Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.

Whole cell screen for inhibitors of pH homeostasis in Mycobacterium tuberculosis.

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pHIB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pHIB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis.

Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of Mycobacterium tuberculosis.

Nitazoxanide (Alinia(®)), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against Mycobacterium tuberculosis (Mtb): disruption of Mtb's membrane potential and pH homeostasis. Both actions were shared by a structurally related anti-mycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb's membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb's ultra-low frequency of resistance against nitazoxanide.

Mycobacterium tuberculosis gene Rv2136c is dispensable for acid resistance and virulence in mice.

The gene Rv2136c is annotated to encode the Mycobacterium tuberculosis (Mtb) homolog of Escherichia coli's undecaprenyl pyrophosphate phosphatase. In previous work, a genetic screen of 10,100 Mtb transposon mutants identified Rv2136c as being involved in acid resistance in Mtb. The Rv2136c:Tn strain was also sensitive to sodium dodecyl sulfate, lipophilic antibiotics, elevated temperature and reactive oxygen and nitrogen intermediates and was attenuated for growth and persistence in mice. However, none of these phenotypes could be genetically complemented, leading us to generate an Rv2136c knockout strain to test its role in Mtb pathogenicity. Genetic deletion revealed that Rv2136c is not responsible for any of the phenotypes observed in the transposon mutant strain. An independent genomic mutation is likely to have accounted for the extreme attenuation of this strain. Identification of the mutated gene will further our understanding of acid resistance mechanisms in Mtb and may offer a target for anti-tuberculosis chemotherapy.

Killing of non-replicating Mycobacterium tuberculosis by 8-hydroxyquinoline.

OBJECTIVES: To determine the effect of 8-hydroxyquinoline (8HQ) on non-replicating Mycobacterium tuberculosis (Mtb) in comparison with its reported effect on replicating Mtb. METHODS: The MIC of 8HQ for replicating H37Rv Mtb was determined by microdilution in 7H9 broth. Bactericidal activity was determined by exposing H37Rv Mtb to 8HQ for 4 days under conditions that otherwise allowed exponential replication (20% O(2), pH 6.6) and conditions under which replication was precluded: 1% O(2), pH 6.6; 20% O(2), pH 5.5; or 20% O(2), pH 5.5, 0.5 mM sodium nitrite. Serial dilutions were plated on 7H11 agar to quantify cfu. Frequency of resistance (FOR) was determined with >10(9) bacteria plated on 7H9 agar plates containing 2x MIC 8HQ. RESULTS: 8HQ was active against replicating Mtb (MIC 2.5 microM, 0.36 mg/L). Under both replicating and non-replicating conditions, cfu were reduced in 4 days by > or = 5 log(10) at the highest concentration tested (10 microM). Bactericidal activity was maximal at low pH, where 8HQ reduced cfu by 1-1.5 log(10) at 1 microM. We were unable to recover any 8HQ-resistant colonies. CONCLUSIONS: This study demonstrates that 8HQ has bactericidal activity of comparable potency against non-replicating and replicating Mtb, a property not observed for anti-infective agents currently approved for treatment of tuberculosis, and a very low FOR. Drugs with these properties are urgently needed to shorten the course of treatment for both active and latent tuberculosis.