Establishing a metabolite extraction protocol and elucidating the fate of pyruvate in Blastocystis

Blastocystis is an extremely prevalent gut colonising eukaryotic microbe which has a global distribution. Blastocystis is extremely genetically diverse and encompasses 17 different subtypes 9 of which have been detected in humans. This genetic diversity along with the fact that most Blastocystis cases are asymptomatic, have resulted in its pathogenicity being hotly disputed. Blastocystis is transmitted via the faecal-oral route and has been linked with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Recently the theory that Blastocystis' impact on the gut microbiome is the basis for its pathogenicity has garnered much support and this is likely to be facilitated by its metabolism. Pyruvate metabolism is central to the metabolic pathways of all eukaryotes and Blastocystis' pyruvate metabolism is extremely unique, as it possesses three enzymes which convert pyruvate to acetyl-CoA. This study establishes a method to analyse Blastocystis' metabolome using 1D 1H and 1D 1C nuclear magnetic resonance (NMR) spectroscopy. A method to lyse the Blastocystis cells and extract its metabolites is developed. Methanol was determined to be a better extraction solvent than ethanol, bead beating was a better lysis method than sonication and temperature did not have an impact on metabolite extraction. The method developed was then applied to analyse Blastocystis' pyruvate metabolism. Blastocystis' metabolic footprint following supplementation with 13C-Glucose and then 13C-pyruvate was also analysed. We extracted evidence of lactate, ethanol and xylitol production in Blastocystis. Lactate in past studies has been demonstrated to have probiotic effects, ethanol has been demonstrated to have negative effects on the gut microbiome and xylitol has anti-adhesion effects which could also have an impact on the gut microbiota. The three different molecules detected could all have an important impact on gastrointestinal health.