MECHANISMS OF MYCOBACTERIUM TUBERCULOSIS COMPLEX SPECIES PERSISTENCE

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.