Journal List > J Bacteriol Virol > v.46(4) > 1034222

J Bacteriol Virol. 2016 Dec;46(4):193-200. Korean.
Published online December 31, 2016.  https://doi.org/10.4167/jbv.2016.46.4.193
Copyright © 2016 The Korean Society for Microbiology and The Korean Society of Virology
Invasion of Mammalian Cells by Rough Variant of Mycobacterium abscessus
Jake Whang, Young Woo Back, Gang-In Lee and Hwa-Jung Kim
Department of Microbiology, and Biomedical Sciences, College of Medicine, Chungnam National University, Daejeon, Korea.

Corresponding author: Hwa-Jung Kim. Department of Microbiology, College of Medicine, Chungnam National University, 6 Munwha-Dong, Jung-Ku, Daejeon, 35015, Korea. Phone: +82-42-580-8242, Fax: +82-42-585-3686, Email: hjukim@cnu.ac.kr
Received September 28, 2016; Revised October 14, 2016; Accepted October 20, 2016.

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/).


Abstract

One of a rapid growing mycobacteria (RGM), Mycobacterium abscessus (MAB), is the most causative agents of RGM pulmonary disease. MAB can change their morphology that smooth (S) type to more virulent type of rough (R). Bacterial invasion into host cells is an important first step to initiate their infection. The phagocytic and invasion mechanisms of Mycobacterium tuberculosis through the host-parasite interaction have been researched. Although MAB causes a wide range of clinical diseases, little is known about their invasion ability or why the R type is more virulent than the S type. To compare invasion ability of R with S types, their infection abilities to dermal fibroblast, HaCaT cells, A549 cells and bone marrow derived macrophages were analyzed. After 2 h of infection, intracellular survival numbers of the R type were significantly higher in all infected cells than S types. The fluorescence-activated cell sorting (FACS) and confocal microscopy assay also revealed that red fluorescent amount and intracellular bacterial numbers in all of the cells infected with MAB R type expressing the red fluorescent protein (RFP) were significantly higher than the S type. Our data suggest that the virulence of MAB is proportionally related to the invasion ability into mammalian cells and macrophages.

Keywords: Mycobacterium abscessus; Rough variant; Invasion; Mammalian cells

Figures


Figure 1
Colony morphotypes and cell wall lipid compositions of Mycobacterium abscessus smooth (S) and rough (R) type. (A) Colony morphologies of cultured M. abscessus (MAB) wild type (smooth), isogenic rough type on 7H10 media plates. (B) Extracted total lipids were analyzed by thin layer chromatography using chloroform : methanol = 9 : 1 as a running solution. The glycopepdolipid (GPL) components were indicated by right side of figure.
Click for larger image


Figure 2
Intracellular survival numbers of M. abscessus in phagocytic or non-phagocytic cells. A549, BMDM, dermal fibroblast, and HaCaT cells were infected with M. abscessus S or R types. After 2 h of infection, the serially diluted cell lysates were plated on 7H10 agar plate to determine the colony forming units. *p < 0.05, ***p < 0.001 cells infected with S type versus cells infected with R type.
Click for larger image


Figure 3
FACS analysis of M. abscessus entrances into phagocytic or non-phagocytic cells. Each cell was infected with M. abscessus S, or R types transformed by pMV261-RFP and then intracellular fluorescents were measured by FACS.
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Figure 4
Confocal microscopic analysis of M. abscessus infected cells. Each cell was infected with RFP over-expressive MAB and stained with beta-tubulin of green and nucleus of blue. Representative confocal images are shown.
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Figure 5
The internalized bacterial numbers per cells. Internal fluorescent bacterial numbers from the 200 cells of images of Figure 4 were counted. Data are mean number per cell. **p < 0.01, ***p < 0.001 cells infected with S type versus cells infected with R type.
Click for larger image

Notes

This work was supported by research fund of Chungnam National University (2014).

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