Inhibitors of mycobacterium tuberculosis DosRST signaling and persistence
Tuberculosis (TB) is one of the most deadly infectious diseases in human history and is caused by the bacterium Mycobacterium tuberculosis (Mtb). Current TB therapy requires 6-9 months of treatment with four different antibiotics, including isoniazid, rifampin, ethambutol and pyrazinamide. However, due to the long course of TB therapy and the evolution of drug-resistant Mtb strains, first-line anti-mycobacterial drugs are not sufficient to control the TB epidemic. Therefore, it is urgent to develop new drugs with novel targets to shorten the course of therapy, control the spread of drug-resistant TB and eradicate this deadly disease. In response to host immune cues, Mtb modulates its metabolism to establish a state of low metabolic activity called non-replicating persistence (NRP). During NRP, Mtb can remain viable in the host without causing disease symptoms, a state known as latent TB. DosRST is a two-component regulatory system that plays an essential role to establish and maintain NRP in Mtb. It is induced by host immune stimuli, such as hypoxia, carbon monoxide and nitric oxide, through the histidine kinase sensors DosS and DosT. The response regulator DosR regulates about 50 genes in the dormancy regulon. NRP bacilli are problematic for two reasons: 1) they are insensitive to several anti-mycobacterial agents and drive the long course of TB therapy; and, 2) they can resuscitate for growth once the immune system weakens, thereby serving as a source for reactivation of disease and infectious transmission of the Mtb. Therefore, inhibiting the DosRST pathway may help reduce the population of NRP bacteria during infection and thus function to reduce drug tolerance and shorten TB treatment. This dissertation presents a whole-cell phenotypic high-throughput screen of a ~540,000 compound small-molecule library. The screen employed a DosR-dependent, hypoxia-inducible fluorescent reporter strain, CDC1551(hspX::GFP), and successfully identified six distinct, novel chemical inhibitors of DosRST signaling, named HC101A-106A. Physiological and mechanistic studies were performed to characterize HC101-104 and HC106A. All five inhibitors are shown to inhibit genes of the DosRST regulon and persistence-associated physiologies, such as triacylglycerol accumulation. HC101A, HC102A, HC103A and HC106A also reduce Mtb survival when cultured under strongly hypoxic conditions. UV-visible spectroscopy studies show that HC101A (artemisinin) and HC106A target the heme group of sensor kinases DosS/T via distinct mechanisms. For example, artemisinin modulates the redox status of DosS/T and alkylates the heme to form artemisinin-heme adducts, whereas HC106A interacts with DosS heme in a similar manner to direct CO-heme or NO-heme interactions. In contrast, HC102A and HC103A do not target the heme group, but instead inhibit sensor kinase autophosphorylation activity. Electrophoretic mobility assays suggest that HC104A functions by directly inhibiting DosR DNA binding activity. Overall, this dissertation provides proof-of-concept that multiple components of the DosRST pathway can be targeted by small molecules to inhibit Mtb persistence and antibiotic tolerance. Additionally, this dissertation presents the discovery of a new chemical inhibitor, HC2091, that kills Mtb by targeting the mycolic acid transporter MmpL3. MmpL3 is an essential protein that functions to transport trehalose monomycolate across mycomembranes for trehalose dimycolate biosynthesis. HC2091 is bactericidal against Mtb in a dose- and time- dependent manner in vitro. It also has activity against Mtb inside of macrophages. Whole genome sequencing spontaneous mutants resistant to HC2091 identified five single nucleotide variants primarily located in the C-terminus of MmpL3, and HC2091-treated Mtb exhibits decreased mycolic acid synthesis, thus supporting that MmpL3 is the target of HC2091.Read
- In Collections
Electronic Theses & Dissertations
- Copyright Status
- Attribution-NonCommercial-NoDerivatives 4.0 International
- Material Type
- Thesis Advisors
Abramovitch, Robert B.
- Committee Members
Hausinger, Robert R.
Mansfield, Linda S.
Waters, Christopher M.
Drug resistance in microorganisms
- Program of Study
Microbiology and Molecular Genetics - Doctor of Philosophy
- Degree Level
- xv, 197 pages