J Bacteriol Virol.  2006 Jun;36(2):59-68. 10.4167/jbv.2006.36.2.59.

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

Affiliations
  • 1Department of Microbiology, College of Medicine, Chungnam National University, Korea. hjukim@cnu.ac.kr

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 MALDITOF. 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.

Keyword

Mycobacterium tuberculosis; Dormant state; Hypoxic response; PhoS1; ScoB

MeSH Terms

Anoxia
Bacteria
Electrophoresis, Polyacrylamide Gel
Granuloma
Hydrogen-Ion Concentration*
Lung
Mycobacterium tuberculosis*
Mycobacterium*
Necrosis
Oxygen
Physiology
Tuberculosis
Oxygen

Figure

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.


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