| Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the most common cause of death worldwide due to a single bacterial pathogen. Within the human host, Mtb is thought to reside primarily within the phagolysosomal compartment of macrophages, where it encounters an acidic environment (pH ∼4.5). Despite this acid stress, Mtb can maintain its intrabacterial pH (pHIB ) and persist in a presumably non-replicating state. Non-replicating populations of Mtb are phenotypically tolerant to most anti-infective agents, therefore complicating the treatment of TB. The ability of Mtb to resist acid stress is due in part to the membrane associated serine hydrolase Rv3671c. Mtb lacking Rv3671c is sensitive to acid and fails to maintain its pHIB in vitro and in activated macrophages. The Rv3671c strain is also severely attenuated in mice raising the possibility that pHIB homeostasis is essential for Mtb's growth and persistence in vivo. In this thesis, we focus primarily on understanding the bacterial mechanisms required to resist acid stress and persist in a non-replicating state using both chemical and genetic approaches.;Our first chemical approach, described in Chapter 2, demonstrates a whole-cell high-throughput screen to identify small molecule inhibitors of pHIB homeostasis in Mtb. We used Mtb expressing a pH-sensitive, ratiometric GFP, which allows for the monitoring of pH IB in response to treatment with small molecule compounds. Screening of a 1,980 natural product library resulted in 20 hit compounds that proceeded through secondary screens to eliminate compounds with protonophoric or toxic properties. Our top four hit compounds decrease pHa, affect survival of Mtb at low pH, and perturb Mtb's membrane potential. These compounds are currently being used as tools to identify their putative biological target(s). We also demonstrate that chemical inhibitors can be used to target the serine protease Rv3671c. We have identified one particular class of compounds, benzoxazinones, that inhibit enzymatic activity of the protease in vivo and phenocopy the Rv3671c Mtb mutant by decreasing pHIB when the bacterium is exposed to low pH.;The second genetic approach, described in Chapter 3, investigates the genetic basis of acid susceptibility in the Rv2136c transposon mutant strain (Rv2136c:Tn). By generating a Rv2136c -deficient strain, we demonstrate that the severe attenuation of Rv2136c:Tn is not the result of disruption of the Rv2136c gene by transposon insertion but is due to an unidentified genomic mutation. Studies to identify the mutated gene responsible for the acid sensitivity phenotype are underway.;Finally, in Chapter 4, we evaluated whether the compound 8-hydroxyquinoline (8HQ) could target Mtb under 3 non-replicating conditions: low oxygen, mild acid, and the generation of small fluxes of NO from mildly acidified nitrite. We demonstrate that 8HQ has bactericidal activity of comparable potency against replicating and non-replicating Mtb, a property not observed for anti-infective agents currently approved for the treatment of tuberculosis. |
