Mycobacterium tuberculosis DevR/DosR Dormancy Regulator Activation Mechanism: Dispensability of Phosphorylation, Cooperativity and Essentiality of α10 Helix - PubMed (original) (raw)

Mycobacterium tuberculosis DevR/DosR Dormancy Regulator Activation Mechanism: Dispensability of Phosphorylation, Cooperativity and Essentiality of α10 Helix

Saurabh Sharma et al. PLoS One. 2016.

Abstract

DevR/DosR is a well-characterized regulator in Mycobacterium tuberculosis which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be 'pre-adapted' to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under 'non-inducing' conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the α10 helix, establishing the α10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of α10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to the mechanism of DevR overexpression-mediated induction of the regulon in the absence of the normal environmental cue and establishes the α10 helix as an universal and pivotal targeting interface for DevR inhibitor development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Overexpression of DevR in Mtb DKO.

(a) Vector map of integrating plasmid overexpressing DevR. (b) Western blotting of lysates from aerobic Mtb cultures. A representative blot is shown. (c) Total DevR levels (DevR + DevR-Myc) in DKO-complemented strains. (d) DevR (endo) level in DKO-complemented strains. (e) RT-qPCR analysis of select DevR regulon genes in DKO-complemented strains. (f) RT-qPCR analysis of additional DevR regulon genes in DKO-Pmsp12DevR. Data is Mean ± SD of 2 biological replicates. SigA was used as a loading control in (b). In panels (c) to (f), protein and transcript levels are shown relative to that in DKO (considered as 1).

Fig 2

Fig 2. Overexpression of WT/mutant DevR in Mtb RKO.

(a) Vector map of integrating plasmid overexpressing WT/mutant DevR proteins. (b) Mtb RKO- Pmsp12DevR, RKO-Pmsp12DevR D54E, RKO-Pmsp12DevR D54V and RKO-Pmsp12DevR T82A strains overexpress DevR variants from msp12 promoter. RKO-POperonDevR strain expresses DevR from its native promoter. Western blotting of aerobic (Aer) and 5 day hypoxic (Hyp) cultures lysates. A representative blot is shown. (c) RT-qPCR analysis of regulon genes in aerobic cultures. (d) RT-qPCR analysis of DevR regulon genes under hypoxia. RT-qPCR data is Mean ± SD of 2 biological replicates. SigA was used as a loading control in (b).

Fig 3

Fig 3. Overexpression of DevRΔα10 in Mtb RKO.

(a) Vector map of integrating plasmid employed to overexpress DevRα10 protein. (b) RT-qPCR analysis of aerobic DevR regulon genes expression. WT/mutant DevR expression in RKO-Pmsp12DevR /RKO-Pmsp12DevRα10 strains is supported by msp12 promoter and WT DevR in RKO-POperonDevR by its native promoter. (c) RT-qPCR analysis of DevR regulon genes under hypoxia. RT-qPCR data is Mean ± SD of 2 biological replicates.

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