Orientia tsutsugamushi inhibits apoptosis of macrophages by retarding intracellular calcium release

Abstract

Orientia tsutsugamushi shows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited by O. tsutsugamushi infection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected with O. tsutsugamushi. Antiapoptotic activities of O. tsutsugamushi in infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.

FIG. 1.

(A) Inhibition of beauvericin-induced internucleosomal DNA fragmentation by O. tsutsugamushi infection. Chromosomal DNA from THP-1 cells was separated at various time points following treatment with beauvericin. (B) Immunoblotting of THP-1 cells with anti-PARP antibody at various time points after treatment with beauvericin. Lysates of THP-1 cells were separated by electrophoresis and stained with anti-PARP antibody. Cells were preconditioned by mock treatment or treatment with live O. tsutsugamushi (LOT) and heat-killed O. tsutsugamushi (HOT) 18 h before treatment with beauvericin.

FIG. 2.

Antiapoptotic activity of O. tsutsugamushi on THP-1 cells undergoing apoptosis caused by chemical inducers. Both live O. tsutsugamushi (LOT) and heat-killed O. tsutsugamushi (HOT) show antiapoptotic activity against beauvericin- or staurosporine-induced apoptosis. (A) Flow cytometric analysis of apoptosis after staining of THP-1 cells with 7-AAD. Live cells were not stained with 7-AAD (red), apoptotic cells were stained weakly (green), and dead cells were stained brightly (blue). (B) Analysis of the duration of the antiapoptotic activity of O. tsutsugamushi for 48 h. Apoptosis of THP-1 cells was induced with beauvericin after they had been conditioned by mock treatment (closed circles) or treatment with LPS (open circles), live O. tsutsugamushi (closed rectangles), or heat-killed O. tsutsugamushi (open rectangles) for 18 h. FSC, forward scatter.

FIG. 3.

Inhibition of increased cytosolic Ca²⁺ concentration by O. tsutsugamushi. (A) Fluorescence ratios of cells mock treated or treated with heat-killed O. tsutsugamushi (HOT) or LPS are plotted as a function of time. Ten micromolar beauvericin in Ca²⁺-free Tyrode solution was introduced at the time indicated by the bar. (B) The cells of the three groups were pseudocolored in accordance with the ratios at the three time points indicated by arrows in panel A.