The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm. Orientia tsutsugamushi, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with O. tsutsugamushi revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.Orientia tsutsugamushi, the causative agent of scrub typhus, has been reported to attach to the susceptible host cells in a heparan sulfate glycosaminoglycan-mediated manner (25). After attachment to the host plasma membrane, O. tsutsugamushi induces its own uptake by a process termed induced phagocytosis (40). After entry into the cells, O. tsutsugamushi escapes from the phagocytic vacuole by an unknown mechanism. This probably occurs shortly after phagocytosis, since phagocytic vacuoles containing O. tsutsugamushi have never been seen far from the cell surface (12). Once free in the host cytoplasm, the bacteria replicate in the perinuclear area. Listeriae, shigellae, and rickettsiae also escape from the phagocytic vacuole and replicate in the cytoplasm. These bacteria have independently developed an apparently similar host cell actin-based mechanism that is essential for their intercellular spread (16,18,33,39). In the case of viruses, such as the common type 2 or 5 adenoviruses, they enter a new host cell by receptor-mediated endocytosis and reach the cytosol by acid-dependent disruption of the endosomal membrane (37). It is known that microtubules (MTs) and dyneins are required for directional transport of cytosolic adenovirus to the nucleus (36). Herpes simplex virus 1 (HSV-1) also overcomes the cytosolic barrier by packaging its genome into a capsid structure that is capable of using the minus-end-directed MT-dependent dynein motor (35). Some bacteria, such as Chlamydia trachomatis and Campylobacter jejuni, utilize the host MTs and dyneins during the early events of infection (9, 24). However, it is not clear whether O. tsutsugamushi uses the host cell cytoskeleton for its intracellular movement and intercellular spread.The MT network of an interphase cell is a dyna...