Cysteamine Inhibits Glycine Utilisation and Disrupts Virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major opportunistic human pathogen which employs a myriad of virulence factors. In people with cystic fibrosis (CF) P. aeruginosa frequently colonises the lungs and becomes a chronic infection that evolves to become less virulent over time, but often adapts to favour persistence in the host with alginate-producing mucoid, slow-growing, and antibiotic resistant phenotypes emerging. Cysteamine is an endogenous aminothiol which has been shown to prevent biofilm formation, reduce phenazine production, and potentiate antibiotic activity against P. aeruginosa, and has been investigated in clinical trials as an adjunct therapy for pulmonary exacerbations of CF. Here we demonstrate (for the first time in a prokaryote) that cysteamine prevents glycine utilisation by P. aeruginosa in common with previously reported activity blocking the glycine cleavage system in human cells. Despite the clear inhibition of glycine metabolism, cysteamine also inhibits hydrogen cyanide (HCN) production by P. aeruginosa, suggesting a direct interference in the regulation of virulence factor synthesis. Cysteamine impaired chemotaxis, lowered pyocyanin, pyoverdine and exopolysaccharide production, and reduced the toxicity of P. aeruginosa secreted factors in a Galleria mellonella infection model. Thus, cysteamine has additional potent anti-virulence properties targeting P. aeruginosa, further supporting its therapeutic potential in CF and other infections.

Backbone chemical shift assignments of the glycine cleavage complex H protein of Escherichia coli.

Glycine cleavage complex H protein (GcvH) is one of the four components that form the glycine cleavage complex (GCS), essential for the synthesis of C1 (one-carbon units) for cell metabolism, by the oxidative cleavage of glycine. The activity of this complex is induced in the presence of exogenous glycine, and is repressed by purines. GCS, in cooperation with GCA (serine hydroxymethyltransferase) regulates the endogenous levels of glycine and C1 units in the cell. GcvH, the lipoamide containing component of the complex, plays an indispensable role in this reaction, as its prosthetic group shuttles between the active site of the three other components of the GCS complex sequentially. In environments rich in exogenous lipoic acid, GcvH is converted to lipoyl-GcvH by Lipoate protein ligase (LplA), by the salvage pathway. When exogenous lipoic acid is deficient, it is post-translationally modified to lipoyl-GcvH by the consecutive action of two enzymes, (a) Lipoate protein ligase B (LipB) and (b) Lipoyl synthase (LipA). Although, the crystal structure has been determined for Escherichia coli GcvH, no information exists for its interaction with LipB or LipA. Therefore, we plan to study its interactions with the aforementioned enzymes. As a first step, we have carried out the complete backbone chemical shift assignments of the E. coli glycine cleavage complex H protein in its apo-form, as well as its C8- intermediate.

Implementation of a Reductive Route of One-Carbon Assimilation in Escherichia coli through Directed Evolution.

Endowing biotechnological platform organisms with new carbon assimilation pathways is a key challenge for industrial biotechnology. Here we report progress toward the construction of formatotrophic Escherichia coli strains. Glycine and serine, universal precursors of one-carbon compounds oxidized during heterotrophic growth, are produced from formate and CO2 through a reductive route. An adaptive evolution strategy was applied to optimize the enzymatic steps of this route in appropriate selection strains. Metabolic labeling experiments with 13C-formate confirm the redirected carbon-flow. These results demonstrate the high plasticity of the central carbon metabolism of E. coli and the applicative potential of directed evolution for implementing synthetic pathways in microorganisms.