Metabolism of Rickettsia typhi and Rickettsia akari in irradiated L cells

Abstract

L cells that had been exposed to 3,000 r of (60)Co 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 mmN-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer and 10% calf serum. At various intervals, cycloheximide (2 mug/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 (14)C-labeled amino acids or adenine-8-(14)C. 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-(14)C 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. (14)C-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.