Metabolism of Rickettsia tsutsugamushi and Rickettsia rickettsi in Irradiated Host Cells

Abstract

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 ...

Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study

“…In vitro studies of mouse lymphoblasts infected with R. tsutsugamushi (2) have also suggested a lack of cell damage, with no marked difference in oxygen uptake or anaerobic glycolysis between infected and control cells. Similarly, incorporation of radioactive thymidine by L cells was little affected during the first few days of in vitro infection with scrub typhus organisms (16). However, prolonged cultivation of infected BSC-1 cells led to severe and characteristic cytopathology (1) that was analogous to the degenerative changes we occasionally observed in mesothelial cells of moribund mice.…”

Rickettsia prowazekii requires host cell serine and glycine for growth

“…Addition of cycloheximide (1 p.g/ml) to Vero cells immediately after infection resulted in increases in RI of about 7-fold for the E strain and 28-fold for the Breinl strain after 48 h (Table 1). Weiss et al (20,21) observed that cycloheximide inhibited protein synthesis by host cells without having any effect on rickettsial protein synthesis and multiplication. This differential susceptibility to cycloheximide may render amino acids in the intracellular pools of the host cell available to the rickettsiae by preventing host cell competition.…”

Comparative biology of intracellular parasitism.

“…Host-free rickettsiae exhibit a low rate of incorporation of labeled methionine (52) and glycine (50) into rickettsial protein. which rickettsiae were growing was measured at several intervals after infection in the presence and absence of cyclohexamide (448,452). Under these conditions, the cyclohexamide-resistant incorporation amounts to as much as 50% of the incorporation observed in its absence.…”