Small-Molecule Inhibition of Choline Catabolism in Pseudomonas aeruginosa and Other Aerobic Choline-Catabolizing Bacteria

Abstract

Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13 C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit cholinedependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

“…The AraC family members canonically regulate genes involved in metabolism (e.g., AraC and XylS for regulation of arabinose and xylene/benzoate metabolism, respectively [59,60]) and/or virulence (61) (e.g., ExsA regulation of type III secretion in P. aeruginosa [62] and ToxT regulation of cholera toxin and the toxincoregulated pilus in Vibrio cholerae [63]). Evidence suggests that GbdR senses GB and dimethylglycine levels in the cell and induces transcription of genes involved in virulence, GB transport, GB catabolism, and detoxification of the catabolic byproducts, hydrogen peroxide and formaldehyde (48,56,(64)(65)(66)(67)(68) (Fig. 2).…”

“…GbdR induces transcription in response to GB and dimethylglycine in the cytosol, leading to induction of choline and GB acquisition components (plcH, pchP, cbcXWV, and betX) and induction of the components of the GB catabolic pathway (gbcA and -B, the dgc operon, soxBDAG, glyA1, and sdaB) (47,48,56,(64)(65)(66)(67) (Fig. 2).…”

“…The GB resulting from choline oxidation is then demethylated, a reaction that is predicted to use the oxygenase activity of GbcAB to demethylate GB to dimethylglycine and formaldehyde (56). The demethylation of dimethylglycine is carried out by a heterodimeric flavin-linked oxidoreductase comprised of DgcA and DgcB (56,64), which are homologous to the proteins encoded by dimethylglycine catabolic genes of Arthrobacter spp. (77).…”

Characterization of the GbdR Regulon in Pseudomonas aeruginosa

“…GbdR was also necessary for the acquisition of choline via the hemolytic phospholipase C (PlcH) virulence factor and the periplasmic phosphorylcholine phosphatase (PchP) (3). Evidence from genetics and chemical biology experiments supports the choline catabolites GB and DMG as inducing molecules for GbdR-dependent transcriptional activation (2,4). GB is used nearly universally as an osmoprotectant and general stress protectant in bacteria, as well as playing a similar role in most eukaryotes and a number of Archaea (5).…”

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting

“…This regulatory scheme in which PlcH activity is repressed by low oxygen conditions is surprising considering the numerous benefits of PlcH in P. aeruginosa colonization of and virulence towards diverse hosts (Fitzsimmons et al, 2011;Hogan & Kolter, 2002;Jackson et al, 2013;Wargo, 2013), acquisition of carbon, nitrogen and osmoprotectants (Son et al, 2007;Wargo et al, 2009), and resistance to host immunity (Terada et al, 1999). Anr regulation of PlcH may reflect the fact that PlcH-released catabolites (choline and lipids) require oxygen for catabolism (Wargo et al, 2008) (Fig.…”