Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation (original) (raw)

Abstract

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

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  1. ALLEN E. G., BOVARNICK M. R., SNYDER J. C. The effect of irradiation with ultraviolet light on various properties of typhus rickettsiae. J Bacteriol. 1954 Jun;67(6):718–723. doi: 10.1128/jb.67.6.718-723.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anacker R. L., Gerloff R. K., Thomas L. A., Mann R. E., Brown W. R., Bickel W. D. Purification of Rickettsia rickettsi by density-gradient zonal centrifugation. Can J Microbiol. 1974 Nov;20(11):1523–1527. doi: 10.1139/m74-238. [DOI] [PubMed] [Google Scholar]
  3. BOVARNICK M. R., MILLER J. C. Oxidation and transamination of glutamate by typhus rickettsiae. J Biol Chem. 1950 Jun;184(2):661–676. [PubMed] [Google Scholar]
  4. Canonico P. G., Van Zwieten M. J., Christmas W. A. Purification of large quantities of coxiella burnetii rickettsia by density gradient zonal centrifugation. Appl Microbiol. 1972 May;23(5):1015–1022. doi: 10.1128/am.23.5.1015-1022.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dubois D. R., Cutchins E. C., Berman S., Lowenthal J. P., Timchak R. L. Preparation of purified suspensions of Coxiella burneti by Genetron extraction followed by continuous-flow ultracentrifugation. Appl Microbiol. 1972 May;23(5):841–845. doi: 10.1128/am.23.5.841-845.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Friis R. R. Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J Bacteriol. 1972 May;110(2):706–721. doi: 10.1128/jb.110.2.706-721.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. GIMENEZ D. F. STAINING RICKETTSIAE IN YOLK-SAC CULTURES. Stain Technol. 1964 May;39:135–140. doi: 10.3109/10520296409061219. [DOI] [PubMed] [Google Scholar]
  8. Goodwin C. S., Tyrrell D. A., Head B., Rees R. J. Inhibition of haemaggregation by lepromin and other mycobacterial substances. Nature. 1967 Dec 9;216(5119):1019–1020. doi: 10.1038/2161019a0. [DOI] [PubMed] [Google Scholar]
  9. HOPPS H. E., HAHN F. E., WISSEMAN C. L., Jr, JACKSON E. B., SMADEL J. E. Metabolic studies of rickettsiae. III. Studies of transamination, oxidative phosphorylation and glutamate-2-C14 incorporation by purified suspensions of Rickettsia mooseri. J Bacteriol. 1956 Jun;71(6):708–716. doi: 10.1128/jb.71.6.708-716.1956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HOYER B. H., ORMSBEE R. A., FISET P., LACKMAN D. B. Differentiation of Phase I and Phase II Coxiella burnetti by equilibrium density gradient sedimentation. Nature. 1963 Feb 9;197:573–574. doi: 10.1038/197573a0. [DOI] [PubMed] [Google Scholar]
  11. Howard L., Orenstein N. S., King N. W. Purification on renografin density gradients of Chlamydia trachomatis grown in the yolk sac of eggs. Appl Microbiol. 1974 Jan;27(1):102–106. doi: 10.1128/am.27.1.102-106.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. PARETSKY D., DOWNS C. M., CONSIGLI R. A., JOYCE B. K. Studies on the physiology of rickettsiae. I. Some enzyme systems of Coxiella burnetii. J Infect Dis. 1958 Jul-Aug;103(1):6–11. doi: 10.1093/infdis/103.1.6. [DOI] [PubMed] [Google Scholar]
  14. PEACOCK A. C., BUNTING S. L., QUEEN K. G. SERUM PROTEIN ELECTROPHORESIS IN ACRYLAMIDE GEL: PATTERNS FROM NORMAL HUMAN SUBJECTS. Science. 1965 Mar 19;147(3664):1451–1453. doi: 10.1126/science.147.3664.1451. [DOI] [PubMed] [Google Scholar]
  15. Palmer E. L., Mallavia L. P., Tzianabos T., Obijeski J. F. Electron microscopy of the cell wall of Rickettsia prowazeki. J Bacteriol. 1974 Jun;118(3):1158–1166. doi: 10.1128/jb.118.3.1158-1166.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. ROTHBLAT G. H. PPLO contamination in tissue cultures. Ann N Y Acad Sci. 1960 Jan 15;79:430–432. doi: 10.1111/j.1749-6632.1960.tb42708.x. [DOI] [PubMed] [Google Scholar]
  17. Ramm L. E., Winkler H. H. Rickettsial hemolysis: adsorption of rickettsiae to erythrocytes. Infect Immun. 1973 Jan;7(1):93–99. doi: 10.1128/iai.7.1.93-99.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. SEVER J. L. Application of a microtechnique to viral serological investigations. J Immunol. 1962 Mar;88:320–329. [PubMed] [Google Scholar]
  19. SNYDER J. C., BOVARNICK M. R., MILLER J. C., CHANG R. S. M. Observations on the hemolytic properties of typhus rickettsiae. J Bacteriol. 1954 Jun;67(6):724–730. doi: 10.1128/jb.67.6.724-730.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thompson H. A., Baca O. G., Paretsky D. Presence of ribosomal ribonucleic acid in the rickettsia Coxiella burneti. Biochem J. 1971 Nov;125(1):365–366. doi: 10.1042/bj1250365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. WEISS E., DRESSLER H. R., SUITOR E. C., Jr Selection of mutant strain of Rickettsia prowazeki resistant to p-aminobenzoic acid. J Bacteriol. 1957 Mar;73(3):421–430. doi: 10.1128/jb.73.3.421-430.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. WISSEMAN C. L., Jr, HAHN F. E., JACKSON E. B., BOZEMAN M. F., SMADEL J. E. Metabolic studies of rickettsiae. II. Studies on the pathway of glutamate oxidation by purified suspensions of Rickettsia mooseri. J Immunol. 1952 Mar;68(3):251–264. [PubMed] [Google Scholar]
  23. WISSEMAN C. L., Jr, JACKSON E. B., HAHN F. E., LEY A. C., SMADEL J. E. Metabolic studies of rickettsiae. I. The effects of antimicrobial substances and enzyme inhibitors on the oxidation of glutamate by purified rickettsiae. J Immunol. 1951 Aug;67(2):123–136. [PubMed] [Google Scholar]
  24. Weiss E., Newman L. W., Grays R., Green A. E. Metabolism of Rickettsia typhi and Rickettsia akari in irradiated L cells. Infect Immun. 1972 Jul;6(1):50–57. doi: 10.1128/iai.6.1.50-57.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wiebe M. E., Burton P. R., Shankel D. M. Isolation and characterization of two cell types of Coxiella burneti phase I. J Bacteriol. 1972 Apr;110(1):368–377. doi: 10.1128/jb.110.1.368-377.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wisseman C. L., Jr, Waddell A. D. In vitro studies on rickettsia-host cell interactions: intracellular growth cycle of virulent and attenuated Rickettsia prowazeki in chicken embryo cells in slide chamber cultures. Infect Immun. 1975 Jun;11(6):1391–1404. doi: 10.1128/iai.11.6.1391-1401.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]