Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

Jamie M Newton,Jose Ortega Roldan,Emma L Betts,Lyto Yiangou,Anastasios D Tsaousis,Gary S Thompson

doi:10.3390/molecules26113285

Jamie M Newton, Jose Ortega Roldan + Show 4 more

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https://doi.org/10.3390/molecules26113285

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Abstract

Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome’s “health” and/or subsequently resolve Blastocystis’ potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for 1H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR.

Highlights

Blastocystis is a genus of anaerobic protozoan that resides in the gastrointestinal tract of many vertebrate species and has historically been classified as a parasite, yet its pathogenicity has been a subject of dispute in recent years
Blastocystis has a unique metabolism and possesses a mitochondrial-related organelle (MRO) with chimeric characteristics of an aerobic mitochondrion and hydrogenosomes [1]. Many of these characteristics have been acquired by lateral gene transfer from prokaryotes and possibly other eukaryotic organisms in the gastrointestinal tract, and these have likely supported the adaptation of Blastocystis to the gut environment [2]
We developed an efficient, reproducible protocol to perform metabolomics studies on Blastocystis species and found that the extraction solvent and lysis method were the most important factors for metabolite extraction

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Blastocystis is a genus of anaerobic protozoan that resides in the gastrointestinal tract of many vertebrate species and has historically been classified as a parasite, yet its pathogenicity has been a subject of dispute in recent years. Blastocystis has a unique metabolism and possesses a mitochondrial-related organelle (MRO) with chimeric characteristics of an aerobic mitochondrion and hydrogenosomes [1]. Many of these characteristics have been acquired by lateral gene transfer from prokaryotes and possibly other eukaryotic organisms in the gastrointestinal tract, and these have likely supported the adaptation of Blastocystis to the gut environment [2]

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