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 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.

Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses

“…We consistently observed that inhibition of ETC results in increased NADH:NAD + levels. These results suggest that the ETC is the primary consumer of NADH, although other ways to consume NADH (Trivedi et al, 2012) or degrade NADH (Wolff et al, 2015) exist in mycobacteria.…”

“…A similar metabolic reprogramming was also suggested by transcriptome profiling of Mtb residing in macrophages (Schnappinger et al, 2003). Studies have suggested that higher levels of NADH:NAD + could lead to an overexpression of the virulence factor serine/threonine kinase PknG, which plays an important role in the intracellular survival of Mtb (Wolff et al, 2015). Moreover, it was recently demonstrated that thiol reductive stress induces Mtb biofilm formation (Trivedi et al, 2016).…”

“…Although a Rex homolog has not yet been described in Mtb, but Mtb uses a response regulator RegX3 that regulates expression of cydABCD (Roberts et al, 2011) and is responsive to oxygen levels (Singh and Kumar, 2015). Furthermore, accumulation of NADH leads to the induction of PknG, which activates the redox homeostatic system of Mycobacterium to regulate biofilm formation and augment survival of Mtb in macrophages (Wolff et al, 2015). NADH is also known to covalently bind to isoniazid to produce isonicotinic-NADH, which exhibits potent antimycobacterial activity (Rozwarski et al, 1998).…”

PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis

“…Δ pknG Mt has previously been reported to have a defect in survival in bone marrow derived mouse macrophages [11]. Unlike Δ garA Mt , Δ pknG Mt was able to replicate in THP-1 macrophages, albeit 10-fold less than parental M .…”

Antibiotic resistance mechanisms in M. tuberculosis: an update

“…Through its regulation on GarA, PknG therefore affects the cellular pool of NADH. Interestingly, PknG was recently found to control a redox homeostatic system, RHOCS, which regulates cellular NADH level through a Nudix hydrolase that specifically degrades NADH (Wolff et al 2015). It remains to be established if PknG affects the bactericidal activity of anti-TB drugs through its regulation of NADH and the consequent production of radicals leading to cell death (Fig.…”