MECHANISMS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX SPECIES PERSISTENCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1014797
• Project Start Date: Feb 14, 2018
• Project End Date: Jan 31, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
The TB problem. Combating the ongoing tuberculosis (TB) epidemic represents one of the major challenges in global health. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb) and treatment with currently available antibiotics requires a multidrug, 6-9 month treatment regimen. This long course of treatment has fueled the emergence of multidrug-resistant TB (MDR-TB), which threatens to exacerbate the global TB crisis. New therapeutics are required to: i) reduce the length of drug therapy and, ii) combat MDR-TB.One reason Mtb is difficult to treat is because it establishes a chronic infection. Driven by the hypoxic environment of the granuloma, Mtb enters a persistent, non-replicating state that is recalcitrant to antibiotic therapy. In response to prolonged low O2 environments Mtb enters a non-replicating persistent state. The DosR regulon plays a key role in hypoxia-driven adaptation but the physiological function of this regulon is not well characterized. A critical barrier to genetic studies of the DosR-regulon has been the lack of a well-defined DosR-dependent phenotype. Our use of a synthetic, biosensor-based phenotype enables new methods for studying the DosR regulon. Identification of additional genes, small molecules and environmental conditions that regulate the DosR regulon, will guide the development of new hypotheses regarding the physiological purpose of the DosR regulon and the relationship between Mtb physiology and hypoxia during persistence.The Bovine TB problem. Mycobacterium bovis causes bovine tuberculosis (bTB) disease in wildlife, livestock and humans throughout the world. M. bovis establishes endemic populations in diverse wildlife species and these natural reservoirs present a threat to the health of domesticated livestock and the people that depend on these animals. Worldwide it is estimated that ~3% of all TB cases are caused by M. bovis, with the highest prevalence in developing countries where it is estimated to account for ~10% of TB cases. If we hope to control bTB, we must bring together veterinary and human medicine to understand how the disease maintains wildlife reservoirs and is transmitted between wildlife, livestock and humans, and the "One Health" approach is an ideal lens through which to view this problem. Unfortunately, little is known about the epidemiology of M. bovis between animal and human hosts or the mechanisms by which M. bovis establishes reservoirs in animals. By understanding the links between animal, humans and bTB, we will be able to interfere with the maintenance of bTB wildlife and livestock reservoirs and stop the transmission of bTB from animals to humans.Human TB as a model for Bovine TB. Bacteria that belong to the Mycobacterium tuberculosis complex, including Mtb and M. bovis, are closely related with many shared physiologies. In the case of hypoxia and persistence, both pathogens share the DosRST pathway and can establish persistence in response to hypoxia (Mak et al. 2012). Therefore, findings made in Mtb can be directly applied and tested in M. bovis. Experimental methods and tools in my lab are best established for Mtb research and it is most efficient to conduct initial studies in Mtb and then translate the discoveries to the animal pathogen. Specifically, in Aim 2, the animal models in the C3Heb/FeJ mice are specifically optimized for Mtb. Small molecules that interfere with M. bovis persistence (using technologies defined in Objectives 1 and 2) could be applied to feed or wildlife to reduce transmission to livestock and humans. Thus, all three objectives are directly relevant to agricultural research.Relevance to United States. The spread of TB is a global health crisis leading to over one million deaths annually. The global burden of TB is a threat to the health of all Americans, given the easy transmission of the disease through the air and the emergence of drug resistant strains that are difficult to treat. Moreover, there exists an increasing population of Americans with enhanced susceptibility to TB due to factors associated with compromised immune systems, including: HIV infection, the use of anti-rejection and anti-inflammatory drugs, as well as natural decreases in immunity associated with an aging population.Relevance to Michigan. Bacteria belonging to the Mycobacterium tuberculosis complex cause tuberculosis (TB) disease in humans and many species of animals. M. bovis causes TB in humans as well as in cattle and deer. M. bovis represents a serious threat to Michigan agriculture because discovery of infected cattle herds can lead to slaughter of the herd as well as possibly limiting the transport of animals to other states. Additionally, the relatively high prevalence of M. bovis in Michigan deer represents a reservoir of the pathogen that makes it difficult to eliminate M. bovis in Michigan. A goal of this project (Aim 3) is to examine the ability of M. bovis to persist in ensiled forages, which represents a potential mechanism of transmission of M. bovis from wildlife to livestock.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

silage

ensiled forages

bovine tuberculosis

human tuberculosis

bacterial pathogenesis

dormancy.

Goals / Objectives
Mycobacterium bovis causes bovine tuberculosis (bTB) disease in wildlife, livestock and humans throughout the world. M. bovis establishes endemic populations in diverse wildlife species and these natural reservoirs present a threat to the health of domesticated livestock and the people that depend on these animals. Worldwide it is estimated that ~3% of all TB cases are caused by M. bovis, with the highest prevalence in developing countries where it is estimated to account for ~10% of TB cases. If we hope to control bTB, we must bring together veterinary and human medicine to understand how the disease maintains wildlife reservoirs and is transmitted between wildlife, livestock and humans, and the "One Health" approach is an ideal lens through which to view this problem. Unfortunately, little is known about the epidemiology of M. bovis between animal and human hosts or the mechanisms by which M. bovis establishes reservoirs in animals. By understanding the links between animal, humans and bTB, we will be able to interfere with the maintenance of bTB wildlife and livestock reservoirs and stop the transmission of bTB from animals to humans.Bacteria that belong to the Mycobacterium tuberculosis complex, including Mycobacterium tuberculosis (Mtb) and M. bovis, are closely related with many shared physiologies. In the case of hypoxia and persistence, both pathogens share the DosRST pathway and can establish persistence in response to hypoxia (Mak et al. 2012). Therefore, findings made in Mtb can be directly applied and tested in M. bovis. Experimental methods and tools in my lab are best established for Mtb research and it is most efficient to conduct initial studies in Mtb and then translate the discoveries to the animal pathogen. Specifically, in Aim 2, the animal models in the C3Heb/FeJ mice are specifically optimized for Mtb. Small molecules that interfere with M. bovis persistence (using technologies defined in Objectives 1 and 2) could be applied to feed or wildlife to reduce transmission to livestock and humans.Using an innovative drug screening strategy we have discovered new compounds that inhibit Mtb non-replicating persistence (NRP) and survival[2]. NRP bacteria are thought to drive the long course of TB treatment[3]. Our goal is to develop these inhibitors further -- characterizing their impact on Mtb persistence and antibiotic tolerance, defining mechanisms of action, testing for synergistic interactions, optimizing leads for potency and pharmacokinetics (PK), and testing optimized leads in vivo for efficacy.Mtb is remarkably successful, in part, due to its ability to become dormant in response to host immune pressures[4]. Mtb has a two-component regulatory system (TCS), DosRST, that when induced by hypoxia and nitric oxide (NO) remodels Mtb physiology to promote NRP[5]. Therefore isolating inhibitors of DosRST-dependent adaptation should reduce survival of drug-tolerant NRP Mtb bacteria. Studies support this premise: dosRST mutants have reduced survival during hypoxia in vitro[2,6], and reduced virulence in rabbits, guinea pigs, non-human primates, and C3HeB/FeJ mice[7-9]. These animal models generate hypoxic granulomas where DosR-dependent persistence is predicted to be required for survival. In addition, disrupting tgs1, a DosR-regulated gene, enhanced sensitivity of Mtb to antibiotics in vitro and during mouse infection[10,11].By an innovative, reporter-based whole-cell phenotypic screen of a 540,288 compound library, we have discovered new inhibitors of the DosRST regulon and other inhibitors independent of the DosRST regulon[2]. These first-in-class chemical probes represent an innovative, strategy to inhibit Mtb persistence physiology. We initially characterized 3 DosRST regulon inhibitors (artemisinin, HC102A and HC103A). Under hypoxia, all three compounds inhibit Mtb NRP-associated physiologies, including triacylglycerol synthesis, survival and antibiotic tolerance[2]. Mechanism of action studies showed they directly inhibit DosS and DosT kinases[2]. Two additional DosRST regulon inhibitors, HC104 and HC106, will be characterized in this project. Our screen also identified several new chemical probes that inhibit Mtb growth independent of DosRST. The unifying goal of Aims 1 and 2 is to define the mechanisms of action and therapeutic potential of chemical probes that modulate Mtb and M. bovis survival via DosRST-dependent mechanisms. This project will define new mechanisms of NRP physiology and generate proof-of-concept data supporting development of new TB drugs. Because our new chemical probes function in whole Mtb cells, we can rapidly translate our findings into new drug candidates. Additionally, we will test the activity of the compounds against M. bovis in vitro and in silage, to determine if these compounds can function to eradicate M. bovis persisters and to determine if difference exist between M. bovis and Mtb transcriptional networks controlling persistence.In an independent project (Aim 3), we will harness models we have developed to study Mtb persistence to examine the survival of M. bovis in ensiled forages. Bovine tuberculosis (bTB) is endemic in Northeast Michigan in wild white tail deer, which serve as a reservoir for transmission of bTB to local cattle herds. Understanding potential modes of transmission of bTB from deer to cattle is critical for mitigating the risk of infection of cattle. Cattle feed contaminated by infected deer can transmit bTB to cattle. Mycobacterium bovis (M. bovis), the causal agent of bTB, can survive on a variety of fresh feedstuffs (i.e., apples, corn, carrots, sugar beets, potatoes, and hay) for at least 16 weeks. Some bacteria pathogenic for cattle survive the ensiling process including enterococci and streptococci and Listeria monocytogenes. Survival of M. bovis in fermented feeds commonly fed to cattle in Northeast Michigan is a concern because those feeds might serve as a potential reservoir of indirect transmission of bTB to cattle. Recently, we demonstrated that M. bovis could be cultured from various fermented feeds (alfalfa, mixed-grass, corn) for 2 to 28 days after the start of the ensiling process and DNA from M. bovis could be detected for 112 days. This last finding is of concern because the PCR assay used cannot differentiate residual DNA from dead M. bovis from DNA present in viable but not culturable dormant M. bovis.Under stressful conditions, many bacteria can enter a state of dormancy, where basic metabolic activity is maintained (e.g. ATP pools and membrane potential) but the bacteria do not grow. These bacteria are referred to as non-growing but metabolically active (NGMA) bacteria. Mycobacterium tuberculosis complex bacteria establish dormancy in response to environmental cues including hypoxia, acidic pH and starvation. NGMA M. tuberculosis can remain viable for decades and become resuscitated upon the appropriate environmental stimuli. Therefore, a sample or culture may appear to be free of viable bacteria using standard culture-based methods, however, in reality viable bacteria may exist in the sample. Identifying NGMA bacteria in complex samples is a major problem in clinical settings, particularly in the diagnosis of active tuberculosis in human sputum. Similarly, given that silage is predicted to have conditions that may induce dormancy, including hypoxia, nutrient limitation and acidic pH, it is possible that NGMA M. bovis may be present in silage, even if appears to be M. bovis-free using culture based methods. The potential resuscitation of NGMA M. bovis represents a risk for transmission; however, assessing the presence of NGMA bacteria remains a major challenge in Mycobacterial research. We propose to develop in vitro models of M. bovis dormancy and methods to assess the presence of NGMA bacteria. These methods will provide us with the tools to address the ultimate question of the presence of NGMA M. bovis in silage.

Project Methods
Specific Aim 1.1 Characterize the impact of HC104 and HC106 on Mtb and M. bovispersistence and drug tolerance.1.1A Transcriptional profiling of Mtb and M. bovistreated with HC104 or HC106. HC104A and HC106A inhibit key DosRST-regulated genes (data not shown). To define the breadth of DosRST inhibition, we will conduct RNA-seq profiling similar to that described in the preliminary data and our published study[2]. Briefly, CDC1551 or CDC1551(ΔdosR) cultures will be treated with 40 μM HC104A, HC106A or DMSO (as a negative control) and grown in rich medium at 37°C without shaking in T-25 vented, standing tissue culture flasks. Following 6 days of incubation, total bacterial RNA will extracted and sequenced and analyzed using the SPARTA software package developed by my lab.The above experiments will be repeated with an M. bovis strain isolated from Michigan deer, with the medium being changed to Dubos medium supplemented with pyruvate. Comparisons of the transcriptional profiles will provide new insights into shared or differential gene expression networks in Mtb or M. bovis.1.1B Inhibition of Mtb and M. bovis survival and drug tolerance during NRP. To determine if HC104A or HC106A inhibit survival during NRP, the hypoxic shift down assay will be used as a model for NRP[2,13]. Briefly, CDC1551, a CDC1551(DdosR) mutant or complemented strain will be pelleted and resuspended in Dubos medium, and inoculated in 24-well plates (1 mL/well). Cells will be treated with 40 mM HC104A or HC106A or equal volume of DMSO, and incubated in an anaerobic chamber (with a BD GasPak) for 12 days. Percent viability will be determined by comparing surviving bacteria at day 10 relative to day 0. For INH tolerance assays, the cells willbe treated with HC104 or HC106A and1, 5 or 25 mM INH or a DMSO control, incubated in the anaerobic chamber for 10 days and CFUs enumerated by plating on solid medium.The above experiments will be repeated with an M. bovis strain isolated from Michigan deer, with the Dubos medium being supplemented with pyruvate.Specific Aim 2.1 Optimize HC103, HC104 and HC106 leads for potency and PK properties.2.1A Optimizing compounds for potency and PK properties. i) Medicinal chemistry optimization strategy: The aromatic character of HC103A, HC104A and HC106F allows the investigation of each via a Topliss Tree evaluation[14], which provides an organized operational scheme for analog design that explores electronic, hydrophobic and steric effects, while minimizing the number of analogs pursued. ii) Monitoring DMPK properties: As the SAR proceeds, analogs will be tested for drug metabolism (DM)PK properties. Optimized compounds will be tested for activity against M. bovis, to define the function in M. bovis.?2.1B. Evaluate in vivo PK and pharmacodynamics (PKPD) of prioritized analogs. Tolerability: Acceptable formulation(s) will be developed suitable for oral administration and the tolerability of representative compounds will be assessed following single oral doses (25 mg/kg to 250 mg/kg). In vivo PK: We will first complete a 'snapshot' PK (oral, 4 time-points up to 5h post-dose) for 3 priority HC103, HC104 and HC106 analogs. For one HC103, HC104 and HC105 analog showing the best PK properties, full PK profiling (3 doses, 6 timepoints up to 24h post-dose) will be defined.Specific Aim 2.2 Test optimized leads in vivo for efficacy and inhibition of DosRST signaling.Evaluate effect of DosR regulon inhibition on Mtb antibiotic tolerance and virulence in Kramnik mice. C3HeB/FeJ mice will be aerosol infected with Mtb Erdman (hspX'::GFP, smyc'::mCherry) reporter strain (low dose of 55 CFU/mouse[15]) in our ABSL3 lab. We will use 10 mice per group to obtain significant results in the inherently variable Kramnik model[15]. The study will include 4 arms: Arm 1) Untreated; Arm 2) Treated with INH, starting 4 weeks post infection (PI); Arm 3) Treated with persistence inhibitor for the duration of the experiment; Arm 4) Treated with persistence inhibitor, with INH starting 4 weeks PI. Four, 8 and 12 weeks PI, lungs and spleens will be harvested for CFUs and histopathology. Specific Aim 3.1: Establishment of in vitro models of M. bovis persistenceExperimental procedure: A clinical M. bovis isolate will be grown in Dubos complete medium (with pyruvate), washed twice and then resuspended in 1 mL of Dubos medium at an OD of 0.2 in a well of a 24 well plate. Control wells will be include the presence of 1.5 mg/ml of methylene blue to monitor the establishment of hypoxia. Inoculated wells will be treated with DMSO, 25 ?M INH or 25 ?M PA-824 and the plates will be sealed in the anaerobic jars and incubated for 10 days. At days 0, 5 and 10, cultures will be dilution plated on 7H11 agar and colony forming units (CFUs) counted.Specific Aim 3.2: Development of methods to detect non-growing metabolically active M. bovis.3.2A Using live/dead stains to detect NGMA M. bovis.Experimental procedure: Several live-dead stains exist that enable the detection of individual viable bacteria by fluorescence microscopy or flow cytometry. Fluorescein diacetate (FDA) is non-fluorescent until it is actively transported into the bacterial cell and metabolized into a fluorescent state. Rhodamine 123 stains bacteria with an energized membrane potential. LIVE/DEAD BacLight kit allows for detection of all bacteria with SYTO9 stain and dead bacteria with the membrane impermeable propidium iodide. NGMA M. bovis bacteria will be generated using the hypoxic downshift assay.3.2B Using a luminescent reporter to detect NGMAM. bovis.Experimental procedure: Luciferase reporters are powerful tools to detect viable bacteria. Because the luciferase enzyme will only be maintained in live cells, the presence of luminescence is a direct measure of viability. The Abramovitch lab has successfully developed luciferase expressing M. tuberculosis strains that exhibit high levels of luminescence. M. bovis will be transformed with the pMV306::hsp60-fluc firefly luciferase (fluc) reporter plasmid. The plasmid is an integrative plasmid that will stably incorporate into the M. bovis genome at the attB site and expresses fluc from a strong constitutive hsp60 promoter. Reporter function will be determined using the Bright-Glo luciferase reagent (Promega) and measuring luminescence using a Perkin Elmer Espire ultra-sensitive luminometer housed in the Abramovitch BSL-3 lab. Once the M. bovis (hsp60'::fluc) is validated, NGMA M. bovis (hsp60'::fluc) bacteria will be generated using the hypoxic downshift assay. In this assay, we will include the presence of 20 ?M INH, to ensure that replicating bacteria are killed. Bacteria will be isolated from the assay at Days 0, 5 and 10 and lumiscence will be monitored.Specific Aim 3.3: Determine viability of M. bovis in laboratory fermentation modelExperimental procedure: A mixed grass forage of the type grown in Northeast MI will be harvested and inoculated with either M. bovis strain MDCH # 358258 or M. bovis (hsp60'::fluc) at a concentration of 1ml/100gms of forage. Exactly 500 grams of inoculated forage will be placed into a 8" x 12" nylon/polyethylene 4-mil high-performance film vacuum pouch. Air will then be evacuated and the bag sealed andheld at 20°C (68°F) until the designated period of opening. In parallel, uninoculated bags will be ensiled serving as controls for the ensiling process. Bags of inoculated and control silage will be opened at the following time points: Day - 0, 1, 7, 14, 28, 56, and 112 (0 = immediately after inoculation). Samples will be taken at each time point and processed for routine culture of M. bovis, analyzed using quantitative PCR for M. bovis DNA, and tested for viability using live/dead stains and ultrasensitive luminescent reporter system described above. In addition, a sub sample of the control silage will be sent for fermentation profile at each time point.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Mycobacterium tuberculosis is a bacterial pathogen that causes tuberculosis (TB) in humans. The spread of TB is a global health crisis leading to over one million deaths annually. The global burden of TB is a threat to the health of all Americans, given the easy transmission of the disease through the air and the emergence of drug resistant strains that are difficult to treat. Moreover, there exists an increasing population of Americans with enhanced susceptibility to TB due to factors associated with compromised immune systems, including: HIV infection, the use of anti-rejection and anti-inflammatory drugs, as well as natural decreases in immunity associated with an aging population. Therefore, my research on TB has a global target audience. Mycobacterium bovis causes bovine tuberculosis (bTB) disease in wildlife, livestock and humans throughout the world. M. bovis has established endemic populations in diverse wildlife species and these natural reservoirs present a threat to the health of domesticated livestock and the people that depend on these animals. bTB is present in wildlife and livestock in most African countries. Livestock mortality caused by M. bovis negatively impacts both food security and income of people in Africa. In developed countries, robust bTB surveillance programs are in place to protect human and livestock health. In Michigan, bTB represents a problem for both agriculture and recreational hunting because in northeastern, lower peninsula townships, M. bovis is found in wild populations of white tailed deer. bTB negatively impacts Michigan's economy, given the costs of ongoing bovine TB surveillance programs, restrictions on interstate movement of livestock, and reduction of hunting- associated tourism. Therefore, my research on bTB has a global target audience with a specific emphasis on those involved in Michigan agriculture and tourism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development opportunities were somewhat limited due to COVID-19 this past year. However, I did present data directly related to this project at one scientific conference and one departmental seminar. March 2020 "Small Molecules Targeting theMycobacterium tuberculosisDosRST Two-Component Regulatory System"Central Michigan University, Dept. of Chemistry and Biochemistry,Departmental Seminar. January 2020 "TargetingM. tuberculosistwo-component regulatory systems"Gordon Research Conference on Sensory Transduction in Microorganisms.Ventura, CA.National meeting. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Our lab recently (Sept. 2020)received an R01 grant to expand our studies on HC106 and DosRST signaling. We plan to conduct additional molecular modeling of DosRST with DosS and DosT GAF domains to conduct structure driven HC106 optimization. In collaboration with the Ellsworth lab, we synthesize up to 50 new analogs and test their activity against Mtb and M. bovis. We will also conduct biochemical and structural characterizations of HC106 interactions with DosS and DosT. Using the newly defined positive selection method, we will screen for mutants in DosS and DosT with reduced signaling in response to hypoxia or nitric oxide. We will identify the mutations and examine the biochemical basis for the altered function of the proteins. Finally, we will use newly developed CRISPRi tools to exmine the impact of conditionally knocking down DosRST genes in Mtb and M. bovis during hypoxia and NO treatment.

Impacts
What was accomplished under these goals? This past year we published our findings about the mechanism of action of HC106. Notably, this manuscript described new analogs and structure-activity relationships of HC106. The analogs have increased potentcy and provided new insights into the potential mechanism of action. With these new data, we initiatedModeling HC106 interactions with the DosS GAF domain. In collaboration withProf. Angela Wilson's team, wemodelledHC106A into the crystal structure of the DosS GAF domain (PDB: 2W3E),minimized using molecular mechanics (Extended Hückel Theory) under Molecular Operating Environment (MOE). Studies identified a binding site, which neighbors both the heme and the Gly117 and HC106A was docked using a pharmacophore approach and the poses scored using Generalized-Born Volume Integral/Weighted Surface area scoring function (GBVI/WSA ?G). The highest scoring pose supports the SAR data presented in our recent publicationincluding, the coordination of the isoxazole nitrogen to the iron center of the heme.Additional efforts docking the analogs in provided a model of the ligand binding environment.It predicts the isoxazole ring to be surrounded by three hydrophobicresidues and the urea "locked" in place by hydrogen-bonding interactions with one of the heme carboxylates, which makes H-binding interactions to the urea NHs.The binding domain then stretches through a narrow channel leading to a lipophilic space that transitions to a polar environment with opportunities to make H-bonding interactions (i.e.; H89 and H93) to residues previously reported to be key in the conformational changes associated with CO on NO-dependent DosS/T kinase activation. Beyond, the binding domain then opens to solvent. The model suggests that with the binding of HC106, the conformations of E87 and H89 are, as described by others, locked in a position that prevents the protein from autophoshorylation.It has been suggested that the upward swing of Glu87 making a H-bonding interaction with H89 is essential for the redox process of Fe(II) to Fe(III).This model, with HC106A bound, blocks this interaction.The lipophilic interactions with V95 and I125 are also viewed as important as they too participate in the mechanistic behavior of the protein. We also compared the docking energies from studies to the publishedEC50valuesfinding that although the model predicts the strongest and weakest binders, there are examples where the calculated binding energies are over- and under-predictive, relative to the observed EC50s.The differential is readily explained by the combination of variable compound permeability (physical properties), the preliminary nature of the model and the potential flexibility of the protein when binding to inhibitors of diverse structure.As the G117L mutant inactivates HC106A activity vs. DosS/T, we docked the most potent inhibitors from Table 1 (MSU-33189, -39445, -39446, -39447, -41462, -41542) into the protein modified with this mutation. The selected analogs saw a reduction of binding energy, relative to the WT protein, supporting the binding model. We have also collaborated with Sean Crosson's lab to optimize expression of the DosS GAF domainwith an H6-sumo tag. Pure DosS-GAF protein fractions were isolated from Superdex 200 16/600 following Ni-NTA affinity chromatography and tag cleavage by ULP1. The final concentration of the purified protein was 34.8 mg/ml (2 mM) and suitable for crystallization studies. Additionally, we have developed a new tool to allow positive genetic selection for signaling mutants in the DosRST signaling. We plan to screen both Mtb and M. bovis using this new tool, to conduct comparitive genomics in DosRST signaling between Mtb and M. bovis.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Martini MC, Zhang T, Williams JT, Abramovitch RB, Weathers PJ, Shell SS (2020). Artemisia annua and Artemisia afra extracts exhibit strong bactericidal activity against Mycobacterium tuberculosis. J. Ethnopharmacology. doi.org/10.1016/j.jep.2020.113191
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zheng H and Abramovitch RB (2020). Inhibiting DosRST as a new approach to TB therapy. Future Medicinal Chemistry. Mar;12(5):457-467.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sanchez KG, Ferrell MJ, Chirakos AE, Nicholson KR, Abramovitch RB, Champion MM, Champion PA (2020). EspM is a conserved transcription factor that regulates gene expression in response to the ESX-1 system in Mycobacterium marinum. mBio. Feb 4;11(1):e02807-19.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Carneiro PAM, Pasquatti T, Takatani HD, Zum�rraga MJ, Marfil MJ, Barnard C, Fitzgerald, S, Abramovitch, RB, Araujo F, Kaneene JB (2020). Molecular Characterization of Mycobacterium bovis infection in Cattle and Buffaloes in Amazon Region, Brazil. Veterinary Medicine and Science. Feb;6(1):133-141.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Williams JT, Haiderer ER, Coulson GB, Conner K, Chen C, Dick T, Ellsworth E, Li W, Jackson MJ, Abramovitch RB (2019). Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance. Antimicrobial Agents and Chemotherapy, Oct 63(10). pii: e00547-19.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zheng H, Williams JT, Aleiwi B, Ellsworth E, and Abramovitch RB (2020). Inhibition of Mycobacterium tuberculosis DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme. ACS Chemical Biology. 15(1):52-62.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Baker JJ, Dechow SJ, Abramovitch RB (2019). Acid Fasting: Modulation of Mycobacterium tuberculosis metabolism at acidic pH. Trends in Microbiology. 27(11):942-953.
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Zheng H, Abramovitch RB (2019). Host-pathogen interactions influencing Mycobacterium tuberculosis persistence and drug tolerance. Persister Cells and Infectious Disease. Edited by Kim Lewis. Springer.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: Mycobacterium tuberculosis is a bacterial pathogen that causes tuberculosis (TB) in humans. The spread of TB is a global health crisis leading to over one million deaths annually. The global burden of TB is a threat to the health of all Americans, and directly relevant to the mission of the National Institute of Allergy and Infectious Diseases, given the easy transmission of the disease through the air and the emergence of drug resistant strains that are difficult to treat. Moreover, there exists an increasing population of Americans with enhanced susceptibility to TB due to factors associated with compromised immune systems, including: HIV infection, the use of anti-rejection and anti inflammatory drugs, as well as natural decreases in immunity associated with an aging population. Therefore, my research on TB has a global target audience. Mycobacterium bovis causes bovine tuberculosis (bTB) disease in wildlife, livestock and humans throughout the world. M. bovis has established endemic populations in diverse wildlife species and these natural reservoirs present a threat to the health of domesticated livestock and the people that depend on these animals. Bovine TB is present in wildlife and livestock in most African countries. Livestock mortality caused by M. bovis negatively impacts both food security and income of people in Africa. In developed countries, robust bovine TB surveillance programs are in place to protect human and livestock health. In Michigan, bovine TB represents a problem for both agriculture and recreational hunting because in northeastern, lower peninsula townships, M. bovis is found in wild populations of white tailed deer. Bovine TB negatively impacts Michigan's economy, given the costs of ongoing bovine TB surveillance programs, restrictions on interstate movement of livestock, and reduction of hunting-associated tourism. Therefore, my research on bTB has a global target audience with a specific emphasis on those involved in Michigan agriculture and tourism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported the training of a graduate student (John Williams) in using the hypoxic shift down model to study persistence and in conducting biochemical studies with DosS and DosT proteins. This work will enable John to extend his work into other mycobacterial pathogens, including M. bovis and M. abscessus. The project also supported the training of a postdoc(Uma Shankar Gautum) in the use of ITC and CRISPRi in mycobacteria. These new methods can be applied for future studies related to this project. Research related to DosRST inhibitors project was presented at the following meetings and department seminars: 05/19"TargetingM. tuberculosistwo-component regulatory systems"Chemistry and Biology of Pathogens Symposium.East Lansing, MI.Local Meeting 04/19"Targeting two component systems to inhibitMycobacterium tuberculosispathogenesis"Rutgers New Jersey Medical School, Dept. of Pharmacology, Physiology and Neuroscience.Departmental Seminar. 03/19"Targeting two component systems to inhibitMycobacterium tuberculosispathogenesis"Vanderbilt Institute of Chemical Biology.Departmental Seminar. 01/19"Targeting two component systems to inhibitMycobacterium tuberculosispathogenesis"Keystone Symposium on Tuberculosis: Mechanisms, Pathogenesis, and Treatment, Banff, Alberta, Canada.International meeting. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are eager to establish CRISPRi technology in M. bovis. Given the similarities between Mtb and M. bovis, it is expected to function, but has not yet been reported. Sequence similiarity between M. bovis and Mtb DosRis identical (100% identity) and nearly identical for DosT (99.9% identical with one SNP), enabling us to use the DosT and DosR CRISPRi constructs in M. bovis. For these studies, we will transform the CRISPRi constructs into M. bovis and then examine the impact of CRISRPi induction on regulation of DosR regulated genes and survival during NRP. We will similarly examine the impact of treatment with HC106 onthe survival of M. bovis in the hypoxic shift-down model. Should we observe an impact with CRISPRi or HC106 treatment, we will see if targeting this pathway can reduce M. bovis survival in ensiled forages, using methods developed and described in the previous year's report.

Impacts
What was accomplished under these goals? Towards the goal of chacterizing HC104 and HC106 we made additional progress this year. To address reviewer critiques for the submitted study, we characterized a new HC106 analog called MSU-39446.For this experiment, we used a more potent analog, MSU-39446 and examined its impact on survival of WT,dosRmutant and the complemented strains in the hypoxic shift down assay. The experiment was repeated in two independent experiments, each with three biological replicates, and the data from all six replicates was combined.The experiments showed that MSU-39446 kills Mtb in the hypoxic shift-down assay and did so more effectively that HC106A. As previously shown, thedosRmutant had significantly reduced survival as compared to the WT, and the phenotype was complemented. There was no significant difference in survival of the dosR mutant between the MSU-39466 treated or DMSO control. Thus, these data are consistent with HC106 targeting DosR to kill Mtb during NRP. We also biochemical experiments examining the impact of HC106 on DosT function. We observed that DosT treated with HC106 exhibited an intermediate peak in a UV-visible spectroscopy assay. This finding is consistent with HC106 binding DosT heme in a manner similar to nitric oxide:heme or carbon monoxide:heme ligand interactions. Thus, we have shown that HC106 interacts with DosS and DosT by a similar mehcanism. In other experiments, we examined the impact of overexpressing HC106-resistant alleles of DosS or DosT in M. tuberculosis and examining their impact on Mtb survival duringpersistence. For these experiments, we tested if overexpression of DosS(G117L) or DosT (G115L) provides resistance to treatment with the HC106 analog MSU-39446 in the hypoxic shift-down assay. Unexpectedly, we observed overexpression of the control DosS or DosT proteins caused a significant 80% and 60% reduction of Mtb survival, respectively. Treatment of the DosS overexpressor with MSU-39446 caused a significant reduction of survival relative to the DMSO control, whereas, no significant difference in survival was observed in the DosS(G117L) overexpressor. These data are suggestive of the DosS (G117L) mutant providing HC106 resistance, but are not conclusive given the confounding survival defect in the DosS overexpressor. The DosT (G115L) overexpressor had significantly reduced survival when treated with MSU-39446, supporting limited resistance provided by this mutation during non-replicating persistence. With the additional studies described above, we prepared a revised version of the HC104/6 study that was accepted for publication at ACS Chemical Biology. We have also initiate a collabaration with our MSU colleague, Prof. Sean Crosson, to obtain a DosS/HC106 crystal structure. We generated a his-tagged version of the DosS GAF domain and optimized conditions for its expression and purification from E. coli. The protein was suitable for crystallization studies and Dr. Crosson is currently working to obtain crystals of DosS with HC106. Obtaining a co-crystal structure would provide important new information regarding the mechanism of action of HC106 and enable structure guided optimizations of these compounds. Additionally, we have conducted biochemical studies examining physical interactions of DosRST inhibitors with full length recombinant DosS protein.Optimization experiments wereundertaken to validate the use of Isothermal Titration Calorimetry (ITC) to study protein-probe interactions. In these studies, we used full length DosS-His protein and HC103A, which inhibits 50% of DosS autophosphorylation at 1 µM. DosS-His (8 µM, in presence or absence of KCl, MgCl2 and ATP cofactors used in the kinase assay shown in Fig. 4D) was tested with 30 automatic injections of HC103A (200 µM) or DMSO. In the presence of cofactors, DosS-His interacted with HC103A with an equilibrium dissociation constant (KD) of 3.2 µM (range 1.7-17 µM) and one binding site (N=1.00), consistent with the autophosphorylation assay. No interaction was observed in the DMSO control or DosS-His in the absence of cofactors, supporting a specific interaction dependent on conditions associated with kinase activity. CRISPRi knockdown constructs forDosR and DosT were also developed. CRISPRi is a powerful new approach to conditionally knockdown gene expression in mycobacteria. The system involves conditionally expressing, by addition of anhydrotetracycline (aTC), a single guide RNA (sgRNA) specific for a gene of interest and a nuclease-dead Cas964. The sgRNA targets Cas9 to a gene of interest and its binding blocks transcription. For the Mtb system, the construct is built on an integrative plasmid with an attP that inserts on the Mtb genome at the attB site. Notably, multiple sgRNAs can be expressed to knockdown expression of multiple genes. For dosR and dosT, we have generated CRISPRi constructs that cause 55-fold and 26-fold, inducible knockdown, respectively. To validate the DosR knockdown, we conducted RNA-seq transcriptional profiling and observed strong and specific downregulation of the DosR pathway, with the most strongly downregulated gene being dosR (69-fold downregulated). dosS, which is downstream of dosR in an operon, is also downregulated. All 48 genes of the DosR regulon were inhibited. The downregulation of the regulon is similar to what we observe with a dosR knockout or HC101, -2 or -3 treatments. Thus, like the chemical inhibitors, the CRISPRi system can be used to conditionally knockdown DosR expression.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Zheng H, Williams JT, Aleiwi B, Ellsworth E, and Abramovitch RB (2019). Inhibition of�Mycobacterium tuberculosis�DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme. ACS Chemical Biology, in press.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Williams JT, Haiderer ER, Coulson GB, Conner K, Chen C, Dick T, Ellsworth E, Li W, Jackson MJ, Abramovitch RB (2019). Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance. Antimicrobial Agents and Chemotherapy, Oct 63(10). pii: e00547-19.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Baker JJ, Dechow SJ, Abramovitch RB (2019). Acid Fasting: Modulation of Mycobacterium tuberculosis metabolism at acidic pH. Trends in Microbiology. In press.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Jeon AB, Ackart DF, Li W, Jackson M, Melander RJ, Melander C, Abramovitch RB, Chicco AJ, Casaraba RJ, Obregon-Henao A (2019). 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain. Scientific Reports. �9 (1), 1513.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Franfater C*, Abramovitch RB*, Purdy GE*, Turk J, Legentil L, Lemiegre L, Hsu FF (2019). Multiple-stage precursor ion separation and high-resolution mass spectrometry toward structural characterization of 2,3-diacyltrehalose family from Mycobacterium tuberculosis. Separations, in press
• Type: Book Chapters Status: Accepted Year Published: 2019 Citation: 1. Zheng H, Abramovitch RB (2019). Host-pathogen interactions influencing Mycobacterium tuberculosis persistence and drug tolerance. Persister Cells and Infectious Disease. Edited by Kim Lewis. Springer. In press.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Carneiro PAM, Pasquatti T, Takatani HD, Zum�rraga MJ, Marfil MJ, Barnard C, Fitzgerald, S, Abramovitch, RB, Araujo F, Kaneene JB (2019). Molecular Characterization of Mycobacterium bovis infection in Cattle and Buffaloes in Amazon Region, Brazil. Veterinary Medicine and Science. In press.

Progress 02/14/18 to 09/30/18

Outputs
Target Audience:Mycobacterium tuberculosis is a bacterial pathogen that causes tuberculosis (TB) in humans. The spread of TB is a global health crisis leading to over one million deaths annually. The global burden of TB is a threat to the health of all Americans, and directly relevant to the mission of the National Institute of Allergy and Infectious Diseases, given the easy transmission of the disease through the air and the emergence of drug resistant strains that are difficult to treat. Moreover, there exists an increasing population of Americans with enhanced susceptibility to TB due to factors associated with compromised immune systems, including: HIV infection, the use of anti-rejection and anti inflammatory drugs, as well as natural decreases in immunity associated with an aging population. Therefore, my research on TB has a global target audience. Mycobacterium bovis causes bovine tuberculosis (bTB) disease in wildlife, livestock and humans throughout the world. M. bovis has established endemic populations in diverse wildlife species and these natural reservoirs present a threat to the health of domesticated livestock and the people that depend on these animals. Bovine TB is present in wildlife and livestock in most African countries. Livestock mortality caused by M. bovis negatively impacts both food security and income of people in Africa. In developed countries, robust bovine TB surveillance programs are in place to protect human and livestock health. In Michigan, bovine TB represents a problem for both agriculture and recreational hunting because in northeastern, lower peninsula townships, M. bovis is found in wild populations of white tailed deer. Bovine TB negatively impacts Michigan's economy, given the costs of ongoing bovine TB surveillance programs, restrictions on interstate movement of livestock, and reduction of hunting-associated tourism. Therefore, my research on bTB has a global target audience with a specific emphasis on those involved in Michigan agriculture and tourism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Work related to this project was presented at the following seminars during the reporting period: 04/18 "Chemical biology of Mycobacterium tuberculosis pathogenesis"MSU Drug Discovery Seminar, Dept. of Pharmacology and Toxicology 11/17 "Small molecules that inhibit Mycobacterium tuberculosis environmental sensing and virulence" Johns Hopkins University, Department of Medicine.Departmental seminar. 11/17 "Inhibitors ofMycobacterium tuberculosis persistence and pathogenesis"Worcester Polytechnic Institute, Department of Biology and Biotechnology. Departmental seminar. 10/17 "Chemical biology of Mycobacterium tuberculosis pathogenesis"University of Tennessee-Knoxville, Department of Microbiology.Departmental seminar. 10/17 "Chemical biology of Mycobacterium tuberculosis pathogenesis"The Ohio State University, Department of Microbial Infection and Immunity. Departmental seminar. How have the results been disseminated to communities of interest?To disseminate the findings from the M. bovis survival in silage study, we held a meeting with representatives in from the state of Michigan with an interest in M. bovis. The meeting was held at the Veterinary Diagnotic Laboratory on June 1, 2018 andwas attended by representatives from the Michigan Department of Agriculture and Rural Development and the state veterinarian. Additionally a summary report was submitted to AgBioResearch as part of the Michigan Animal Agriculture Alliance grant that supported the M. bovis surivival in silage study. What do you plan to do during the next reporting period to accomplish the goals?For Aims 1 and 2, we will continue to study the function of HC106 and its analogs in M. tuberculosis and plan to extend our studies to M. bovis and other mycobacterial species, including work with collaborators on nontuberculous mycobacteria. This work, pending additional funding, will include additional structure activity relationship studies of HC106/DosST interactions, attempts to obtain a HC106/DosS co-crystal structure and efforts to examine HC106 activity during animal infection. Efforts on HC104 will be deprioritized given its partial impact on DosRST signaling. As a substitute, we will be begin characterizaiton of an unrelated DosRST inhibitor, called HC105A. For Aim 3, we plan to repeat the experiment examining M. bovis survival in silage to obtain a replicate set of data suitable for publication.

Impacts
What was accomplished under these goals? For Aims 1 and 2 we have made substantial progress in characterizing the function of HC104 and HC106 as inhibitors of the DosR regulon. As described in a manuscript under review (and available as a preprint athttps://www.biorxiv.org/content/early/2018/09/11/411793), we have conducted mehcanism of action and structure activity relationsship studies for both compounds. Using RNA-seq transcriptional profiling, we found that HC104 and HC106 inhibit DosR regualted genes, with HC106 strongly inhbiting the full DosR regulon and HC104 inhibiting only a portion of DosR regulated genes. HC106 was shown to inhibit Mtb survival during non-replicating persistence and found to act synergistically with other DosRST inhibitors. HC104A did not inhibit survival during non-replicating persistence and had limited synergistic interactions. Using biochemical assays, it was found that HC106 targted sensor kinase heme, while HC104 functioned to inhibit DosR binding of promoter DNA. Medicinal chemistry optimizations of HC106 resulted in over 20 new analogs, several of which had significantly enhanced potency. For Aim 3, we completed a study examining M. bovis survival in silage.The objective of this study is to determine if M. bovis is destroyed during the enisling process or if it is transformed into a not culturable, dormant state. The specific Aims of this study are to:? Development of methods to detect live vs. dead M. bovis:To develop this reporter, a M. tuberculosis codon optimized luciferase was employed. This reporter plasmid contains a firefly luciferase expressed under the control of a constitutive hsp60 promoter. The plasmid is an integrative plasmid, that can be stably integrated into the M. bovis genome. As part of this project, M. bovis (strain MDCH 358258)electrocompetent cells were generated and the luciferase reporter plasmid was electroporated into M. bovis to generate the reporter strain M. bovis (hsp60::Ffluc). The reporter strain was initially validated in vitro. Bacteria were grown in rich medium (7H9, OADC, without glycerol and supplemented with pyruvate) to log phase and then diluted across an 8-point 10-fold dilution curve from 20 CFU to 2x108 CFU, as 100 µL in each well of a 96 well plate. One hundred µl of BrightGlo (Promega) luciferin reagent was added to each well and imaged in an Enspire plate reader with an ultrasensitive luminometer over the course of 20 minutes. The reporter showed strong luminescent signal, with an over 1000-fold dynamic range, and a linear signal-to-CFU ratio up to the limit of detection of around ~50,000 CFU. From these data, we conclude that we have successfully developed a reporter strain that can detect viable bacteria. ? Determine viability of M. bovis in laboratory fermentation model:?The experiment was begun in August 2017. Three mL of M. bovis (hsp60'::Ffluc) at ~ 108 CFU/mL was added to a dialysis cassette and added to a freezer bag containing 250 g of freshly cut alfalfa grass and 10 mL of sterile Tryptose broth. Two biological reps were included for each experimental condition. A control to examine survival of M. bovis in the absence of silage was included, where the bacteria were added to a dialysis cassette and placed in a freezer bag without any grass. To monitor the ensiling process, a control was included with grass and broth, but no M. bovis. The freezer bags were sealed and incubated at room temperature. Sampling was conducted following 1, 2, 4, 8, and 16 weeks of incubation, M. bovis was removed from the cassettes and the samples were examined for: luciferase signal, cultivability, direct staining and PCR. Fermentation reports from the no-bacteria silage were obtained from Dairy One. For the luminescence assay, following 1 week of exposure in silage no bioluminescent bacteria could be detected. Similarly, no bacteria could be detected following 2 weeks of incubation in silage. Based on these observations, luminescence of bacteria was not monitored beyond week 2. In the no silage controls, luminescent bacteria were detected in both week 1 and 2, with ~1 log reduced survival following each week of incubation at room temperature in a sealed bag in a dialysis cassette. Given the initial input, we can conclude that within 1 week of treatment in silage there is >3 logs of bacterial killing, to levels below our level of detection. We also monitored the samples for the presence of bacteria by direct staining and quantitative PCR and viability by direct culture and enrichment culture. In the no silage control (bacteria in a dialysis cassette with no grass) viable bacteria were detectable at all time points, except week 6 and 16, suggesting, in the absence of silage, the bacteria could remain viable in the dialysis cassettes. In the bacteria incubated in silage viable M. bovis was detected by direct culture robustly in week 1 and 2, but was not detected in one of the replicates (Rep A) in week 4 and only 1 colony was observed in Rep B. By week 6, bacteria could no longer be detected by direct culture in the silage. Enrichment culture enables more sensitive detection of bacteria in culture and was used to further assess viability of the bacteria. We observed viable bacteria by enrichment culture through week 6, but no viable bacteria were detected in week 8 or 16. Notably, the time to positivity, increased as the bacteria were incubated in silage, supporting that the ensiling process was functioning to kill the bacteria. At all time points bacteria could be detected by staining and quantitative PCR, however, these methods do not distinguish live versus dead bacteria. Live dead staining was also conducted, the samples were fixed and will be analyzed by flow cytometry following the completion of the second experimental replicate for this experiment, planned for the summer of 2018. At each time point the silage was also analyzed and the fermentation reports showed that the silage had acidified to pH 5 following 1 week of incubation and remained acidified through week 8. Thus, the experimental silage used in this experiment functioned as anticipated. Conclusions These experiments show that the M. bovis is rapidly killed in silage and following 8 weeks cannot be cultured, even when high numbers of bacteria were inoculated (>100 million bacteria) and the bacteria are known to be recovered as they are all contained in the dialysis cassette. These data support that silage is an effective mechanism of killing M. bovis. However, with these data, we cannot rule out that there may exist an hidden subpopulation of M. bovis that cannot be recovered or cultured using standard methods. ?

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Baker JJ, Abramovitch RB (2018). Genetic and metabolic regulation of Mycobacterium tuberculosis acid growth arrest. Scientific Reports. 8;8(1): 4168.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zheng H, Williams JT, Coulson GB, Haiderer ER, Abramovitch RB (2018). HC2091 kills Mycobacterium tuberculosis by targeting the MmpL3 mycolic acid transporter. Antimicrobial Agents and Chemotherapy. Jun 26;62(7). pii: e02459-17.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Abramovitch RB (2018). Mycobacterium tuberculosis reporter strains as tools for drug discovery and development. IUBMB Life. 70(9):818-825.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Zheng H, Aleiwi B, Ellsworth E, Abramovitch RB (2018) Inhibition of Mycobacterium tuberculosis DosRST two-component regulatory system signaling by targeting response regulator DNA binding and sensor kinase heme. Under Review