Validation of RelMtb as a target for Mycobacterium tuberculosis persisters

The lengthy and complicated multidrug therapy currently available to treat active tuberculosis (TB) infection has contributed to medical non-adherence and the emerging problems of multidrug-resistant (MDR)- and extensively drug-resistant (XDR)-TB, which are particularly deadly in the setting of HIV co-infection. The prolonged therapy required to eradicate TB infection is believed to reflect the ability of Mycobacterium tuberculosis (Mtb) to persist within host necrotic granulomas in a non-replicating state characterized by antibiotic tolerance to bactericidal drugs, which predominantly target actively dividing tubercle bacilli. The stringent response enzyme, RelMtb, is essential for Mtb survival under physiologically relevant stress conditions in vitro and in the lungs of mice and guinea pigs. A library of over 2 million compounds was screened in RelMtb-inhibition assays and whole-cell screens under conditions in which RelMtb is essential. A total of 178 RelMtb inhibitor candidates, representing 18 unique scaffolds, were identified and 39 compounds were tested against nutrient-starved wild-type Mtb. The antibiotic susceptibility of a relMtb deletion mutant (Δrel) was studied during nutrient starvation and chronic infection in mice, and isoniazid and RelMtb inhibitor candidates were tested for synergy against nutrient-starved wild-type Mtb and Δrel using checkerboard assays. The minimum bactericidal concentration of isoniazid was 500-fold lower against Δrel relative to wild type during nutrient starvation, and the potent bactericidal activity of isoniazid was maintained against Δrel during chronic infection in the lungs of mice. Inhibition of RelMtb appears to be a promising new approach to target Mtb persisters, with the potential to shorten the duration of treatment for drug-susceptible and drug-resistant TB. We used microarray to characterize the transcriptomic profiles of the wild type and Δrel during nutrient starvation. The RelMtb inhibitor, (E)-4-(3-methyl-4-(2-(4-methylthiazol-5-yl)ethoxy)styryl)benzoic acid, killed wild-type Mtb and prevented isoniazid tolerance during nutrient starvation. Treatment of nutrient-starved wild-type with the RelMtb inhibitor led to downregulation of the RelMtb regulon. Transcriptomic analysis revealed an altered gene expression in Δrel and wild-type treated with RelMtb inhibitor during nutrient starvation. Microarrays were used to study the global gene expression profile of wild type Mtb exposed to the candidate Rel inhibitor (final concentration= 2µM) in 1x PBS and the transcriptional pattern was compared to that of ΔrelA in PBS at Day 1 and Day 7.