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. 2009 Mar;89(2):169-74.
doi: 10.1016/j.tube.2008.12.003. Epub 2009 Feb 13.

DevR-mediated adaptive response in Mycobacterium tuberculosis H37Ra: links to asparagine metabolism

Vandana Malhotra  1 , Jaya Sivaswami Tyagi, Josephine E Clark-Curtiss
Affiliations

DevR-mediated adaptive response in Mycobacterium tuberculosis H37Ra: links to asparagine metabolism

Vandana Malhotra et al. Tuberculosis (Edinb). 2009 Mar.

Abstract

The DevR transcriptional switch that defines the response of Mycobacterium tuberculosis to the lack of oxygen is now well established and likely helps the bacteria shift to a state of persistence. The M. tuberculosis two component signal transduction system (TCS), DevR-DevS, implicated in this transition to latency, is differentially expressed in H37Ra and H37Rv strains. Despite originating from the H37 ancestral strain, H37Ra and H37Rv have significant differences in their growth, physiology, and virulence. To further dissect the role of DevR in growth adaptive processes of M. tuberculosis, we investigated the hypoxic response of the avirulent H37Ra strain. Our results show that the DevR-DevS TCS in H37Ra is responsive to hypoxia and capable of target gene regulation, indicating similar DevR-DevS signaling pathways in H37Ra and H37Rv. A key finding of this study was the constitutive expression of the Rv3134c-devR-devS operon and a subset of sentinel DevR-regulated genes in aerobic cultures of H37Ra but not H37Rv grown in Dubos-Tween-albumin medium. Asparagine and/or catabolites of asparagine metabolism were implicated in aerobic induction of the DevR-DevS TCS in H37Ra. This is the first report of medium-specific constitutive expression of the DevR regulon in an avirulent strain and suggests a potential role for metabolite(s) in the activation of the DevR-DevS TCS.

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Figures

Figure 1
Figure 1. Differential aerobic expression of Rv3134c-devR-devS operon in M. tuberculosis cultivated in 7H9T and DTA media
qRT-PCR analyses of Rv3134c-devR-devS operon during exponential growth of H37Rv (Rv) and H37Ra (Ra) in 7H9T and DTA media. The normalized expression of these genes with respect to expression of 16S rRNA is presented as mean ± standard deviations (SD) of three independent experiments. *** represents P < 0.001 for the differences in expression of the operon between H37Ra and H37Rv in DTA medium. The decrease in the expression of devR (***, P < 0.001) and devS (*, P < 0.05) genes in H37Rv grown in DTA versus 7H9T medium is significant.
Figure 2
Figure 2. Aerobic expression of DevR and a subset of sentinel DevR-regulated genes in M. tuberculosis grown in 7H9T and DTA media
(A) Fold induction of devR, hspX, narK2, fdxA, Rv1738, Rv2626c, and Rv3130c genes in H37Ra grown aerobically in 7H9T (white bars) and DTA (black bars) media with respect to H37Rv (baseline expression set to 1.0). *** represents P < 0.001, for the differences in expression between H37Ra and H37Rv strains in DTA medium. (B) Western blot analyses of M. tuberculosis H37Ra and H37Rv grown in 7H9T (Lanes 1 and 2) and DTA (Lanes 3 and 4) media using anti-DevR and anti-HspX antibodies. Lane 5 is purified HspX protein. The levels of DevR in H37Ra cultivated in 7H9T (RaT) and DTA (RaDTA) media are reported as percentage expression with respect to H37Rv (RvT or RvDTA, fixed at 100%) based on densitometric measurement of band intensities (below Figure 2B).
Figure 3
Figure 3. Asparagine: A metabolic signal for aerobic induction of the DevR regulon in M. tuberculosis H37Ra
(A) devR expression in H37Rv (Rv, black bars), H37Ra (Ra, gray bars) grown in DTA medium with (hatched lines, DTA+G) or without glycerol (solid, DTA) and in H37Ra grown in DTA supplemented with 300 mM D-asparagine (vertical lines, DTA+Dasn). *** represents P < 0.001. (B) Western blot analyses of DevR and HspX expression in 30 μg (Lanes 1 and 3) and 45 μg (Lanes 2 and 4) of H37Ra cell lysates prepared from DTA and DTA+Dasn media respectively. Lane 5 represents cell lysate of H37Ra grown in DTA+G medium (C) Fold induction of devR, devS, Rv3134c, hspX, narK2 and Rv3130c genes in H37Ra grown in 7H9T medium with L-asparagine (7H9T+Lasn) with respect to 7H9T control (set at 1.0).
Figure 4
Figure 4. Hypoxic response of M. tuberculosis H37Rv and H37Ra
Fold induction at 24 hr hypoxia of (A) devR expression in H37Rv (black bars) and H37Ra (white bars) cultured in DTA and 7H9T media and of (B) Rv3134c, devR, devS, hspX and narK2 genes in H37Ra grown in 7H9T (white bars) and DTA+G (black bars) media with respect to their aerobic controls (set at 1.0). **, * represents P < 0.01 and 0.05 respectively for devR induction in H37Rv and H37Ra in the respective media compared to their aerobic controls. The hypoxic induction of the DevR regulon in DTA+G media is significant (P < 0.001).
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