A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

The alteration of serum bile acid profile among traumatic brain injury patients: a small-scale prospective study.

Traumatic brain injury is one of the major causes of morbidity and mortality worldwide. With the development of bile acids as a potential treatment, to identify the influence of traumatic brain injury on bile acid metabolism shows growing importance. This present study did a preliminary exploration of the bile acid profile alteration among traumatic brain injury patients. In total, 14 patients and 7 healthy volunteers were enrolled. The bile acid profile of the blood samples were detected by an Ultra-performance Liquid Chromatography Mass Spectrometer/Mass Spectrometer system. It was found that 6 bile acids were statistically decreased in traumatic brain injury patients comparing with healthy volunteers: glycocholic acid (median level 44.4 ng/ml vs 98.7 ng/ml, p = 0.003), taurocholic acid (median level 10.9 ng/ml vs 19.5 ng/ml, p = 0.006), glycoursodeoxycholic acid (median level 17.4 ng/ml vs 71.4 ng/ml, p = 0.001), ursodeoxycholic acid (median level <1 ng/ml vs 32.4 ng/ml, p = 0.002), taurochenodeoxycholic acid (median level <1 ng/ml vs 53.6 ng/ml, p = 0.003) and glycochenodeoxycholic acid (GCDCA, median level 160 ng/ml vs 364 ng/ml, p<0.001). In conclusion, traumatic brain injury events are able to induce bile acid metabolism alteration in plasma and might cause reduction in glycocholic, taurocholic, glycoursodeoxycholic, ursodeoxycholic, taurochenodeoxycholic and glycochenodeoxycholic acid levels.

Inhibiting DosRST as a new approach to tuberculosis therapy.

Progress against tuberculosis (TB) requires faster-acting drugs. Mycobacterium tuberculosis (Mtb) is the leading cause of death by an infectious disease and its treatment is challenging and lengthy. Mtb is remarkably successful, in part, due to its ability to become dormant in response to host immune pressures. The DosRST two-component regulatory system is induced by hypoxia, nitric oxide and carbon monoxide and remodels Mtb physiology to promote nonreplicating persistence (NRP). NRP bacteria are thought to play a role in the long course of TB treatment. Therefore, inhibitors of DosRST-dependent adaptation may function to kill this reservoir of persisters and potentially shorten therapy. This review examines the function of DosRST, newly discovered compounds that inhibit DosRST signaling and considers future development of DosRST inhibitors as adjunct therapies.

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

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.

HC2091 Kills Mycobacterium tuberculosis by Targeting the MmpL3 Mycolic Acid Transporter.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills M. tuberculosis in a time- and dose-dependent manner in vitro and inhibits M. tuberculosis growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mmpL3 mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized Mycobacterium membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating M. tuberculosis, thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of M. tuberculosis treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.

Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics.

Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here, we report that the compound AC2P36 selectively kills Mtb at acidic pH and potentiates the bactericidal activity of isoniazid, clofazimine, and diamide. We show that AC2P36 activity is associated with thiol stress and causes an enhanced accumulation of intracellular reactive oxygen species at acidic pH. Mechanism of action studies demonstrate that AC2P36 directly depletes Mtb thiol pools, with enhanced depletion of free thiols at acidic pH. These findings support that Mtb is especially vulnerable to thiol stress at acidic pH and that chemical depletion of thiol pools is a promising target to promote Mtb killing and potentiation of antimicrobials.

Inhibitors of Mycobacterium tuberculosis DosRST signaling and persistence.

The Mycobacterium tuberculosis (Mtb) DosRST two-component regulatory system promotes the survival of Mtb during non-replicating persistence (NRP). NRP bacteria help drive the long course of tuberculosis therapy; therefore, chemical inhibition of DosRST may inhibit the ability of Mtb to establish persistence and thus shorten treatment. Using a DosRST-dependent fluorescent Mtb reporter strain, a whole-cell phenotypic high-throughput screen of a ∼540,000 compound small-molecule library was conducted. The screen discovered novel inhibitors of the DosRST regulon, including three compounds that were subject to follow-up studies: artemisinin, HC102A and HC103A. Under hypoxia, all three compounds inhibit Mtb-persistence-associated physiological processes, including triacylglycerol synthesis, survival and antibiotic tolerance. Artemisinin functions by disabling the heme-based DosS and DosT sensor kinases by oxidizing ferrous heme and generating heme-artemisinin adducts. In contrast, HC103A inhibits DosS and DosT autophosphorylation activity without targeting the sensor kinase heme.