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Kim, Kim, Lim, and Kang: Stabilizing Microtubular Network Facilitates the Intracellular Growth of Orientia tsutsugamushi
Original Article

J Bacteriol Virol 2006;36(2):51-57.

Published online: 18 February 2006

DOI: https://doi.org/10.4167/jbv.2006.36.2.51

Stabilizing Microtubular Network Facilitates the Intracellular Growth of Orientia tsutsugamushi

Mee-Kyung Kim2, Mi-Jeong Kim, Byung-Uk Lim, Jae-Seung Kang1, ∗

Department of Microbiology, Inha University College of Medicine, Republic of Korea

1Incheon 400-712, Republic of Korea

2Inha Research Institute for Medical Sciences, Republic of Korea

∗Corresponding author: Jae-Seung Kang. Department of Microbiology, Inha University College of Medicine, Jungsuk B/D, 3rd street, Shinheung-Dong, Choong-Gu, Inchon 400-712, Republic of Korea. Phone: 82-32-860-0952, Fax: 82-32-881-8559 e-mail: jaeskang@inha.ac.kr

Received 20 March 2006       Accepted 13 June 2006

Copyright © 2006 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Microtubule network provides many intracellular microbes with an efficient way to move within host cells. Orientia tsutsugamushi move from the cell periphery to the microtubule organizing center (MTOC) by dynein-dependent mechanism. In this study, we investigated the role of microtubule on the growth of O. tsutsugamushi. The treatment of infected cells with taxol as well as daunomycin enhanced the bacterial growth in contrast to colchicine. Immunofluorescent (IF) staining of taxol-treated cells exhibited that O. tsutsugamushi clustered tightly near the nucleus with thick bundles of microtubules, whereas dispersed in the cytoplasm in colchicine-treated cells. These results suggest that microtubule network facilitate the growth of O. tsutsugamushi.

Keywords: Orientia tsutsugamushi, Microtubule, Taxol, Daunomycin, Colchicine

References

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Figure 1.
The effect of microtubule-modifying agents on the growth of O. tsutsugamushi. ECV304 cells were grown to subconfluence on 24-well culture plates. After infection, cells were incubated further for 3 days in the presence of 50 nM taxol (Tax), 10 ng/ml colchicine (Col), 40 ng/ml daunomycin (Dau) and without agent (C). The number of viable cells was counted using trypan blue exclusion assay. Growth index was calculated as described in Materials and Methods. Representative result of three independent experiments is shown.
jbv-36-51f1.tif
Figure 2.
The stimulating effect of taxol on the growth of O. tsutsugamushi. (A) The infected cells treated with taxol or colchicine for 3 days were solubilized in lysis buffer. The lysates (25 μg) was separated on an 11% SDS-PAGE and was further analyzed with the immunoblot analysis using M686–8 for the estimation of bacterial growth. Lane 1, untreated control; lane 2, 2.5 nM taxol; lane 3, 25 nM taxol; lane 4, 100 nM taxol; lane 5, 5 ng/ml colchicine; lane 6, 10 ng/ml colchicine; lane 7, 100 ng/ml colchicine. (B) The cells treated with the indicated concentration of taxol or colchicine were stained using an anti-α-tubulin MAb and FS15 to stain tubulin and O. tsutsugamushi simultaneously. Magnification, ×200.
jbv-36-51f2.tif
Figure 3.
Immunoblot analysis of acetylated tubulin after treatment of cells with chemicals. The infected cells treated with indicated chemicals were solubilized in lysis buffer. (A) 25 μg of lysate was separated on 11% SDS-PAGE and was analyzed with M686–8 for bacterial protein, anti-acetylated α-tubulin, and anti-β-actin. Lane 1, uninfected ECV304 cells; lane 2, infected cells without treatment; lane 3, 50 nM taxol; lane 4, 10 ng/ml colchicine; lane 5, 5 μM nocodazole; lane 6, 40 ng/ml daunomycin. (B) The density of each band was quantified using Quantaty One software and amount of acetylated tubulin was normalized with the density of β-actin.
jbv-36-51f3.tif
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