Apoptosis of Endothelial Cell Line ECV304 Persistently Infected with Orientia tsutsugamushi

Abstract

Endothelial cells are major targets of Orientia tsutsugamushi. To examine the consequences of the infection of endothelial cells with O. tsutsugamushi, we used human endothelial cell line ECV304. Persistent infection was established and infected cultures could be maintained for over seven months without the addition of normal cells. The heavily infected cells became round and floated in the culture medium, harboring large numbers of organisms inside them. Some of the infected ECV304 cells showed features of apoptotic cells, as determined by the terminal deoxytransferase-mediated dUTP nick end-labeling reaction and DNA fragmentation.We also found that O. tsutsugamushi increased transcription of the mRNAs of proinflammatory cytokines such as IL-6 and IL-8. These results show the first evidence of in vitro-persistent infection by O. tsutsugamushi, which may be related to in vivo persistence reported previously.

Orientia tsutsugamushiInhibits Apoptosis of Macrophages by Retarding Intracellular Calcium Release

“…Apoptosis of the host cells would deprive the obligate intracellular pathogens of their intracellular hideouts, while apoptosis of infected macrophages or lymphocytes may seriously compromise the host defense mechanisms (22). Previous studies on the apoptosis of O. tsutsugamushi-infected cells (12,14) have shown that apoptosis is a relatively late event and predominantly involves cells that are heavily laden with intracellular bacteria. The mechanisms of delayed apoptosis induced by O. tsutsugamushi have been elucidated in this study.…”

“…It has been shown previously that O. tsutsugamushi can induce apoptosis in a variety of host cells, including lymphocytes and endothelial cells (11,12,14). Many intracellular bacteria have evolved mechanisms by which to down-regulate apoptosis of the cells they infect (9).…”

“…The Boryong serotype of O. tsutsugamushi (3) was cultivated in ECV304 cells as described previously (14). When infected ECV304 cells showed a maximum cytopathic effect, the infected cells were disrupted with glass beads (diameter, 1.0 mm) and centrifuged at 300 ϫ g for 5 min.…”

Characterization of Monoclonal Antibody Reacting Exclusively against Intracellular Orientia tsutsugamushi

“…In a previous study, we found that the ECV304 cells that were heavily infected became round and floated in the culture medium, whereas other cells still adhered to the culture flask and had only a relatively small number of bacteria inside the cell (10). In this study, we found that there were more dead bacteria in the nonadherent cells than in the adherent cells, as judged by bacterial viability count of preparations from each of the cell groups.…”

“…tsutsugamushi. The Boryong serotype of O. tsutsugamushi (4) was cultivated in ECV304 cells as described previously (10). The cells, once infected, were cultivated until the heavily infected cells detached from the culture substrate.…”