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Inhibiting Mycobacterium tuberculosis DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme.

ABSTRACT: Mycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of nonreplicating persistence (NRP). NRP bacteria are tolerant to several antimycobacterial drugs in vitro and are thought to play a role in the long course of tuberculosis therapy. Previously, we reported the discovery of six novel chemical inhibitors of DosRST, named HC101A-106A, from a whole cell, reporter-based phenotypic high throughput screen. Here, we report functional and mechanism of action studies of HC104A and HC106A. RNaseq transcriptional profiling shows that the compounds downregulate genes of the DosRST regulon. Both compounds reduce hypoxia-induced triacylglycerol synthesis by ?50%. HC106A inhibits Mtb survival during hypoxia-induced NRP; however, HC104A did not inhibit survival during NRP. An electrophoretic mobility assay shows that HC104A inhibits DosR DNA binding in a dose-dependent manner, indicating that HC104A may function by directly targeting DosR. In contrast, UV-visible spectroscopy studies suggest HC106A directly targets the sensor kinase heme, via a mechanism that is distinct from the oxidation and alkylation of heme previously observed with artemisinin (HC101A). Synergistic interactions were observed when DosRST inhibitors were examined in pairwise combinations with the strongest potentiation observed between artemisinin paired with HC102A, HC103A, or HC106A. Our data collectively show that the DosRST pathway can be inhibited by multiple distinct mechanisms.

SUBMITTER: Zheng H

PROVIDER: S-EPMC6970277 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Inhibiting Mycobacterium tuberculosis DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme.

Zheng Huiqing H Williams John T JT Aleiwi Bilal B Ellsworth Edmund E Abramovitch Robert B RB

ACS chemical biology 20191014 1

Mycobacterium tuberculosis (Mtb) possesses a two-component regulatory system, DosRST, that enables Mtb to sense host immune cues and establish a state of nonreplicating persistence (NRP). NRP bacteria are tolerant to several antimycobacterial drugs in vitro and are thought to play a role in the long course of tuberculosis therapy. Previously, we reported the discovery of six novel chemical inhibitors of DosRST, named HC101A-106A, from a whole cell, reporter-based phenotypic high th ...[more]

PMID: 31556993

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Project description:The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 ?M(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.

Dataset Information

Inhibiting Mycobacterium tuberculosis DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme.

Publications

Inhibiting <i>Mycobacterium tuberculosis</i> DosRST Signaling by Targeting Response Regulator DNA Binding and Sensor Kinase Heme.

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