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

Abstract

Sensing and response to changes in nutrient availability are essential for the lifestyle of environmental and pathogenic bacteria. Serine/threonine protein kinase G (PknG) is required for virulence of the human pathogen Mycobacterium tuberculosis, and its putative substrate GarA regulates the tricarboxylic acid cycle in M. tuberculosis and other Actinobacteria by protein-protein binding. We sought to understand the stimuli that lead to phosphorylation of GarA, and the roles of this regulatory system in pathogenic and non-pathogenic bacteria. We discovered that M. tuberculosis lacking garA was severely attenuated in mice and macrophages and furthermore that GarA lacking phosphorylation sites failed to restore the growth of garA deficient M. tuberculosis in macrophages. Additionally we examined the impact of genetic disruption of pknG or garA upon protein phosphorylation, nutrient utilization and the intracellular metabolome. We found that phosphorylation of GarA requires PknG and depends on nutrient availability, with glutamate and aspartate being the main stimuli. Disruption of pknG or garA caused opposing effects on metabolism: a defect in glutamate catabolism or depletion of intracellular glutamate, respectively. Strikingly, disruption of the phosphorylation sites of GarA was sufficient to recapitulate defects caused by pknG deletion. The results suggest that GarA is a cellular target of PknG and the metabolomics data demonstrate that the function of this signaling system is in metabolic regulation. This function in amino acid homeostasis is conserved amongst the Actinobacteria and provides an example of the close relationship between metabolism and virulence.

Targeting the messengers: Serine/threonine protein kinases as potential targets for antimycobacterial drug development

“…Reports from O'Hare et al . suggested that in response to the amino acid availability, PknG regulates glutamate and TCA cycle metabolism by phosphorylation dependent inhibition of GarA, a central metabolic regulator . Reports from Wolff et al .…”

Tuberculosis: Smart manipulation of a lethal host

“…pknG mutant BCG, which is unable to resist lysosomal transfer, survives and proliferates in vivo . During infection in mice, pknG knock out Mtb show only partial defects in replication, whereas GarA‐deficient Mtb are completely unable to replicate . These findings indicate that some functions of PknG may be dispensable and compensated for by other kinases in vivo .…”

The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway

“…In M. tuberculosis, amino acids serve as the primary nitrogen donors for most anabolic processes (27,28). Additionally, optimal M. tuberculosis growth, both in vitro and in vivo, requires the uptake of exogenous amino acids as a nitrogen source (29)(30)(31)(32). It has therefore been hypothesized that the products of bulk proteolysis by the M. tuberculosis proteasome could be an important source of nitrogen under nutrient-limiting conditions.…”