.
Materials and Methods Acholeplasmas and viruses. Acholeplasma laidlawii, strain JA 1, furthermore a strain of A.laidlawii, recovered from the human oral cavity, the bullet shaped Mycoplasmatales virus-laidlawii 1 (group 1) and the enveloped, spherical group 2 virus were used (16). The JA J-strain and the viruses were kindly provided by]. Maniloff of the University of Rochester, Rochester, N. Y. The oral strain of A. laidlawii had been obtained from R. M. Chanock, National Institutes of Health, Bethesda, Md. (7). Growth medium and culture conditions. One liter of fluid growth medium contained 20 g tryptose, 5 g tris-(hydroxymethyl)-aminomethane, 5 g sodium-chloride, 10 g glucose and 10 ml PPLO-serum fraction (Difco-Laboratories, Detroit, Michigan). Agar-medium contained in addition 11 g special agar Noble (Difco) and 0.05% thallium-acetate. In order to obtain organisms in the log-growth-phase, daily 4.5 ml of the tryptose-broth were inoculated with 0.1 ml of an overnight culture of A.laidlawii. After 24-hours of incubation at 37°C this resulted in a suspension, containing 1 to 2 X 108 colony forming units (CFU) if A. laidlawii per m!. Propagation of mycoplasma-viruses. Group 1 and group 2 viruses were grown on JA 1 lawns as described by Maniloff and Liss (17). The suspension, containing JA1 and the virus, was filtered through an 0.2 ,urn pore size Millipore filter. This yielded a virus suspension of approximately 1 to 2 X 1010 plaque forming units (PFU) per m!. The viruses were plaqued on JA 1 lawns as described by Maniloff and Liss (17). Light microscopy. Colonies were photographed with a Zeiss photomicroscope. Transmission electron microscopy. A. laidlawii colonies were cut out of the agar and fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 2 hrs at 4°C, postfixed with 1.5% osmium tetroxide in the same buffer for additional 2 hrs, washed with
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distilled water, treated with 0.5% uranyl acetate in distilled water for 20 min, washed again, dehydrated in acetone and embedded in Durcupan (Fluka, Basel). Thin sections were stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop 102 at 80 kV (5). Scanning Electron Microscopy. Colonies with and without blebs were cut out of the agar, fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer, postfixed with 1.5% osmium tetroxide in the same buffer, and dehydrated in various concentrations (30,50,70, 90 and 100%) of acetone in 0.05 M cacodylate buffer for 10 mil'. each. The acetone was exchanged with amylacetate in four 10 min-steps (25, 50, 75 and 100%), the agar blocks fixed on a specimen stud, coated with gold by a Balzers Union sputtering device and examined in a Leitz Scanning electron microscope AMR 1600 T.
Results
Light microscopy. Overnight cultures of A.laidlawii JA1, grown in tryptose broth, which had not been infected with a virus, were inoculated on tryptose-agar. After incubation at 37°C for 2 to 6 days colonies were examined by light microscopy at a magnification of 40 X. Already after 48 hrs incubation, when visible colonies appeared, some of these young colonies showed alterations of their morphology. In the centers of the colonies multiple bleb-like changes could be seen (Fig. 1). These blebs later spread over the entire colony. By day 4 to 5 approximately 50 per cent of the colonies were covered with these vesicles (Fig. 1). In the remaining colonies morphological alterations were not seen during the observation period of 6 days. In contrast to JA1, colonies of the oral strain of A.laidlawii did not exhibit any blebs. Scanning electron microscopy. In the scanning electron microscope "craters" were seen in the surface of the colonies (Fig. 2). In addition, large forms of mycoplasmas could be observed in the vicinity of the "craters" (Fig. 2).
Fig. 1. Photomicrograph of A. laidlawii ]A 1, not artificially infected with a virus, Part of a normal colony (left) and of a colony with multiple blebs can be seen (Bar: 10 ,urn).
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Fig. 2. Scanning electron micrograph of a colony of A. laidlatoii, strain ]A 1. A "crater" can be seen on the surface of the colony. In the vicinity of the "crater" large and normal cells of A.laidlawii are visible (Bar: 2 {lm).
Transmission electron microscopy. In addition to normal mycoplasmas, electron microscopic investigation of altered colonies revealed large forms bound by a unit membrane (Fig. 3, 4, 5). The size of these giant cells was on the average 2-10,um, some measured up to 14,um (Fig. 3 and 4). Compared to the many acholeplasma cells of normal size, the cytoplasma of the giant-cells appeared often less dense. A few cells showed finger-like projections sometimes resembling "tumorous" growth of tissue (Fig. 4 and 5). In later stages giant forms appeared as membrane bound vesicles (Fig. 6).
Fig. 3. Section of cells of A.laidlawii, not artificially infected with a virus. A giant cell, surrounded by a unit membrane and several cells of normal size can be seen (Bar: 1 {lm).
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Fig. 4. Longitudinal section of a colony of A. Iaidlawii JA 1 showing a giant cell with filamentous extrusions (Bar: 1 pm).
Fig. 5. Longitudinal section through a "cratered" colony of A.laidlawii JA 1 showing tumor-like growth of a giant cell (Bar: 1 pm).
Appearance of ] A 1 colonies after infection with group 1 or group 2 Viruses. Colonies of A. laidlawii JA1 which had been infected with group 1 or group 2 mycoplasma viruses showed by far more blebs than non-virus infected colonies. Three days after incubation at 37 °C all colonies exhibited morphological alterations with various numbers of blebs per colony. In the vicinity of these blebs, virus could be seen sporadically by electron microscopy (Figs. 6 and 7).
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Fig. 6. Section of part of a colony of JAI infected with group I-virus. Virus particles are seen as thin rods within a large cell (Bar: 250 nm),
Fig. 7. Section of part of a colony of A. laidlawii infected with group 2-virus. Several spherical, enveloped virus particles are seen in close proximity to a giant cell and in the cell (Bar: 250 nm).
Influence of penicillin or thallium acetate. When penicillin G or thallium acetate were omitted from the growth medium a reduction in the frequency of bleb-formation in JA 1 colonies was not observed, indicating that neither penicillin nor thallium acetate were responsible for the morphological alterations, described above.
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Discussion Striking alterations of the colony-morphology of A. laidlatoii, not arti ficially infected with a virus, are described. Blebs can be ob served by light microscopy in a high percent age of the colonies. In th e scanning electron micro scop e " craters" can be detected in the surface of colonies containing blebs. When altered colonies are studi ed by tr ansmission electron micro scopy, large cells, up to 20 tim es the size of a normal cell can be seen. These morphological alterations can be fo und in strain JA 1 but not in the or al stra in of A. laidlatoii. Strain JA 1 is susceptible to infection with Mycoplasmatales-viruses whereas th e ora l stra in of A. laidlatoii is not. The reason for the development of giant-cells in JA 1, not arti ficially infected with viruses, may be that JA 1 carri es a virus in so me cryptic form. Thi s conclusion can be drawn from the fact that th e three kn own Mycoplasmatales-viruses were isolated from either spontaneous plaques or from washes of lawns of cert ain str ains of A. laidlaioii (9, 10, 11,20). Large bodies of L-form s of bacteria and of M. hominis have been seen before (1, 13, 14,22,25). The large cells of M. hominis measured up to 10,um in diameter (1). After artificial infection with group 1 virus, Milne et aI. (18) described A.laidlatoii colonies which appeared collapsed or "cratered" and which contained numerous rod shaped viru s particles. We could also detect group 1 and gro up 2 viruses within host cells. "Craters" cou ld be th e result of destruction of mycopl asma cells within a colony during th e development of th e viruses. An impor ta nt mechanism of pa thogenicity of Mycoplasma pneumoniae is the ability of the organisms to adhere to epithelial cells of th e respirator y tract (24). Gabridge et aI. (8) recentl y pr omoted the hypothesis th at adherence leads to fusion of th e cell membrane of th e par asite with that of its host. Fusion of M . gallisepticum with erythrocy tes and of A. laidlawii with isolated lipid vesicles has been shown (2, 25). Me mbra ne fusion has also been assumed for M . homin is (1). Thus, th e formation of giant cells of A. laidlawii as reported here, could be the result of cell fusion or inh ibition of binar y fission (21, 22). The development of p olykaryocytes by cell fusion is a common cytop athi c effect in cells undergoing pr oductive infectio n with viruses of the herpes-, param yxo-, and poxviru s gro ups (15, 23). On the othe r hand , th e possibility th at geno me replication occur s whereas binar y fission is inhibited, cannot be excluded. Gr owth of A. laidlawii strai n JA 1 is easy to achieve. This strai n of A . laidlawii is th e host of three basically different viruses. The system would appe ar to be very suita ble for the study of surface changes of the host cell during membrane fusion, since studies with influenza- and herp es-virus have suggested th at th e viral structure involved in the early events of cell fusion is located on the surface of the membrane (15,23). Although fusion of biological membranes is an important and fundamental event in cell biology only very few stu dies have been dir ectly concerned with the mechanism of this process (19).
Acknowledgments The authors thank ] . Malliloff for his advice. The excellent technical assistance of Gerhard Wiesner is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Br 346 und SFB 47.
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References 1. Anderson, D.R. and M.F.Barile: Ultrastructure of Mycoplasma bominis. J. Bact. 90 (1965) 180-192 2. Apostolov, K. and G. D. Windsor: The interaction of Mycoplasma gallisepticum with erythrocytes. I. Morphology. Microbios 13 (1975) 205-215 3. Barksdale, I. and S.B.Arden: Persisting bacteriophage infections, lysogeny, and phage conversions. Ann. Rev. Microbiol. 28 (1974) 265-299 4. Barksdale, I., I. Garmise, and R. Rivera: Toxinogeny in Corynebacterium diphtheriae. J. Bact. 81 (1961) 527-540 5. Brunner, H., H.Krauss, H.Schaar, and H.-G. Schiefer: Electron microscopic studies on the attachment of Mycoplasma pneumoniae to guinea pig erythrocytes. Infect. Immun. 24 (1979) 906-911 6. Congdon, A.L. and G.E.Kenny: Alteration of colonial morphology of Acholeplasma laidlawii and Acholeplasma modicum by infection with Mycoplasmatales viruses. J. Bact. 138 (1979) 962-968 7. Dorner, I., H. Brunner, H.-G. Schiefer, and H.-J. Wellensiek: Complement-mediated killing of Acholeplasma laidlawii by antibodies to various membrane components. Infect. Immun.13 (1976) 1663-1670 8. Gabridge, M. G., Y.D. Barden-Stahl, R. B. Polisky, and J. A. Engelhardt: Differences in the attachment of Mycoplasma pneumoniae cells and membranes to tracheal epithelium. Infect. Immun. 16 (1977) 766-772 9. Gourlay, R. N.: Mycoplasmatales virus-laidlawii 2, a new virus isolated from Acholeplasma laidlawii. J. gen. Virol. 12 (1971) 65-67 10. Gourlay, R.N., J.Bruce, and D.J.Garwes: Characterization of Mycoplasmatales viruslaidlawii 1. Nature (Lond.) New BioI. 229 (1971) 118-119 11. Gourlay, R. N., D.]. Garwes, ]. Bruce, and S. G. Wyld: Further studies on the morphology and composition of Mycoplasmatales virus-laidlawii 2. J. gen. Virol. 18 (1973) 127-133 12. Groman, N. B.: Evidence for the active role of bacteriophage in the conversion of non toxigenic Corynebacterium diphtheriae to toxin production. J. Bact. 69 (1955) 9-15 13. lnosca, H.: Genetique sur une propriere de Bacillus megaterium liee a la presence d'un prophage. C.R. Acad. Sci. 237 (1953) 1794-1795 14. Katz, I. N., S. A. Gulevskaya, and S. V. Prozorovsky: Morphological characteristics of large bodies in the cultures of L-forms of bacteria according to scanning electron microscopy. Zh. Mikrobiol. (Mosk.) 10 (1977) 108-111 15. Ludwig, H., H. Becht, and R. Rott : Inhibition of Herpes Virus-induced cell fusion by concanavalin A, Antisera, and 2-deoxy-D-glucose. J. Virol. 14 (1974) 307-314 16. Maniloff, J... J. Das, and J. R. Christensen: Viruses of Mycoplasmas and Spiroplasmas. Advanc. Virus Res. 21 (1977) 343-380 17. Maniloff, J. and A. Liss: Comparative structure, chemistry and evolution of mycoplasmaviruses. In: Viruses, evolution and cancer, p. 583-604, E. Kurstak and K. Maramorosch, ed. Academic Press Inc., New York (1974) 18. Milne, R. G., G. W. Thompson, and D. Taylor-Robinson: Electron microscope observations on Acholeplasma laidlawii viruses. Arch. ges. Virusforsch. 37 (1972) 378-385 19. Poste, G.: Mechanisms of virus-induced cell fusion. Rev. Cytol. 33 (1972) 157-252 20. Putzrath, R. M. and J. Maniloff: Growth of an eveloped mycoplasmavirus and establishment of a carrier state. J. Virol. 22 (1972) 308-314 21. Razin, S.: The Mycoplasmas. Microbiol. Rev. 42 (1978) 414-470 22. Razin, S. and O. Oliver: Morphogenesis of Mycoplasma and bacterial L-form colonies. J. gen. Microbiol. 24 (1961) 225-237 23. Raft, R., H. Becht, H. Hammer, H. D. Klenk, and C. Scholtissek: Changes of the surface of the host cell after infection with enveloped viruses. In: R. D. Barry and B.W. J. Mahy (ed.), Negative strand viruses. Academic Press Inc., New York (1974)
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24. Sobeslavsky, 0., B.Prescott, and R.M.Chanock: Adsorption of Mycoplasma pneumoniae to neuraminic acid receptors of various cells and possible role in virulence. J. Bact. 96 (1968) 695-705 25. Teuber, M. and [, Bader: Action of polymyxin B on bacterial membranes: phosphatidyglycerol- and cardiolipin-induced susceptibility to polymyxin B in Acholeplasma laidlawii B. Antimicrob. Agents Chemother. 9 (1976) 26-35 26. Zabriskie, ]. B.: The role of temperate bacteriophage in the production of erythrogenic toxin by Group A streptococci. J. expo Med. 119 (1964) 761-780
Professor Dr. med. Helmut Brunner, Institut fur Medizinische Mikrobiologie und Virologie der Universirat, Moorenstr. 5, D-4000 Dusseldorf, Federal Republic of Germany
Giant Cell Formation in Acholeplasma laidlawii, Strain JA 1 Riesenzellbildung von Acholeplasma laidlawii, Stamm JAl HELMUT BRUNNER!, HERBERT SCHAAR 2, and HARTMUT KRAUSS3
With 7 Figures· Received March 18, 1980
Abstract Striking alterations, which looked like blebs, were observed in colonies of Acholeplasma laidlawii, strain JA 1. "Craters" were seen on the surface of these colonies by scanning electron microscopy. In addition, giant celIs of acholeplasma, up to 14,um in diameter, i.e. approximately 20 times the size of a normal A. laidlawii cell, were visible in these colonies. The large forms were surrounded by a unit membrane. After infection with group 1 and group 2 mycoplasma-viruses, the proportion of altered colonies and the number of large celIs within these colonies increased. A strain of A. laidlau/ii, which was not susceptible to infection with the three known acholeplasma-viruses did not exhibit comparable morphological changes. The development of the giant cells can be explained either by cell fusion or by a lag of cell division behind genome replication. Blebs and "craters" may result from the destruction of mycoplasma organisms within the complex structure of the colony. There is also suggestive evidence that strain JA 1 carries a virus in some cryptic form.
Zusammenfassung Auffallige Veranderungen, die wie Blaschen imponierten, wurden in den Kolonien von Acholeplasma laidlawii, Stamm JA 1, beobachtet. Im Rasrerelektronenmikroskop wurcn "Krater" in der Oberflache dieser Kolonien zu sehen. Aufserdem zeigten sich in den veranderten Kolonien Riesenzellen mit einem Durchmesser bis zu 14,um, d. h. der ungefahr 20fachen Grage einer normalen Acholeplasma-Zelle. Die grofsen Formen waren vollstandig von einer doppelschichtigen Membran umgeben. Nach Infektion mit Mycoplasma-Viren der Gruppen 1 und 2 nahm der AnteiI veranderrer Kolonien und die Zahl von Riesenzellen in diesen Kolonien zu. Ein Stamm von A. laidlawii, der nicht empfanglich war fur eine Infektion mit den drei bekannten Acholeplasma-Viren, zeigte keine vergleichbaren morphologischen Veranderungen. Die Entwicklung der Riesenzellen kann man entweder durch eine Zellfusion oder durch eine Verzogerung der Zellteilung gegenuber der Genomrepli-
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kation erklaren. Blaschen und "Krater" konnten das Resultat einer Zerstorung von Mycoplasmen innerhalb der komplexen Struktur einer Kolonie sein. Es gibt Hinweise dafiir, dag der Stamm JA 1 latent mit einem Virus infiziert ist.
Introduction Recently, Congdon and Kenny, observed strikingly altered and distinct colonies of Acholeplasma laidlawii, infected with Mycoplasmatales virus-laidlawii MV-Ll and MV-L3 (6). This is of special interest, since Mycoplasmatales are important pathogens for man, animals, plants and insects. Our knowledge about the pathogenic mechanisms of mycoplasmas is scarce. It should therefore be kept in mind, that the virulence of some pathogenic mycoplasma species may be determined by the presence of a phage, as has already been demonstrated for Corynebacterium diphtheriae and for group A Streptococci (3, 4, 12, 26). In addition, mycoplasmas are of special importance for basic research in microbiology, because they are the smallest free living organisms known. They lack a rigid cell wall and therefore expose their cytoplasmic membrane directly to the environment. In this publication, we provide evidence that A. laidlawii, strain JA 1, not artificially infected with a virus, forms colonies with bleb-like alterations. In scanning electron microscopy these blebs appear as "craters". In addition, we have seen giant cells of A.laidlawii, up to 14.um in diameter, in blebbed colonies. After infection with group 1 or group 2 acholeplasma-viruses the number of these giant cells increased.
Materials and Methods Acholeplasmas and viruses. Acholeplasma laidlawii, strain JA 1, furthermore a strain of A.laidlawii, recovered from the human oral cavity, the bullet shaped Mycoplasmatales virus-laidlawii 1 (group 1) and the enveloped, spherical group 2 virus were used (16). The JA J-strain and the viruses were kindly provided by]. Maniloff of the University of Rochester, Rochester, N. Y. The oral strain of A. laidlawii had been obtained from R. M. Chanock, National Institutes of Health, Bethesda, Md. (7). Growth medium and culture conditions. One liter of fluid growth medium contained 20 g tryptose, 5 g tris-(hydroxymethyl)-aminomethane, 5 g sodium-chloride, 10 g glucose and 10 ml PPLO-serum fraction (Difco-Laboratories, Detroit, Michigan). Agar-medium contained in addition 11 g special agar Noble (Difco) and 0.05% thallium-acetate. In order to obtain organisms in the log-growth-phase, daily 4.5 ml of the tryptose-broth were inoculated with 0.1 ml of an overnight culture of A.laidlawii. After 24-hours of incubation at 37°C this resulted in a suspension, containing 1 to 2 X 108 colony forming units (CFU) if A. laidlawii per m!. Propagation of mycoplasma-viruses. Group 1 and group 2 viruses were grown on JA 1 lawns as described by Maniloff and Liss (17). The suspension, containing JA1 and the virus, was filtered through an 0.2 ,urn pore size Millipore filter. This yielded a virus suspension of approximately 1 to 2 X 1010 plaque forming units (PFU) per m!. The viruses were plaqued on JA 1 lawns as described by Maniloff and Liss (17). Light microscopy. Colonies were photographed with a Zeiss photomicroscope. Transmission electron microscopy. A. laidlawii colonies were cut out of the agar and fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 2 hrs at 4°C, postfixed with 1.5% osmium tetroxide in the same buffer for additional 2 hrs, washed with
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distilled water, treated with 0.5% uranyl acetate in distilled water for 20 min, washed again, dehydrated in acetone and embedded in Durcupan (Fluka, Basel). Thin sections were stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop 102 at 80 kV (5). Scanning Electron Microscopy. Colonies with and without blebs were cut out of the agar, fixed with 5% glutaraldehyde in 0.05 M cacodylate buffer, postfixed with 1.5% osmium tetroxide in the same buffer, and dehydrated in various concentrations (30,50,70, 90 and 100%) of acetone in 0.05 M cacodylate buffer for 10 mil'. each. The acetone was exchanged with amylacetate in four 10 min-steps (25, 50, 75 and 100%), the agar blocks fixed on a specimen stud, coated with gold by a Balzers Union sputtering device and examined in a Leitz Scanning electron microscope AMR 1600 T.
Results
Light microscopy. Overnight cultures of A.laidlawii JA1, grown in tryptose broth, which had not been infected with a virus, were inoculated on tryptose-agar. After incubation at 37°C for 2 to 6 days colonies were examined by light microscopy at a magnification of 40 X. Already after 48 hrs incubation, when visible colonies appeared, some of these young colonies showed alterations of their morphology. In the centers of the colonies multiple bleb-like changes could be seen (Fig. 1). These blebs later spread over the entire colony. By day 4 to 5 approximately 50 per cent of the colonies were covered with these vesicles (Fig. 1). In the remaining colonies morphological alterations were not seen during the observation period of 6 days. In contrast to JA1, colonies of the oral strain of A.laidlawii did not exhibit any blebs. Scanning electron microscopy. In the scanning electron microscope "craters" were seen in the surface of the colonies (Fig. 2). In addition, large forms of mycoplasmas could be observed in the vicinity of the "craters" (Fig. 2).
Fig. 1. Photomicrograph of A. laidlawii ]A 1, not artificially infected with a virus, Part of a normal colony (left) and of a colony with multiple blebs can be seen (Bar: 10 ,urn).
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Fig. 2. Scanning electron micrograph of a colony of A. laidlatoii, strain ]A 1. A "crater" can be seen on the surface of the colony. In the vicinity of the "crater" large and normal cells of A.laidlawii are visible (Bar: 2 {lm).
Transmission electron microscopy. In addition to normal mycoplasmas, electron microscopic investigation of altered colonies revealed large forms bound by a unit membrane (Fig. 3, 4, 5). The size of these giant cells was on the average 2-10,um, some measured up to 14,um (Fig. 3 and 4). Compared to the many acholeplasma cells of normal size, the cytoplasma of the giant-cells appeared often less dense. A few cells showed finger-like projections sometimes resembling "tumorous" growth of tissue (Fig. 4 and 5). In later stages giant forms appeared as membrane bound vesicles (Fig. 6).
Fig. 3. Section of cells of A.laidlawii, not artificially infected with a virus. A giant cell, surrounded by a unit membrane and several cells of normal size can be seen (Bar: 1 {lm).
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Fig. 4. Longitudinal section of a colony of A. Iaidlawii JA 1 showing a giant cell with filamentous extrusions (Bar: 1 pm).
Fig. 5. Longitudinal section through a "cratered" colony of A.laidlawii JA 1 showing tumor-like growth of a giant cell (Bar: 1 pm).
Appearance of ] A 1 colonies after infection with group 1 or group 2 Viruses. Colonies of A. laidlawii JA1 which had been infected with group 1 or group 2 mycoplasma viruses showed by far more blebs than non-virus infected colonies. Three days after incubation at 37 °C all colonies exhibited morphological alterations with various numbers of blebs per colony. In the vicinity of these blebs, virus could be seen sporadically by electron microscopy (Figs. 6 and 7).
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Fig. 6. Section of part of a colony of JAI infected with group I-virus. Virus particles are seen as thin rods within a large cell (Bar: 250 nm),
Fig. 7. Section of part of a colony of A. laidlawii infected with group 2-virus. Several spherical, enveloped virus particles are seen in close proximity to a giant cell and in the cell (Bar: 250 nm).
Influence of penicillin or thallium acetate. When penicillin G or thallium acetate were omitted from the growth medium a reduction in the frequency of bleb-formation in JA 1 colonies was not observed, indicating that neither penicillin nor thallium acetate were responsible for the morphological alterations, described above.
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Discussion Striking alterations of the colony-morphology of A. laidlatoii, not arti ficially infected with a virus, are described. Blebs can be ob served by light microscopy in a high percent age of the colonies. In th e scanning electron micro scop e " craters" can be detected in the surface of colonies containing blebs. When altered colonies are studi ed by tr ansmission electron micro scopy, large cells, up to 20 tim es the size of a normal cell can be seen. These morphological alterations can be fo und in strain JA 1 but not in the or al stra in of A. laidlatoii. Strain JA 1 is susceptible to infection with Mycoplasmatales-viruses whereas th e ora l stra in of A. laidlatoii is not. The reason for the development of giant-cells in JA 1, not arti ficially infected with viruses, may be that JA 1 carri es a virus in so me cryptic form. Thi s conclusion can be drawn from the fact that th e three kn own Mycoplasmatales-viruses were isolated from either spontaneous plaques or from washes of lawns of cert ain str ains of A. laidlaioii (9, 10, 11,20). Large bodies of L-form s of bacteria and of M. hominis have been seen before (1, 13, 14,22,25). The large cells of M. hominis measured up to 10,um in diameter (1). After artificial infection with group 1 virus, Milne et aI. (18) described A.laidlatoii colonies which appeared collapsed or "cratered" and which contained numerous rod shaped viru s particles. We could also detect group 1 and gro up 2 viruses within host cells. "Craters" cou ld be th e result of destruction of mycopl asma cells within a colony during th e development of th e viruses. An impor ta nt mechanism of pa thogenicity of Mycoplasma pneumoniae is the ability of the organisms to adhere to epithelial cells of th e respirator y tract (24). Gabridge et aI. (8) recentl y pr omoted the hypothesis th at adherence leads to fusion of th e cell membrane of th e par asite with that of its host. Fusion of M . gallisepticum with erythrocy tes and of A. laidlawii with isolated lipid vesicles has been shown (2, 25). Me mbra ne fusion has also been assumed for M . homin is (1). Thus, th e formation of giant cells of A. laidlawii as reported here, could be the result of cell fusion or inh ibition of binar y fission (21, 22). The development of p olykaryocytes by cell fusion is a common cytop athi c effect in cells undergoing pr oductive infectio n with viruses of the herpes-, param yxo-, and poxviru s gro ups (15, 23). On the othe r hand , th e possibility th at geno me replication occur s whereas binar y fission is inhibited, cannot be excluded. Gr owth of A. laidlawii strai n JA 1 is easy to achieve. This strai n of A . laidlawii is th e host of three basically different viruses. The system would appe ar to be very suita ble for the study of surface changes of the host cell during membrane fusion, since studies with influenza- and herp es-virus have suggested th at th e viral structure involved in the early events of cell fusion is located on the surface of the membrane (15,23). Although fusion of biological membranes is an important and fundamental event in cell biology only very few stu dies have been dir ectly concerned with the mechanism of this process (19).
Acknowledgments The authors thank ] . Malliloff for his advice. The excellent technical assistance of Gerhard Wiesner is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Br 346 und SFB 47.
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24. Sobeslavsky, 0., B.Prescott, and R.M.Chanock: Adsorption of Mycoplasma pneumoniae to neuraminic acid receptors of various cells and possible role in virulence. J. Bact. 96 (1968) 695-705 25. Teuber, M. and [, Bader: Action of polymyxin B on bacterial membranes: phosphatidyglycerol- and cardiolipin-induced susceptibility to polymyxin B in Acholeplasma laidlawii B. Antimicrob. Agents Chemother. 9 (1976) 26-35 26. Zabriskie, ]. B.: The role of temperate bacteriophage in the production of erythrogenic toxin by Group A streptococci. J. expo Med. 119 (1964) 761-780
Professor Dr. med. Helmut Brunner, Institut fur Medizinische Mikrobiologie und Virologie der Universirat, Moorenstr. 5, D-4000 Dusseldorf, Federal Republic of Germany