A potential sensor-effector biocircuit to facilitate selective killing of resistant bacteria

This project focused on an ecological solution to the problem of antimicrobial resistance that depended on promoting the selection of susceptible bacteria over resistant bacteria. If achievable, this could reduce the likelihood of infections being caused by resistant pathogens, thus preserving the life-saving utility of antibiotics. The specific aims were to design and construct genetic switches that were predicted to sense and respond to the presence of resistance-related mRNA, and to validate the selective nature of these switches using gene-reporter systems. Ultimate proof-of-concept would require the building of resistance-sensing lysogenic-lytic switches into the bacteriophage λ genome to generate „anti-resistance‟ phages that selectively kill resistant bacteria. A reporter plasmid encoding cI857 governed by λ PRM promoter and a ß-galactosidase gene expressed from the λ CI857-responsive PR promoter was constructed. Production of the CI857 repressor blocked expression of ß-galactosidase. However, multiple distinct manipulations of the sequence between PRM and the start of cI857, to allow for subsequent introduction antisense orientated DNA fragments, resulted in loss of CI857 production and non-functional CI-Reporters. Only insertion of a restriction site and a Shine-Dalgarno sequence between truncated and intact versions of cI857 and the introduction of a restriction site downstream of cI857 led to functional CI-reporters. Analysis of various E. coli DH5α derivatives, simultaneously harbouring a CI-reporter bearing an antisense-orientated integron-derived DNA fragment and a second plasmid expressing sense integron RNA failed to reveal reduced CI857 production, suggesting negligible antisense effects for the candidate switches and target RNA sequences investigated. Nevertheless, data from an in vitro protein expression experiment utilizing a CI-reporter and an antisense oligonucleotide supported the notion that relevant antisense RNA effects could occur, perhaps only when optimum sense:antisense RNA ratios were achieved. The tools constructed and data derived have laid the groundwork for future pursuit of the proposed anti-resistance phages

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha-ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α-ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non-pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient-dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha-ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α-ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non-pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient-dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways

Mycobacterium tuberculosis RNA Polymerase-binding Protein A (RbpA) and Its Interactions with Sigma Factors

RNA polymerase-binding protein A (RbpA), encoded by Rv2050, is specific to the actinomycetes, where it is highly conserved. In the pathogen Mycobacterium tuberculosis, RbpA is essential for growth and survival. RbpA binds to the β subunit of the RNA polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. Here we report the solution structure of RbpA and identify the principle sigma factor σ[superscript A] and the stress-induced σ[superscript B] as interaction partners. The protein has a central ordered domain with a conserved hydrophobic surface that may be a potential protein interaction site. The N and C termini are highly dynamic and are involved in the interaction with the sigma factors. RbpA forms a tight complex with the N-terminal domain of σB via its N- and C-terminal regions. The interaction with sigma factors may explain how RbpA stabilizes sigma subunit binding to the core RNA polymerase and thereby promotes initiation complex formation. RbpA could therefore influence the competition between principal and alternative sigma factors and hence the transcription profile of the cell

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

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

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

<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

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>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