William Bishai and colleagues report that cyclic-di-adenosine monophosphate produced during infection with Mycobacterium tuberculosis induces IFN-β and contributes to the innate sensing of tuberculosis. Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit type I interferon (IFN) responses, which are crucial to antimicrobial defense1,2,3. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)4-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)5-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS)6,7, but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen-associated molecular pattern (PAMP) driving host type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against tuberculosis.

Mycobacterium tuberculosis residing within pulmonary granulomas and cavities represents an important reservoir of persistent organisms during human latent tuberculosis infection. We present a novel in vivo model of tuberculosis involving the encapsulation of bacilli in semidiffusible hollow fibers that are implanted subcutaneously into mice. Granulomatous lesions develop around these hollow fibers, and in this microenvironment, the organisms demonstrate an altered physiologic state characterized by stationary-state colony-forming unit counts and decreased metabolic activity. Moreover, these organisms show an antimicrobial susceptibility pattern similar to persistent bacilli in current models of tuberculosis chemotherapy in that they are more susceptible to the sterilizing drug, rifampin, than to the bactericidal drug isoniazid. We used this model of extracellular persistence within host granulomas to study both gene expression patterns and mutant survival patterns. Our results demonstrate induction of dosR (Rv3133c) and 20 other members of the DosR regulon believed to mediate the transition into dormancy, and that rel_Mtb is required for Mycobacterium tuberculosis survival during extracellular persistence within host granulomas. Interestingly, the dormancy phenotype of extracellular M. tuberculosis within host granulomas appears to be immune mediated and interferon-γ dependent.

The sevenMycobacterium tuberculosis whiB-like genes encode small proteins postulated to be transcriptional regulators. A systematic real-time reverse transcription-PCR analysis following exposure to antibiotics and a variety of growth and in vitro stress conditions indicates differential, and in some cases dramatic, transcription modulations for the differentM. tuberculosis whiBfamily members. This information together with biochemical analyses of thewhiB1towhiB7gene products will be important for understanding the biology of this novel family of proteins in mycobacteria and related actinomycetes.

Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis . We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

Rationale: A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. Objectives: Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4, which is induced by rifampin, we evaluated the pharmacokinetic/pharmacodynamic relationships of verapamil and rifampin coadministration in mice. Methods: Using doses that achieve human bioequivalent levels matched to those of standard verapamil, but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild-type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20, and 24 weeks of treatment in mice. Measurements and Main Results: We determined that a dose adjustment of verapamil by 1.5-fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization, and significantly lowers relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone. Conclusions: These data demonstrate treatment shortening by verapamil adjunctive therapy in mice, and strongly support further study of verapamil and other efflux pump inhibitors in human TB.