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. 2001 Jan;69(1):494-500.
doi: 10.1128/IAI.69.1.494-500.2001.

Microtubule- and dynein-mediated movement of Orientia tsutsugamushi to the microtubule organizing center

S W Kim  1 , K S Ihn, S H Han, S Y Seong, I S Kim, M S Choi
Affiliations

Microtubule- and dynein-mediated movement of Orientia tsutsugamushi to the microtubule organizing center

S W Kim et al. Infect Immun. 2001 Jan.

Abstract

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.

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Figures

FIG. 1
FIG. 1
O. tsutsugamushi moves to the perinuclear regions, as shown by conventional immunofluorescence microscopy of ECV304 cells infected with 1 × 106 ICU of O. tsutsugamushi (about 50 orientiae per cell were found). The cells were fixed in methanol and labeled with KI-37, a MAb against the O. tsutsugamushi Boryong 56-kDa protein, and an FITC-conjugated secondary antibody. The nucleus stained by propidium iodide. The yellowish, round spots at the perinucleus are oriential accumulation. (A) Magnification, ×20; (B) magnification, ×100.
FIG. 2
FIG. 2
Subcellular distribution of cytosolic O. tsutsugamushi. Representative immunofluorescence confocal microscopic images of ECV304 cells infected with O. tsutsugamushi for 30 min, 60 min, and 48 h are shown. Cells were fixed in methanol and double-labeled with human polyserum against O. tsutsugamushi (tetramethylrhodaminyl isothiocyanate-conjugated anti-human) and anti-tubulin MAb (FITC-conjugated anti-mouse). (A) ECV304 cells infected at 1 × 105 ICU (about 5 to 10 orientiae per cell were found) for 30 min. The orientiae were scattered throughout the cytoplasm. (B) ECV304 cells infected for 60 min. They began to accumulate at the MTOC. Most intracellular orientiae colocalized with MTOC. The brightest area of MT network in the perinucleus is the MTOC. (C) ECV304 cells infected for 48 h, with numerous orientiae located at the MTOC. (D) Orientiae located quite close to MTs under the nuclear region (arrowheads) at 30 min postinfection. Magnification, ×63.
FIG. 3
FIG. 3
Inhibitory effects of oriential movement in the presence of MT-disrupting agents; immunofluorescence confocal microscopy of ECV304 cells infected with 2 × 105 ICU (about 10 to 20 orientiae per cell were found) of O. tsutsugamushi for 90 min. O. tsutsugamushi and MT were fixed and stained as described for Fig. 2. Under control conditions (A-1 and A-2) most cytosolic O. tsutsugamushi accumulated around the MTOC. If the cells were infected in the presence of nocodazole (10 μg/ml) (B-1 and B-2) or colchicine (1 μg/ml) (C-1 and C-2), there was no accumulation of O. tsutsugamushi at the MTOC. Orientiae were scattered throughout the entire cytoplasm. Magnification: for panels A-, B-, and C-1, ×40; for panels A-, B-, and C-2, ×100.
FIG. 4
FIG. 4
Nocodazole reversibility and MT disruption after movement to the MTOC. O. tsutsugamushi and MT were fixed and stained as described for Fig. 2. In the presence of nocodazole (10 μg/ml) the number of orientiae localized to the MTOC was drastically reduced (A-1 and A-2), whereas after a 4-h chase in nocodazole-free medium, the MT network was reconstituted and the orientiae accumulated around the MTOC (B-1 and B-2). Orientiae replicated in the perinuclear region after 24 h of infection (C-1 and C-2), but if the cells were treated with colchicine (1 μg/ml), after 24 h of infection, the orientiae were scattered throughout the cytoplasm. (D-1 and D-2). Magnification: for panels A-1, B-1, C-1, and D-1, ×40; for panels A-2, B-2, C-2, and D-2, ×100.
FIG. 5
FIG. 5
Dynamitin overexpression inhibits cytosolic movement of O. tsutsugamushi. HeLa cells transiently transfected with a plasmid DNA expressing GFP (A) or with a dynamitin-expressing plasmid (B). The orientiae were internalized for 90 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin MAb and FITC-goat anti-mouse IgG. (A) O. tsutsugamushi accumulated in the cytosol both in nontransfected cells (arrows) and GFP-transfected cells. (B) O. tsutsugamushi did not accumulate in the cytosol in dynamitin-overexpressing cells. O. tsutsugamushi accumulate in the cytosol both in moderately overexpressed cells (arrowhead) and nontransfected cells (arrow). Magnification, ×63.
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