Cysteamine inhibits glycine utilisation and disrupts virulence in pseudomonas aeruginosa. [Dataset]

Abstract

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 reported activity blocking the glycine cleavage system in human cells. Despite the inhibition of glycine metabolism, cysteamine inhibits hydrogen cyanide (HCN) production by P. aeruginosa, implicating interference in the regulation of virulence. Cysteamine impaired chemotaxis and reductions in pyocyanin and exopolysaccharide production in cysteamine-treated P. aeruginosa and reduced toxicity of secreted factors in a Galleria mellonella model. Thus, cysteamine has additional potent anti-virulence properties targeting P. aeruginosa, further supporting its therapeutic potential in CF and other infections.

Citation

FRASER-PITT, D.J., DOLAN, S.K., TOLEDO-APARICIO, D., HUNT, J.G., SMITH, D.W., LACY-ROBERTS, N., NUPE HEWAGE, P.S., STOYANOVA, T.N., MANSON, E., MCCLEAN, K., INGLIS, N.F., MERCER, D.K. and O’NEIL, D.A. 2021. Cysteamine inhibits glycine utilisation and disrupts virulence in pseudomonas aeruginosa. [Dataset]. Frontiers in cellular and infection microbiology [online], 11, article 718213. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2021.718213/full#supplementary-material

• Publication Date: Dec 31, 2021
• Deposit Date: Oct 21, 2021
• Publicly Available Date: Oct 26, 2021
• DOI: https://doi.org/10.3389/fcimb.2021.718213
• Keywords: Virulence; Biofilm; Pseudomonas aeruginosa; Novel therapeutic; Glycine cleavage complex
• Public URL: https://rgu-repository.worktribe.com/output/1500455
• Type of Data: Supplementary tables, a figure and raw data files.
• Collection Date: Jul 7, 2021
• Collection Method: Six separate 10 ml cultures of P. aeruginosa PAO1 in MHB were prepared in sterile Universal tubes using single colonies grown from frozen stock revived overnight on MHA plates. Cultures were incubated statically for 20 hours and diluted in fresh media if necessary, to achieve growing cultures with an optical density of 0.3 at 625 nm. Each culture was then sub-cultured 1 ml into 9 ml of fresh MHB and incubated for 4 hours at 37°C to achieve mid-logarithmic growth. Bacterial cells were then pelleted by centrifugation at 5,000 g for 5 min. Following this, three of these cultures were resuspended in MHB media alone and three were resuspended in media containing sub-inhibitory 250 mg/L cysteamine and all cultures were incubated for a further 4 hours at 37°C. Bacterial cells were then pelleted by centrifugation at 5,000 g for 5 min at 4°C and washed once with ice cold PBS. Bacterial cell pellets were resuspended and lysed in 200 µl of BugBuster (Merck Millipore, MA, USA) containing a protease inhibitor cocktail (Complete Mini, Roche Diagnostics GmbH, Basel, Switzerland) at the manufacturer’s recommended concentration. Lysate protein concentrations were determined by bicinchoninic acid (BCA) assay (Smith et al., 1985) and equal amounts 5 mg total protein of whole cell lysates were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a precast 4-20% gradient Tris-glycine gel. Gel visualised with Coomassie based EZBlue (Sigma, MO, USA) stain. Each of 6 lanes of whole cell proteins was fractionated into 21 slices. Each slice was subjected to in-gel tryptic digestion, reduction, alkylation and trypsinolysis, reversed-phase liquid chromatographic separation of tryptic peptides using rapid monolithic column chromatography and ESI-MS/MS using a fast-scanning three-dimensional ion trap tandem mass spectrometer (Bruker Daltonics amaZon-ETD). LC was performed using an Ultimate 3000 nano-HPLC system (Dionex, LC-Packings) comprising a WPS-3000 well-plate micro autosampler, a FLM-3000 flow manager and column compartment, a UVD3000 UV detector, an LPG-3600 dual-gradient micropump and an SRD-3600 solvent rack controlled by Chromeleon software. A final flow rate of 3 mL/min through a 200 mm i.d. monolithic column maintained at a constant 50°C. Samples of 1 mL were applied to the column by direct injection. For the chromatography, the following solvents were used: solvent A (98% H2O, 2% acetonitrile, 0.1% formic acid), 0–55% solvent B (80% acetonitrile, 20% H2O, 0.08% formic acid). Peptides were eluted by the application of a 4 min linear from 8% to 45% solvent B (80% acetonitrile, 0.1% formic acid) and directed through a 3 nL UV detector flow cell. The LC system was interfaced directly with a 3D high-capacity ion trap mass spectrometer (Esquire HCTplusTM, Bruker Daltonics) utilizing a low-volume (50mL/min max.) stainless steel nebulizer (Agilent). Mass spectrum peak lists for peptides and fragmentation ions were compared with in-silico published sequence for P. aeruginosa PAO1 utilising the Mascot™ V2.5.1 (Matrix Science) search engine. Mascot search parameters were set to, fixed (carbamidomethyl “C”) and variable (oxidation “M” and deamidation “N,Q”) modifications were selected along with peptide (MS) and secondary fragmentation (MS/MS) tolerance values of 0.5Da whilst allowing for a single 13C isotope. Protein identifications obtained from each of the 21 individual gel slices per lane were compiled using the Meta-score protein compilation feature within the ProteinScape bioinformatics platform. From the compiled protein lists individual protein identifications were inspected manually and considered significant only if a) two peptides were matched for each protein, b) peptides were represented by a sequence coverage of >5% and c) each matched peptide contained an unbroken “b” or “y” ion series represented by of a minimum of four contiguous amino acid residues.