INFECTION AND IMMUNITY, Dec. 1977, p. 857-859 Copyright X 1977 American Society for Microbiology
Vol. 18, No. 3 Printed in U.S.A.
NOTES Anabolic Potential of Virulent Treponema pallidum JOEL B. BASEMAN* AND NANCY S. HAYES Department of Bacteriology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 Received for publication 29 July 1977
Acrylamide gel autoradiography of 3H-labeled proteins from Treponema pallidum demonstrates that virulent treponemes incubated in vitro synthesize a spectrum of high-molecular-weight proteins. A comparison of the protein profiles of T. pallidum with the Reiter treponeme shows that T. pallidum possesses significant anabolic competence. Recent information concerning metabolic and energy-generating pathways in virulent Treponema pallidum reveals a considerable degree of catabolic activity (3, 9, 11, 12). In addition, we earlier reported that T. pallidum was not defective in its ability to synthesize proteins based upon the incorporation of radioactive amino acids into trichloroacetic acid-precipitable material (2). Other evidence indicates that coincubation of virulent treponemes with animal cells in monolayer culture under specified conditions permits the retention of motility and virulence of T. pallidum for 2 to 3 weeks (6). Based upon these data and its estimated in vivo generation time of 30 to 33 h (5, 10), this spirochete should be capable of in vitro multiplication. However, none has been observed, or reports suggesting growth remain unsubstantiated. A summary of data relating to coincubation and other aspects of T. pallidum research has been published (1). In contrast to the above and consistent with potential deficiencies in T. pallidum metabolism, we reported that degradation of radioactive pyruvate and glucose by virulent treponemes was not accompanied by incorporation of carbon into structural or macromolecular components (3, 11). We also indicated that ribonucleic acid synthesis and processing by T. pallidum appeared abnormal during extended in vitro incubation (1; J. C. Nichols and J. B. Baseman, submitted for publication) and that treponemes demonstrated reduced attachment to host cell surfaces under similar conditions (7). It is possible that previous reports concerning the in vitro behavior of T. pallidum merely reflect the endogenous maintenance of resting treponemes in relatively nontoxic environments. Then how is the degree of anabolic competence in T. pallidum determined when the treponeme cannot
be grown in vitro? Serious deficiencies in macromolecular synthesis would readily explain the inability to cultivate this spirochete. We have attempted to circumvent this problem by using the technique of gel autoradiography to clarify capabilities of T. pallidum. Conceptually we felt that metabolically competent microorganisms, with growth potential, should synthesize numerous high-molecular-weight proteins and that this profile should compare favorably to patterns obtained from microorganisms proven capable of growth. Virulent treponemes were prepared from infected rabbit testes in extraction medium as previously described (3), except that manipulations were performed under normal atmospheric conditions. After two low-speed centrifugations (500 x g for 5 min at room temperature) to remove the majority of tissue components, the supernatant containing treponemes and some contaminating host cells was adjusted to a density of 108 treponemes per ml of extraction medium. 3H-labeled amino acid mixture (5 ,uCi/ml;
Schwarz/Mann, Orangeburg, N.Y.) was added, and incubation was performed at 340C for 21 h. To establish appropriate controls, cycloheximide or erythromycin at 5 pg/ml was included in specific test samples to inhibit eucaryotic or procaryotic protein synthesis (2). Treponema phagedenis biotype Reiter was grown in Spirolate broth supplemented with 10% heat-inactivated rabbit serum at 350C in an anaerobic hood (Coy Manufacturing, Ann Arbor, Mich.). During the mid-log phase of growth, the Reiter culture was centrifuged at 18,000 x g for 15 min, and the pellet was resuspended to a cell density of 108 treponemes per ml in T. pallidum extraction medium which had been pre-equilibrated in an
Ar-N2-H2-CO2 atmosphere (3). Radioisotope was
857
858
INFECT. IMMUN.
NOTES
added as described above, and the culture was incubated at 350C for 21 h in the anaerobic hood. Then T. pallidum and T. phagedenis biotype Reiter were centrifuged at 18,000 x g and washed twice with cold phosphate-buffered saline. Pellets were resuspended in cold phosphate-buffered saline, and trichloroacetic acid was added to a final concentration of 10%. After at least 4 h at 40C, the trichloroacetic acidprecipitated material was washed once with cold phosphate-buffered saline and dissolved in solubilizing buffer consisting of 0.0625 M
A
B
C
D
tris(hydroxymethyl)aminomethane-hydrochloride (pH 6.8), 2% mercaptoethanol, and 10% glycerol to which 2% sodium dodecyl sulfate was introduced after the pellets were suspended. Bromophenol blue was included as tracking dye, and the samples were boiled for 3 min. Undissolved residues were removed by centrifugation before sample application (50 to 75 ,ug of protein in a 100-,ul volume) to gels. Stacking and separating gels consisted of 3 and 7.5% acrylamide, respectively, and electrophoresis was performed as previously outlined (8). Gels were stained with Coomassie brilliant blue, destained, and sliced longitudinally. Certain gels were immediately dried under vacuum, and others were processed for autoradiography using the dimethyl sulfoxide-diphenyloxazole procedure (4). X-ray film (RP/S-14, Eastman Kodak Co., Rochester, N.Y.) was exposed to dried gels for 2 weeks at -70°C before development. Figure 1 presents the gel profile of T. pallidum proteins. Gel A is the Coomassie brilliant blue pattern, and gels B, C, and D are autoradiographs prepared from treponemal samples that had been radiolabeled in extraction medium alone (B) or with cycloheximide (C) or erythromycin (D). It is clear that T. pallidum synthesizes numerous proteins of varying molecular weights. Based upon the positioning of known marker proteins in similar gels, the slower migrating bands in gels B and C are in the 100,000molecular-weight class. Furthermore, the observed synthesis is totally negated by erythromycin but unaffected by cycloheximide, indicating that T. pallidum is responsible for the anabolic activity. A comparison of gel autoradiographs between T. pallidum and the Reiter treponeme shows that, although differences in protein patterns exist, the number and molecular weight range of the radiolabeled proteins are similar (Fig. 2). These results demonstrate that T. pallidum freshly extracted from rabbit tissue possesses significant biosynthetic capabilities. The lack of success in cultivating these microorganisms is unclear at present but may be attributed to the
0
0_
FIG. 1. Gel profiles of proteins from T. pallidum. Virulent treponemes were exposed to 3H-labeled amino acids for 21 h at 34°C before processing as described in the text. Gel A represents total proteins as stained with Coomassie brilliant blue. Gels B, C, and D are autoradiographs of test samples that were incubated during the radiolabeling period in extraction medium alone (B), or with cycloheximide (C) or
erythromycin (D).
NOTES
VOL. 18, 1977
A
B
859
occurrence of early and potentially irreversible biological damage arising during or shortly after extraction from tissue. It is difficult to identify these technical deficiencies. However, we believe that sensitive assays for determining metabolic competence of T. pallidum under a variety of experimental in vitro conditions will be beneficial to the development of appropriate procedures. Relative to the use of gel autoradiography described here, the increased or decreased synthesis of particular protein species by virulent treponemes may be an early signal of growth potential and permit essential modifications in the environment to optimize anabolic activity of T. pallidum. This work was supported by Public Health Service grant AI-11283 and research career development award 1-K04-AI00178 from the National Institute of Allergy and Infectious Diseases to J.B.B.
0o
FIG. 2. Comparative autoradiographs of T. pallidum and the Reiter treponeme. Gel A: 3H-labeled proteins of T. pallidum. Gel B: 3H-labeled proteins of T. phagedenis biotype Reiter. Experimental conditions are outlined in the text.
LITERATURE CITED 1. Baseman, J. B. 1977. Report of a workshop: the biology of Treponema pallidum. J. Infect. Dis. 136:308-311. 2. Baseman, J. B., and N. S. Hayes. 1974. Protein synthesis by Treponema pallidum extracted from infected rabbit tissue. Infect. Immun. 10:1350-1355. 3. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976. Virulent Treponema pallidum: aerobe or anaerobe. Infect. Immun. 13:704-711. 4. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 5. Cumberland, M. C., and T. B. Turner. 1949. The rate of multiplication of T. pallidum in normal and immune rabbits. Am. J. Syph. Gonorrhea Vener. Dis. 33:201-212. 6. Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977. Prolonged survival of virulent Treponema pallidum (Nichols strain) in cell-free and tissue culture systems. Infect. Immun. 18:173-182. 7. Hayes, N. S., K. E. Muse, A. M. Collier, and J. B. Baseman. 1977. Parasitism by virulent Treponema pallidum of host cell surfaces. Infect. Immun. 17:174-186. 8. Hu, P. C., A. M. Colier, and J. B. Baseman. 1977. Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. J. Exp. Med. 145:1328-1343. 9. Lysko, P. G., and C. D. Cox. 1977. Terminal electron transport in Treponema pallidum. Infect. Immun. 16:885-890. 10. Magnuson, H. J., H. Eagle, and R. Fleischman. 1948. The minimal infectious inoculum of Spirochaeta pallida (Nichols strain), and a consideration of its rate of multiplication in vivo. Am. J. Syph. Gonorrhea Vener. Dis. 32:1-18. 11. Nichols, J. C., and J. B. Baseman. 1975. Carbon sources utilized by virulent Treponema pallidum. Infect. Immun. 12:1044-1050. 12. Schiller, N. L., and C. D. Cox. 1977. Catabolism of glucose and fatty acids by virulent Treponema pallidum. Infect. Immun. 16:60-68.
Vol. 18, No. 3 Printed in U.S.A.
NOTES Anabolic Potential of Virulent Treponema pallidum JOEL B. BASEMAN* AND NANCY S. HAYES Department of Bacteriology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 Received for publication 29 July 1977
Acrylamide gel autoradiography of 3H-labeled proteins from Treponema pallidum demonstrates that virulent treponemes incubated in vitro synthesize a spectrum of high-molecular-weight proteins. A comparison of the protein profiles of T. pallidum with the Reiter treponeme shows that T. pallidum possesses significant anabolic competence. Recent information concerning metabolic and energy-generating pathways in virulent Treponema pallidum reveals a considerable degree of catabolic activity (3, 9, 11, 12). In addition, we earlier reported that T. pallidum was not defective in its ability to synthesize proteins based upon the incorporation of radioactive amino acids into trichloroacetic acid-precipitable material (2). Other evidence indicates that coincubation of virulent treponemes with animal cells in monolayer culture under specified conditions permits the retention of motility and virulence of T. pallidum for 2 to 3 weeks (6). Based upon these data and its estimated in vivo generation time of 30 to 33 h (5, 10), this spirochete should be capable of in vitro multiplication. However, none has been observed, or reports suggesting growth remain unsubstantiated. A summary of data relating to coincubation and other aspects of T. pallidum research has been published (1). In contrast to the above and consistent with potential deficiencies in T. pallidum metabolism, we reported that degradation of radioactive pyruvate and glucose by virulent treponemes was not accompanied by incorporation of carbon into structural or macromolecular components (3, 11). We also indicated that ribonucleic acid synthesis and processing by T. pallidum appeared abnormal during extended in vitro incubation (1; J. C. Nichols and J. B. Baseman, submitted for publication) and that treponemes demonstrated reduced attachment to host cell surfaces under similar conditions (7). It is possible that previous reports concerning the in vitro behavior of T. pallidum merely reflect the endogenous maintenance of resting treponemes in relatively nontoxic environments. Then how is the degree of anabolic competence in T. pallidum determined when the treponeme cannot
be grown in vitro? Serious deficiencies in macromolecular synthesis would readily explain the inability to cultivate this spirochete. We have attempted to circumvent this problem by using the technique of gel autoradiography to clarify capabilities of T. pallidum. Conceptually we felt that metabolically competent microorganisms, with growth potential, should synthesize numerous high-molecular-weight proteins and that this profile should compare favorably to patterns obtained from microorganisms proven capable of growth. Virulent treponemes were prepared from infected rabbit testes in extraction medium as previously described (3), except that manipulations were performed under normal atmospheric conditions. After two low-speed centrifugations (500 x g for 5 min at room temperature) to remove the majority of tissue components, the supernatant containing treponemes and some contaminating host cells was adjusted to a density of 108 treponemes per ml of extraction medium. 3H-labeled amino acid mixture (5 ,uCi/ml;
Schwarz/Mann, Orangeburg, N.Y.) was added, and incubation was performed at 340C for 21 h. To establish appropriate controls, cycloheximide or erythromycin at 5 pg/ml was included in specific test samples to inhibit eucaryotic or procaryotic protein synthesis (2). Treponema phagedenis biotype Reiter was grown in Spirolate broth supplemented with 10% heat-inactivated rabbit serum at 350C in an anaerobic hood (Coy Manufacturing, Ann Arbor, Mich.). During the mid-log phase of growth, the Reiter culture was centrifuged at 18,000 x g for 15 min, and the pellet was resuspended to a cell density of 108 treponemes per ml in T. pallidum extraction medium which had been pre-equilibrated in an
Ar-N2-H2-CO2 atmosphere (3). Radioisotope was
857
858
INFECT. IMMUN.
NOTES
added as described above, and the culture was incubated at 350C for 21 h in the anaerobic hood. Then T. pallidum and T. phagedenis biotype Reiter were centrifuged at 18,000 x g and washed twice with cold phosphate-buffered saline. Pellets were resuspended in cold phosphate-buffered saline, and trichloroacetic acid was added to a final concentration of 10%. After at least 4 h at 40C, the trichloroacetic acidprecipitated material was washed once with cold phosphate-buffered saline and dissolved in solubilizing buffer consisting of 0.0625 M
A
B
C
D
tris(hydroxymethyl)aminomethane-hydrochloride (pH 6.8), 2% mercaptoethanol, and 10% glycerol to which 2% sodium dodecyl sulfate was introduced after the pellets were suspended. Bromophenol blue was included as tracking dye, and the samples were boiled for 3 min. Undissolved residues were removed by centrifugation before sample application (50 to 75 ,ug of protein in a 100-,ul volume) to gels. Stacking and separating gels consisted of 3 and 7.5% acrylamide, respectively, and electrophoresis was performed as previously outlined (8). Gels were stained with Coomassie brilliant blue, destained, and sliced longitudinally. Certain gels were immediately dried under vacuum, and others were processed for autoradiography using the dimethyl sulfoxide-diphenyloxazole procedure (4). X-ray film (RP/S-14, Eastman Kodak Co., Rochester, N.Y.) was exposed to dried gels for 2 weeks at -70°C before development. Figure 1 presents the gel profile of T. pallidum proteins. Gel A is the Coomassie brilliant blue pattern, and gels B, C, and D are autoradiographs prepared from treponemal samples that had been radiolabeled in extraction medium alone (B) or with cycloheximide (C) or erythromycin (D). It is clear that T. pallidum synthesizes numerous proteins of varying molecular weights. Based upon the positioning of known marker proteins in similar gels, the slower migrating bands in gels B and C are in the 100,000molecular-weight class. Furthermore, the observed synthesis is totally negated by erythromycin but unaffected by cycloheximide, indicating that T. pallidum is responsible for the anabolic activity. A comparison of gel autoradiographs between T. pallidum and the Reiter treponeme shows that, although differences in protein patterns exist, the number and molecular weight range of the radiolabeled proteins are similar (Fig. 2). These results demonstrate that T. pallidum freshly extracted from rabbit tissue possesses significant biosynthetic capabilities. The lack of success in cultivating these microorganisms is unclear at present but may be attributed to the
0
0_
FIG. 1. Gel profiles of proteins from T. pallidum. Virulent treponemes were exposed to 3H-labeled amino acids for 21 h at 34°C before processing as described in the text. Gel A represents total proteins as stained with Coomassie brilliant blue. Gels B, C, and D are autoradiographs of test samples that were incubated during the radiolabeling period in extraction medium alone (B), or with cycloheximide (C) or
erythromycin (D).
NOTES
VOL. 18, 1977
A
B
859
occurrence of early and potentially irreversible biological damage arising during or shortly after extraction from tissue. It is difficult to identify these technical deficiencies. However, we believe that sensitive assays for determining metabolic competence of T. pallidum under a variety of experimental in vitro conditions will be beneficial to the development of appropriate procedures. Relative to the use of gel autoradiography described here, the increased or decreased synthesis of particular protein species by virulent treponemes may be an early signal of growth potential and permit essential modifications in the environment to optimize anabolic activity of T. pallidum. This work was supported by Public Health Service grant AI-11283 and research career development award 1-K04-AI00178 from the National Institute of Allergy and Infectious Diseases to J.B.B.
0o
FIG. 2. Comparative autoradiographs of T. pallidum and the Reiter treponeme. Gel A: 3H-labeled proteins of T. pallidum. Gel B: 3H-labeled proteins of T. phagedenis biotype Reiter. Experimental conditions are outlined in the text.
LITERATURE CITED 1. Baseman, J. B. 1977. Report of a workshop: the biology of Treponema pallidum. J. Infect. Dis. 136:308-311. 2. Baseman, J. B., and N. S. Hayes. 1974. Protein synthesis by Treponema pallidum extracted from infected rabbit tissue. Infect. Immun. 10:1350-1355. 3. Baseman, J. B., J. C. Nichols, and N. S. Hayes. 1976. Virulent Treponema pallidum: aerobe or anaerobe. Infect. Immun. 13:704-711. 4. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 5. Cumberland, M. C., and T. B. Turner. 1949. The rate of multiplication of T. pallidum in normal and immune rabbits. Am. J. Syph. Gonorrhea Vener. Dis. 33:201-212. 6. Fieldsteel, A. H., F. A. Becker, and J. G. Stout. 1977. Prolonged survival of virulent Treponema pallidum (Nichols strain) in cell-free and tissue culture systems. Infect. Immun. 18:173-182. 7. Hayes, N. S., K. E. Muse, A. M. Collier, and J. B. Baseman. 1977. Parasitism by virulent Treponema pallidum of host cell surfaces. Infect. Immun. 17:174-186. 8. Hu, P. C., A. M. Colier, and J. B. Baseman. 1977. Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. J. Exp. Med. 145:1328-1343. 9. Lysko, P. G., and C. D. Cox. 1977. Terminal electron transport in Treponema pallidum. Infect. Immun. 16:885-890. 10. Magnuson, H. J., H. Eagle, and R. Fleischman. 1948. The minimal infectious inoculum of Spirochaeta pallida (Nichols strain), and a consideration of its rate of multiplication in vivo. Am. J. Syph. Gonorrhea Vener. Dis. 32:1-18. 11. Nichols, J. C., and J. B. Baseman. 1975. Carbon sources utilized by virulent Treponema pallidum. Infect. Immun. 12:1044-1050. 12. Schiller, N. L., and C. D. Cox. 1977. Catabolism of glucose and fatty acids by virulent Treponema pallidum. Infect. Immun. 16:60-68.
