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Lee, Park, Lim, Kim, Shin, Yang, Oh, Kwon, Song, Jo, and Kim: Identification of Proteins Induced at Hypoxic and Low pH Conditions in Mycobacterium tuberculosis H37Rv
Original Article

J Bacteriol Virol 2006;36(2):59-68.

Published online: 18 February 2006

DOI: https://doi.org/10.4167/jbv.2006.36.2.59

Identification of Proteins Induced at Hypoxic and Low pH Conditions in Mycobacterium tuberculosis H37Rv

Kil-Soo Lee, Jeong-Kyu Park, Jae-Hyun Lim, Su-Young Kim, A-Rum Shin, Chul-Su Yang, Jae-Hee Oh, Yu-Mi Kwon, Chang-Hwa Song, Eun-Kyeong Jo, Hwa-Jung Kim

Department of Microbiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-ku, Daejeon 301-747, Korea

Received 18 April 2006       Accepted 8 May 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

Mycobacterium tuberculosis likely reside within a granuloma as a dormant state. An area of necrosis forms at the center of lung granulomas. Within this area, the bacteria are deprived of nutrients and exposed to harsh conditions, including low pH and anoxia. The response of M. tuberculosis to low pH and low oxygen conditions was investigated in both cellular and extracellular proteins by two-dimensional polyacrylamide gel electrophoresis analysis and MALDI-TOF. Several proteins intensively expressed under low pH and/or hypoxic conditions were found. In the culture filtrate, PhoS1 (Rv0934) and ScoB (Rv2503c) were found in significant amounts under both the low oxygen and acidic stress conditions. These results indeed extend our understanding of acidic response as well as hypoxic in M. tuberculosis and provide an important insight into physiology of the latent bacilli.

Keywords: Mycobacterium tuberculosis, Dormant state, Hypoxic response, PhoS1, ScoB

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Figure 1.
Two-dimensional electrophoretic analysis of the culture filtrate (CF) of M. tuberculosis H37Rv grown under 20% (A), 13% (B), and 0% (C) oxygen conditions. The CF proteins were separated by isoelectric focusing (pH 4–7) in the first dimension and by SDS-PAGE (15% polyacrylamide gel) in the second dimension. The gels were stained with 0.25% Coomassie brilliant blue R250.
jbv-36-59f1.tif
Figure 2.
2-DE analysis of the lysate of M. tuberculosis H37Rv grown under 20% (A), 13% (B), and 0% (C) oxygen conditions. The lysate proteins were separated by isoelectric focusing (pH 4–7) in the first dimension and by 15% SDS-PAGE in the second dimension. The gels were stained with Coomassie brilliant blue R250.
jbv-36-59f2.tif
Figure 3.
2-DE analysis of the CF of M. tuberculosis H37Rv grown at different oxygen and pH conditions. The CF proteins were separated by isoelectric focusing (pH 4.7–5.9) in the first dimension and by SDS-PAGE (12% polyacryl-amide gel) in the second dimension. The second dimensional gels were stained with Coomassie brilliant blue R250. The bacteria were cultured with an oxygen supply of 20% (A), 13% (B), and 0% (C), respectively. PhoS1 and ScoB are highly induced under low oxygen and low pH conditions than other dormancy induced protein spots. The numbers on the left indicate molecular mass markers and pI values at the bottom of the panel.
jbv-36-59f3.tif
Figure 4.
Analysis of 38 kDa antigen with Coomassie blue staining and Western blot. A. Purified 38 kDa antigen separated by IEF on pH 4.7–5.9 IPG strip in the first dimension and 12% SDS-PAGE in the second dimension. The gel was stained by Coomassie blue. B. The CF obtained from M. tuberculosis grown in pH 6.0, 0% oxygen tension (b) and pH 7.2, 20% oxygen tension 20 (a) were separated by isoelectric focusing (pH 4.7 to 5.9) and SDS-PAGE (12% polyacrylamide gel), and then analyzed by immunoblotting with rabbit anti-38 kDa polyclonal antibody.
jbv-36-59f4.tif
Figure 5.
SDS-PAGE analysis of CF of M. tuberculosis H37Rv grown at 8 different conditions. The CF proteins were separated first dimension by 12% SDS-PAGE. Empty box indicate 38 kDa regions. Lane 1, 3, 5, and 7 (pH 6.0) are showed more dense band than Lane 2, 4, 6, and 8 (pH 7.2). Open circles represent protein bands highly increased under hypoxia and low pH condition with different media composition respectively (The arrow indicates the position of the region of 38 kDa antigen.) Proteins were visualized by Coomassie blue staining.
jbv-36-59f5.tif
Figure 6.
2-DE analysis of the CF of M. tuberculosis H37Rv grown at 8 different conditions. M. tuberculosis were cultured in Sautons medium at 20% O2 (A) and at 0% O2 tension (B) with following the conditions. a, pH 6.0 Sauton medium with MgSO4; b. pH 7.2 Sauton medium with MgSO4; c. pH 6.0 Sauton medium with K2SO4; d. pH 7.2 Sauton medium with K2SO4. All condition samples were separated by isoelectric focusing (pH 4.7–5.9) and by 12% SDS-PAGE. The gels were stained with Coomassie blue.
jbv-36-59f6.tif
Table 1.
Culture condition and composition of Sauton media
No. pH Sauton media Culture condition
1 6.0 Sautona) 20% O2
2 7.2 Sautona) 20% O2
3 6.0 Modifiedb) 20% O2
4 7.2 Modifiedb) 20% O2
5 6.0 Sautona) 0% O2
6 7.2 Sautona) 0% O2
7 6.0 Modifiedb) 0% or 13%∗ O2
8 7.2 Modifiedb) 0% O2

a) Sauton media: 4.0 g L-asparagine, 2.0 g citric acid, 0.5 g K2HPO4, 0.5 g MgSO4, 0.05 g ferric ammonium citrate, 60 ml glycerol to 1L DDW. Adjust pH to 7.3 with ammonia.

b) Modified Sauton media: Sauton media has replaced 0.5 g MgSO4 by 4.9 g K2SO4. Adjust pH to 6.0 with HCl.

∗ Modified Sauton media used for 13% O2 culture condition

Table 2.
M. tuberculosis H37Rv proteins significantly induced by low-oxygen condition and/or low pH on 2-DE
Identified protein Gene Theoretical mass (kDa) pI value Putative protein function
Rv0934 phoS1 38.4 5.1 protein antigen B, phosphate-binding protein
Rv2503c scoB 23.1 5.1 succinyl CoA:3-oxoacid CoA-transferase
Rv3029 fixA 28.1 4.7 electron transfer flavoprotein β subunit
Rv3028c fixB 31.7 4.7 electron transfer flavoprotein α subunit
Rv3295   24.9 5.3 hypothetical protein Rv3295
Rv3841 bfrB 18.3 4.2 bacterioferritin
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