Metabolism of Rickettsia typhi and Rickettsia akari in Irradiated L Cells

Abstract

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...

Metabolism of Rickettsia tsutsugamushi and Rickettsia rickettsi in Irradiated Host Cells

“…on August 1, 2020 by guest http://iai.asm.org/ Downloaded from more rapid host cell injury than do other rickettsiae. This difference is reflected in the effect of infection on thymidine incorporation by the host cells which is described in a previous paper (12) and observed in these experiments as well.…”

“…In a previous publication (12) we analyzed the methodology used in our studies. We con-PULSE LABELING cluded that we were closely approximating , , .…”

“…The usefulness of the methods described here for the study of the nutritional requirements of rickettsiae and for the production of labeled rickettsial fractions have already been discussed (12). Obviously, such studies depend on the achievement of a relatively high rickettsial cell density and can be carried out more easily with R. tsutsugamushi than with 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.…”

Energy Metabolism of Rickettsia typhi : Pools of Adenine Nucleotides and Energy Charge in the Presence and Absence of Glutamate

“…Analysis of base, nucleoside, and nucleotide pools. R. typhi were labeled with [U-'4C]adenine (19) during infection of irradiated L-cells. Irradiated cells (3,000 R with '"Co) in monolayer culture were inoculated 5 days postirradiation with R. typhi and incubated at 32°C for 7 days in the presence of [U-'4C] adenine (1 ,tCi/ml of tissue culture medium).…”