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 homeostatic 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.
Highlights
A critical determinant defining pathogenicity of Mycobacterium tuberculosis (Mtb) is its survival in host macrophages
We show that the redox regulatory molecule NADH induces the expression of protein kinase G (PknG), which phosphorylates the ribosomal protein L13 at a unique residue, threonine 11 (T11)
Disruption of the PknG-L13-RenU pathway causes: (i) increased oxidative stress susceptibility, (ii) accumulation of NADH and FAD during oxidative stress, (iii) impaired biofilm growth, and notably, (iv) reduced survival of Mtb in host macrophages. These results suggest that biofilm growth and host persistence are both regulated through the PknG-modulated RHOCS, which regulates levels of nucleoside diphosphate derivatives such as NADH and FAD in mycobacteria
Summary
A critical determinant defining pathogenicity of Mycobacterium tuberculosis (Mtb) is its survival in host macrophages. Upon internalization by the host phagocytic cell, Mtb and related pathogenic mycobacteria block the fusion of their resident phagosome to the destructive lysosome, thereby establishing a niche within the bactericidal macrophage [1,2]. This ability of pathogenic mycobacteria requires the eukaryotic-type serine/threonine protein kinase G (PknG) [3]. In the absence of PknG, both pathogenic Mtb and non-pathogenic M. smegmatis display increased susceptibility to multiple antibiotics [4]. The role of PknG in Mtb survival in host macrophages remains ambiguous
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