GarA phosphorylation in <i>M</i>. <i>smegmatis</i> was regulated by nutrient availability.

<p>The reporter strain of <i>M</i>. <i>smegmatis</i> was cultured in different media and cell lysates analysed by Western blot and densitometry. (<b>A</b>) Glutamate and related amino acids triggered phosphorylation of GarA: the nitrogen source is indicated and the carbon source was glucose. (<b>B</b>) The supplied carbon source affected phosphorylation of GarA: the carbon source is indicated and the nitrogen source was NH₄Cl. (<b>C</b>) Phosphorylation of GarA occurred rapidly when cells were cultured in poor medium and then given supplementary nutrients (initially 1% glucose with 10 mM NH₄Cl and 0.05% tyloxapol, then 1% v/v glycerol and 30 mM asparagine were added at time zero). (<b>D</b>) Dephosphorylation of GarA occurred slowly when cells switched from rich to poor medium (initially 1% glycerol with 30 mM asparagine and 0.05% Tween 80 then switched to 1% glucose with 10 mM NH₄Cl and 0.05% tyloxapol). (<b>E</b>) GarA was predominantly unphosphorylated when <i>M</i>. <i>smegmatis</i> were in stationary phase or starved in PBS. The reporter strain of <i>M</i>. <i>smegmatis</i> was grown in Sauton’s medium with shaking for 5 days. For the starvation experiment exponentially growing <i>M</i>. <i>smegmatis</i> were washed with PBS and incubated in PBS with 0.05% tyloxapol for 5 days. Values represent mean and standard deviation of at least three independent replicates.</p

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

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

<i>pknG</i> disruption in <i>M</i>. <i>tuberculosis</i> caused a nutrient-dependent growth defect.

<p>(<b>A</b>) All strains grew at the same rate on minimal medium supplemented with glycerol 0.2%. Δ<i>pknG</i>_Mt (blue diamonds) grew more slowly than wild type <i>M</i>. <i>tuberculosis</i> (black circles) when the sole carbon source was (<b>B</b>) glutamate 10 mM or (<b>C</b>) asparagine 10 mM. Plasmid-encoded PknG (empty circles) partially restored the growth defect. Graphs show the mean and standard deviation of three independent experiments.</p

Investigation of GarA phosphorylation in <i>M</i>. <i>smegmatis</i> and <i>M</i>. <i>tuberculosis</i>.

<p>(<b>A</b>) Addition of Phos-tag reagent to SDS-PAGE allowed separation of phosphorylated GarA (GarA-P) from GarA. Cell extracts were prepared from <i>M</i>. <i>smegmatis</i> and <i>M</i>. <i>tuberculosis</i> wild type and <i>pknG</i> deletion strains and complemented strains. (<b>B</b>) The same cell extracts of <i>M</i>. <i>tuberculosis</i> wild type and <i>pknG</i> deletion strain were analysed by LC-MS/MS to detect the tryptic peptide of GarA carrying the ETTS phosphorylation motif. The abundance of the peptide with no phosphorylation is shown in black and the T21-phosphorylated peptide is shown in red. A peptide from another part of the protein was also measured as a control (blue). (<b>C</b>) A reporter strain of Δ<i>garA</i>_Ms carrying plasmids encoding hexahistidine-tagged <i>garA</i>, or variants of <i>garA</i>, confirmed that most phosphorylation occurred at the first threonine in the ETTS motif. GarA from cell lysates was visualised by Western blotting. Images shown are representative of three or more independent replicates.</p

GarA phosphorylation in <i>M</i>. <i>smegmatis</i> was regulated by nutrient availability.

<p>The reporter strain of <i>M</i>. <i>smegmatis</i> was cultured in different media and cell lysates analysed by Western blot and densitometry. (<b>A</b>) Glutamate and related amino acids triggered phosphorylation of GarA: the nitrogen source is indicated and the carbon source was glucose. (<b>B</b>) The supplied carbon source affected phosphorylation of GarA: the carbon source is indicated and the nitrogen source was NH₄Cl. (<b>C</b>) Phosphorylation of GarA occurred rapidly when cells were cultured in poor medium and then given supplementary nutrients (initially 1% glucose with 10 mM NH₄Cl and 0.05% tyloxapol, then 1% v/v glycerol and 30 mM asparagine were added at time zero). (<b>D</b>) Dephosphorylation of GarA occurred slowly when cells switched from rich to poor medium (initially 1% glycerol with 30 mM asparagine and 0.05% Tween 80 then switched to 1% glucose with 10 mM NH₄Cl and 0.05% tyloxapol). (<b>E</b>) GarA was predominantly unphosphorylated when <i>M</i>. <i>smegmatis</i> were in stationary phase or starved in PBS. The reporter strain of <i>M</i>. <i>smegmatis</i> was grown in Sauton’s medium with shaking for 5 days. For the starvation experiment exponentially growing <i>M</i>. <i>smegmatis</i> were washed with PBS and incubated in PBS with 0.05% tyloxapol for 5 days. Values represent mean and standard deviation of at least three independent replicates.</p

<i>garA</i> is required for growth of <i>M</i>. <i>tuberculosis in vitro</i>, survival in macrophages, and virulence in mice.

<p>(<b>A</b>) <i>M</i>. <i>tuberculosis</i> lacking <i>garA</i> was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne <i>garA</i> restored the defect, but variants of <i>garA</i> with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. (<b>B</b>) <i>M</i>. <i>tuberculosis</i> lacking <i>garA</i> (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental <i>M</i>. <i>tuberculosis H37Rv</i> (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. (<b>C</b>) <i>M</i>. <i>tuberculosis</i> lacking <i>garA</i> was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10⁵ bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ<i>garA</i>_Mt (red squares) was significantly lower than those infected with <i>M</i>. <i>tuberculosis H37Rv</i> (black circles), or complemented Δ<i>garA</i>_Mt (black triangles) at all time points from day 7 (p<0.005, t test). (<b>D</b>) <i>M</i>. <i>tuberculosis</i> lacking <i>garA</i> failed to disseminate to the spleen by day 28 (symbols match panel <b>C</b>). The minimum number of bacteria that could be detected was 45 CFU/organ, marked by a dashed black line in <b>C</b> and <b>D</b>.</p

Deletion of <i>garA</i> or disruption of GarA phosphorylation caused changes in intracellular glutamate and other changes in the metabolome of <i>M</i>. <i>smegmatis</i>.

<p>Intracellular metabolites from wild type <i>M</i>. <i>smegmatis</i> and mutants were analysed by mass spectrometry using an untargeted metabolomics approach. <i>M</i>. <i>smegmatis</i> lacking <i>garA</i> has lower intracellular glutamate and metabolites related to glutamate than the parental strain (<b>A</b>) but plasmid encoded GarA reversed this change (<b>B</b>). <i>M</i>. <i>smegmatis</i> expressing truncated GarA that lacks phosphorylation sites had higher intracellular ornithine than wild type (<b>C</b>). Intracellular glutamate concentrations for the mutant strains are compared to wild type in panel (<b>D</b>), together with those metabolites that were significantly changed in >1 mutant strain but not in complemented strains. pSer is O-phosphoserine, Orn is ornithine, Cit is citrulline, MaltoP is maltopentaose. (<b>A-C</b>) Each point on the scatter plots represents a single metabolite. Metabolites with the greatest fold-change and statistical significance are highlighted (log2(fold change)>0.5 and q-value<0.05), thresholds marked as dashed lines on graphs): metabolites at lower concentration are blue and those at higher concentration are red. These data were taken from cells growing in Middlebrook 7H9 in early exponential phase and represent the mean from at least 3 independent experiments.</p

GarA was required during stationary phase for the maintenance of intracellular glutamate and for survival.

<p>(<b>A</b>) <i>M</i>. <i>smegmatis</i> lacking <i>garA</i> failed to maintain intracellular glutamate and glutamine pools during extended stationary phase in 7H9 medium. Intracellular glutamate and glutamine were measured for wild type <i>M</i>. <i>smegmatis</i> (black circles), Δ<i>garA</i>_Ms (red squares), and complemented Δ<i>garA</i>_Ms (black triangles). Inset graphs show intracellular metabolites for the same experiment at day 28 (<b>B</b>) <i>M</i>. <i>smegmatis</i> lacking <i>garA</i> gradually lost viability during prolonged stationary phase. Cells were cultured in 7H9 medium over a time period of five months. Aliquots were withdrawn at regular intervals and surviving cells were plated on 7H10 to calculate CFU ml^-1. All experiments were repeated at least 3 times and data show the mean with standard deviation.</p

Summary comparing the intracellular metabolome of mutant strains with parental <i>M</i>. <i>smegmatis</i>.

<p>Metabolites that are significantly changed in more than one strain are in <b>bold</b> type. Metabolites related to amino acid metabolism are highlighted with asterisks *. Metabolites are listed in order of fold-change compared to <i>M</i>. <i>smegmatis</i>, and those that were >2-fold changed are shaded.</p

Disruption of <i>pknG</i> or removal of the phosphorylation motif of <i>garA</i> caused a nutrient-dependent growth defect in <i>M</i>. <i>smegmatis</i>.

<p>(<b>A</b>) All strains grew at the same rate on standard Middlebrook 7H9 medium. (<b>B</b>) Δ<i>garA</i>_Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH₄Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” <i>garA</i>). (<b>C + D</b>) Δ<i>pknG</i>_Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon (<b>C</b>) or nitrogen source (<b>D</b>) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. (<b>E</b>) Δ<i>pknG</i>_Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve (<b>D</b>) imaged at 60 hours. Growth of Δ<i>garA</i>_Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ<i>pknG</i>_Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.</p