A Redox Regulatory System Critical for Mycobacterial Survival in Macrophages and Biofilm Development

Abstract

Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostati c system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

Author Summary

Nearly one-third of the world’s population is infected with Mycobacterium tuberculosis ( Mtb), the causative agent of TB. A key factor that contributes to the widespread infection of Mtb is its capacity to survive inside the host macrophage. Understanding how Mtb withstands the hostile intracellular environment of this phagocytic cell may reveal targets for development of therapeutics that enhance the innate anti- Mtb activities of the macrophage. We discovered a novel signaling pathway in mycobacteria which regulates cellular redox homeostasis through NADH and FAD, regulators of metabolism and redox balance. NADH induces the expression of a protein kinase, PknG, which then phosphorylates the ribosomal protein L13 and promotes its presence in the cytoplasm. L13 therein forms a complex with RenU, a Nudix ( Nucleoside diphosphate linked moiety X) hydrolase that degrades NADH and FAD. Genetic disruption of this signaling cascade leads to cellular accumulation of these molecules, increased mycobacterial sensitivity to oxidative stress, impaired surface biofilm growth, and most importantly, reduced survival of Mtb in macrophages.