Glyoxylate Shunt

The glyoxylate shunt as a target for antibacterial intervention in Gram-negative bacteria (WP3)

Prof. Martin Welch
University of Cambridge

The glyoxylate shunt (Figure 1) is an anaplerotic pathway that allows micro-organisms to grow on acetate or fatty acids as a sole C source. Indeed, aceA is conditionally essential during growth on these substrates, which are presumably abundant in the host because human-adapted lineages of P. aeruginosa often display constitutive up-regulation of aceA. The glyoxylate shunt allows growth on acetate by providing a route from isocitrate to gluconeogenic precursors that bypasses the decarboxylative steps of the TCA cycle. For these reasons it is widely considered to be one of the most important metabolic branchpoints in the whole of microbial metabolism, and much work has been put into understanding how carbon flux is redirected from the TCA cycle through the glyoxylate shunt. In E. coli, decisive control is exercised by the bifunctional kinase/phosphatase AceK acting on isocitrate dehydrogenase (ICD). Phosphorylation inactivates ICD, favouring the glyoxylate pathway, whereas dephosphorylation (triggered by growth on a preferred C source such as glucose) restores flux through the TCA cycle. The P. aeruginosa genome encodes homologues of ICL, ICD, and AceK, but unlike E. coli it also encodes a highly-expressed second isoenzyme of isocitrate dehydrogenase (IDH) which is widespread in other bacterial genera and is almost certainly insensitive to AceK. Metabolic regulation of the P. aeruginosa glyoxylate shunt cannot therefore be simply extrapolated from the E. coli model.

Figure 1. Regulation of the glyoxylate shunt in P. aeruginosa. (A) The enzymes at or near the branchpoint. PDH, pyruvate dehydrogenase; CS, citrate synthase; ACN, aconitase; ICD/IDH, isocitrate dehydrogenases; ICL, isocitrate lyase; GlcB, malate synthase. (B) Known and anticipated branchpoint enzymology [kinetic constants determined by Welch lab (unpublished)]. IDH and ICL are proven targets for allosteric regulation. AceK is likely to be  subject to allosteric regulation. ICD is likely subject to inhibition by AceK-mediated phosphorylation.

Objectives: To characterize the enzymology controlling carbon flux through the TCA cycle / glyoxylate shunt branchpoint of the Gram-negative pathogen, Pseudomonas aeruginosa. To identify and characterize LMW compounds, which suppress carbon flux through the glyoxylate, shunt and thereby impair microbial fitness and virulence. To test these compounds for antimicrobial activity, determine their detailed MoA, and identify/characterize endogenous resistance mechanism(s), which reduce their potency.

Expected Results: One or more LMW compounds that act either to inhibit the glyoxylate shunt enzyme(s) directly, or which elicit a redirection of flux away from the shunt. It is anticipated that these compounds will strongly suppress virulence factor production (especially Type III Secretion) and will also diminish the fitness of the organism in vivo.

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This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No  642620
European Union