Abstract
Sensing and response to environmental cues, such as pH and chloride (Cl−), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl− levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure–activity relationship studies on the hit compound “C6,” or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl− response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl− response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl−-responsive genes in the simultaneous presence of cholesterol and high external Cl− concentration, versus transcript levels observed during exposure to high external Cl− concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery.
Highlights
Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), is a major global health concern that results in 1.4 million deaths annually [1]
A second compound with shared structural components to C6 (PubChem SID: 340463409) that was called as having antitubercular activity in cholesterol media in that assay set (PubChem AID 1259343) did not demonstrate dysregulation of Mtb Cl− response in our original rv2390c0::GFP screen
Further studies will be required to tease this aspect apart and to delineate the relative contribution of the impact of C6 on Mtb Cl− response dysregulation versus cholesterol utilization on the in vivo bacterial growth inhibition phenotype. This is of particular interest as the compound screens conducted here revealed compounds that dysregulate Mtb Cl− response but did not affect growth of the bacteria in J774 macrophage-like cells
Summary
Tuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), is a major global health concern that results in 1.4 million deaths annually [1]. 10,100-bis[3-carboxylpropyl]-9,90-biacridinium; BMDM, bone marrow–derived macrophage; CMC, carboxymethyl cellulose; HPLC, high-performance liquid chromatography; LC–MS, liquid chromatography–mass spectrometry; mKO, monomeric Kusabira Orange; MS/MS, tandem proper colonization of the host, with disruption of the two-component regulatory system PhoPR, which is essential for pH response and important for Cl− response [9,19], leading to significant attenuation of Mtb growth in vivo [11,12] Other abundant ions, such as potassium (K+), play an important role in Mtb host adaptation, as disruption of the Mtb Trk K+ uptake system results in a dampening of Mtb response to acidic pH and high [Cl−], and attenumass spectrometry; Mtb, Mycobacterium tuberculosis; PBS, phosphate buffered saline; PFA, paraformaldehyde; PK, pharmacokinetic; qRT-PCR, quantitative real-time PCR; RT, room temperature; SAR, structure–activity relationship. Oral administration of C6 in a C3HeB/FeJ murine infection model resulted in significant reduction in bacterial load, indicating its utility both as a chemical probe and a starting point for the development of novel antitubercular drugs
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