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.
Highlights
Mycobacterium tuberculosis (Mtb) can establish a dormant state known as nonreplicating persistence (NRP) where the bacterium modulates its metabolism in response to environmental and host immune cues, such as hypoxia, acidic pH, and nutrient starvation.[1,2]
NRP Mtb is tolerant to several antimycobacterial drugs in vitro.[15−17] The NRP population of bacteria is thought to be responsible, in part, for the 6-month long course of TB treatment. dosRST mutants are attenuated in in vitro models of hypoxia-driven NRP18 and in animal models that generate hypoxic granulomas, including nonhuman primates, guinea pigs, and C3HeB/FeJ mouse models of TB infection.[3,19−21] deletion of DosR-regulated gene tgs[1], which is involved in triacylglycerol (TAG) synthesis, causes reduced antibiotic tolerance.[22,23]
Mtb was treated with 40 μM HC104A, HC106A, or dimethyl sulfoxide (DMSO) control for 6 d in a standing flask, and following incubation RNA was extracted, sequenced, and analyzed for differential gene expression relative to the DMSO control
Summary
Mycobacterium tuberculosis (Mtb) can establish a dormant state known as nonreplicating persistence (NRP) where the bacterium modulates its metabolism in response to environmental and host immune cues, such as hypoxia, acidic pH, and nutrient starvation.[1,2] DosRST is a two-component regulatory system that regulates Mtb persistence.[3−5] It consists of two sensor histidine kinases, DosS and DosT, and the cognate response regulator DosR, which regulates expression of about 50 genes in the DosRST regulon.[5−7] The pathway can be induced by host intracellular stimuli, such as nitric oxide (NO), carbon monoxide (CO), and hypoxia, through DosS and DosT.[8−10] DosS is an oxygen and redox sensor, whereas DosT acts as an oxygen sensor.[11−13] Both kinases sense ligands via the heme group and are inactive when the heme exists as either the Met (Fe3+) form (DosS) or the oxy (Fe2+-O2) form (DosT) in the presence of O2.12 hypoxic conditions activate the kinases by inducing the conversion of DosS to the ferrous form and DosT to the deoxy form. DosRST mutants are attenuated in in vitro models of hypoxia-driven NRP18 and in animal models that generate hypoxic granulomas, including nonhuman primates, guinea pigs, and C3HeB/FeJ mouse models of TB infection.[3,19−21] deletion of DosR-regulated gene tgs[1], which is involved in triacylglycerol (TAG) synthesis, causes reduced antibiotic tolerance.[22,23] antivirulence strategies to inhibit the DosRST pathway may function to reduce virulence and deplete the reservoir of drug-tolerant NRP Mtb.[24]. In the Received: September 14, 2018 Accepted: September 26, 2019 Published: September 26, 2019
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