L cells that had been exposed to 3,000 r of "tCo the previous day were used to study the growth and metabolism of Rickettsia typhi and R. akari. Viable (unirradiated) L cells were used to study the effect of rickettsial infection on host-cell metabolism. Monolayers were infected with a rickettsial multiplicity of 1.2 and given Eagle's minimal essential medium containing 25 mm N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer and 10% calf serum. At various intervals, cycloheximide (2 ,g/ml) was added to one set of cultures, to inhibit eukaryotic protein and deoxyribonucleic acid (DNA) metabolism; phosphate-buffered saline (PBS) was added to another set. After 1 hr, the cultures received a mixture of 15 14C_ labeled amino acids or adenine-8-_4C. The cultures were harvested 16 hr later and were tested for incorporation of labeled carbon into the fraction precipitated by cold trichloroacetic acid. Viable cells were exposed to thymidine-2-14C for 2-hr periods. Infectivity of R. typhi increased to a peak of 150 to 400 hemolytic units/ culture on day 4; the titer remained approximately the same on days 5 and 6, and declined rapidly on day 7. Total amino acid incorporation was about the same in infected and uninfected cultures up to day 6, but metabolic activity was reduced to a negligible level on day 7 in infected cells. Cycloheximide-resistant activity was higher in the infected cultures, with a peak equivalent to one-half the total activity at day 4 to 5. Total as well as cycloheximide-resistant adenine incorporation was higher in the infected cells between days 3 and 5 after infection, with a peak at day 3 to 4. Somewhat similar results were obtained with R. akari, except that the cycle of infection and of cycloheximide-resistant activity proceeded and was completed more rapidly. 14C-DNA of both rickettsiae was isolated from infected cultures that had received labeled adenine. With labeled thymidine, which was not incorporated by the rickettsiae, it was shown that R. typhi and R. akari differ considerably in their effects on the host cell. R. typhi elicited moderate inhibition, whereas R. akari infection led to a complete inhibition of thymidine incorporation by the third day, at the time of highest rickettsial activity. It is concluded that rickettsiae have the necessary enzymes for protein and nucleic acid synthesis, but, thus far, these enzymes have been activated or induced only in an intracellular environment. Alexander (1, 2) has clearly shown that cycloheximide-an inhibitor of eukaryotic, but not of prokaryotic protein synthesis (6, 12)-is an excellent tool for the study of the metabolism of chlamydiae multiplying in cell cultures. We have used this compound to study the metabolism of Rickettsia typhi and R. akari grown in L cells. Because the growth of rickettsiae is considerably slower than that of chlamydiae, and because rickettsiae are highly susceptible to antibacterial antibiotics commonly used in cell cultures, three modifications were introduced in the procedure of Alexander (1). (i) To prov...
The metabolism of Rickettsia tsutsugamushi (Gilliam strain) multiplying in irradiated L cells was investigated bymethods involving the use of "4C-labeled substrates and cycloheximide, an inhibitor of eukaryotic metabolism. Cycloheximide-resistant amino acid and adenine incorporations were appreciably higher in infected than in uninfected cultures during the period from 3 to 5 or 6 days postinoculation. The metabolism of R. rickettsi was similarly studied in primary duck embryo cells, which are more susceptible to infection with this rickettsia than are L cells. A difference in cycloheximide-resistant activity between infected and uninfected cultures was also noted, but was small. This finding is attributed to the more limited growth of R. rickettsi.Previous work from this laboratory (12) has provided a broad outline of the metabolism of Rickettsia typhi and R. akari multiplying in irradiated L cells. This information was obtained by the use of cycloheximide (an inhibitor of eukaryotic metabolism) and by pulse labeling the infected cells with "4C-labeled amino acids and adenine. The effect of infection on the host cells was investigated by the addition to viable (unirradiated) infected and uninfected L cells of'4C-thymidine, which was not incorporated by the rickettsiae. These investigations have now been extended to R. tsutsugamushi and R. rickettsi. For the latter rickettsia, which grows in L cells only to a limited extent, primary duck embryo cells were used (7). MATERIALS AND METHODSA supply of ampoules of a yolk sac suspension of R. tsutsugamushi (Gilliam strain) in its 178th egg passage was obtained from the Walter Reed Army Institute of Research. The titer of this rickettsial suspension, determined before receipt, was 1.4 x 107 mouse median lethal doses or 3.2 x 101 mouse medium infective doses per 0.2 ml of yolk sac. R. rickettsi, (Bitterroot strain), in its 55th egg passage, was also obtained from the Army Institute. It was passed once in our laboratory in the yolk sac of duck embryos from which a suspension of heavily infected yolk sacs was prepared.Primary duck cells derived from 13-day-old embryos were suspended in medium 199 with 10% calf serum and placed in 32-ounce bottles (about 0.946 liter) to form monolayers. Four to six days later the cells were resuspended in medium of the same compo-4 sition, subjected to 3,000 R in a 60Co irradiator, and dispensed in 2-ounce plastic flasks, about 600,000 cells per flask. The resulting monolayer cultures were infected or used as uninfected control cultures 2 or 3 days after irradiation.Methods not described above were identical to those of previous experiments (12). RESULTSIrradiated L cells inoculated with an estimated multiplicity > 1 < 10 of infectious scrub typhus rickettsiae supported profuse growth of the infecting organisms. Microscopically, the majority of cells were infected on day 1, and numerous intracellular organisms were seen on day 3. Compact masses of rickettsiae located in the cytoplasm near the nucleus and increasing numbers of extracellular ...
The plaque technique for three strains of Rickettsia tsutsugamushi in chicken embryo cell cultures was greatly improved by modifying the trypsinizing procedure and employing homologous chicken serum in the overlay medium.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.