In vitro studies on Rickettsia-host cell interactions: lag phase in intracellular growth cycle as a function of stage of growth of infecting Rickettsia prowazeki, with preliminary observations on inhibition of rickettsial uptake by host cell fragments

Abstract

Two Rickettsia prowazeki seeds, an "early" seed in the logarithmic or exponential growth phase and a "late" seed in the stationary or possibly early decline phase, were prepared in chicken embryo (CE) cell cultures and compared with respect to morphology and infection cycle in CE cells in culture. Differences in size and ultrastructure of the organisms in the two seeds were similar to those seen in other gram-negative bacteria at comparable stages of growth. Vacuolar structures, rare in log-phase organisms, were common in stationary-phase organisms. Minute spherical forms reminiscent of minicells were seen in the stationary-phase preparations. In quantitative uptake experiments, organisms, typical in size and morphology of each preparation, had comparable capacity per plaque-forming unit to penetrate into CE cells in suspension when the seeds had been depleted of host cell membrane fragments and other debris. This suggests that host cell fragments, presumably of membrane origin, competitively inhibit rickettsial uptake by intact CE cells. Organisms of the log-phase seed, upon entry into a host cell, entered the logarithmic or exponential phase of intracellular growth without a measurable lag phase, whereas stationary-phase organisms displayed a lag phase of about 7.5 h, during which they enlarged and increased in intensity of staining, before entering the log phase of growth. R This equation is in fact a modification of the one given by Lodge and Hinshelwood (9), simply making use of the information contained in the equation for INFECT. IMMUN. on July 15, 2020 by guest http://iai.asm.org/ Downloaded from

Rickettsial interactions with human endothelial cells in vitro: adherence and entry

“…Rickettsia prowazekii is an obligate intracellular bacterium. Because R. prowazekii parasitizes a number of cell types in vitro, studies on rickettsial "penetration" into host cells have utilized such diverse model systems as erythrocytes (21-23, [37][38][39]41), lymphoblasts (6), macrophage cell lines (28), and fibroblasts (6,26,32,42,44). At least three observations have consistently arisen from these reports.…”

Improved plaque assays for Rickettsia prowazekii in Vero 76 cells

“…The data in Table 2 indicate that R. prowazekii inocula show higher percentages of rickettsiae that are able to generate plaques in 10 days compared with the percentages generating a distinct infected cell in 3 to 4 days. Wisseman et al (33) noted that R. prowazekii cells harvested from an infected CEF monolayer during the logarithmic phase of growth were capable of reinitiating the logarithmic phase of growth with little or no delay upon inoculation on new host cells. When harvested during the stationary or late phase, rickettsiae showed a lag period before initiating a new round of logarithmic-phase growth.…”

“…When harvested during the stationary or late phase, rickettsiae showed a lag period before initiating a new round of logarithmic-phase growth. This lag period was estimated to be 7.5 h (33). The status of the rickettsiae at the time of harvest, when they were reinoculated onto a new monolayer, may influence the number of progeny at 4 days p.i.…”

“…Spotted fever group rickettsiae demonstrate rapid cell-to-cell spread via actin-based intercellular motility (9,27). This movement to culture medium and to uninfected cells seems to result in cytopathic effects in the absence of massive intracellular accumulation (23,26,33). Typhus group organisms, including R. canada, do not demonstrate this motility.…”

“…As a result, R. prowazekii accumulates intracellularly during the primary round of chicken embryo fibroblast (CEF) infection and causes host cell lysis at 2 to 4 days postinfection (p.i.). Subsequent rounds of infection and proliferation result in asynchronous lysis of additional host cells (32,33).…”