Nitric oxide induces the distinct invisibility phenotype of Mycobacterium tuberculosis

During infection Mycobacterium tuberculosis (Mtb) forms physiologically distinct subpopulations that are recalcitrant to treatment and undetectable using standard diagnostics. These difficult to culture or differentially culturable (DC) Mtb are revealed in liquid media, their revival is often stimulated by resuscitation-promoting factors (Rpf) and prevented by Rpf inhibitors. Here, we investigated the role of nitric oxide (NO) in promoting the DC phenotype. Rpf-dependent DC Mtb were detected following infection of interferon-γ-induced macrophages capable of producing NO, but not when inducible NO synthase was inactivated. After exposure of Mtb to a new donor for sustained NO release (named NOD), the majority of viable cells were Rpf-dependent and undetectable on solid media. Gene expression analyses revealed a broad transcriptional response to NOD, including down-regulation of all five rpf genes. The DC phenotype was partially reverted by over-expression of Rpfs which promoted peptidoglycan remodelling. Thus, NO plays a central role in the generation of Rpf-dependent Mtb, with implications for improving tuberculosis diagnostics and treatments. Mechanistic study reveals nitric oxide as the trigger of distinct persister-like phenotypes of Mycobacterium tuberculosis during infection and highlights the importance of resuscitation promoting factor for this phenomenon.