High-resolution metabolomics to discover potential parasite-specific biomarkers in a Plasmodium falciparum erythrocytic stage culture system

Abstract

BackgroundCurrent available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. It was proposed that malaria parasite-specific low molecular-weight metabolites could be used as biomarkers for the development of a malaria diagnostic tool aimed to address this diagnostic challenge. In this study, high resolution metabolomics (HRM) was employed to identify malaria parasite-specific metabolites in Plasmodium falciparum in vitro culture samples.MethodsSupernatants were collected at 12 hours interval from 3% haematocrit in vitro 48-hour time-course asynchronized culture system of P. falciparum. Liquid chromatography coupled with high resolution mass spectrometry was applied to discover potential parasite-specific metabolites in the cell culture supernatant. A metabolome-wide association study was performed to extract metabolites using Manhattan plot with false discovery rate (FDR) and hierarchical cluster analysis. The significant metabolites based on FDR cutoff were annotated using Metlin database. Standard curves were created using corresponding chemical compounds to accurately quantify potential Plasmodium-specific metabolites in culture supernatants.ResultsThe number of significant metabolite features was 1025 in the supernatant of the Plasmodium infected culture based on Manhattan plot with FDR q=0.05. A two way hierarchical cluster analysis showed a clear segregation of the metabolic profile of parasite infected supernatant from non-infected supernatant at four time points during the 48 hour culture. Among the 1025 annotated metabolites, the intensities of four molecules were significantly increased with culture time suggesting a positive association between the quantity of these molecules and level of parasitaemia: i) 3-methylindole, a mosquito attractant, ii) succinylacetone, a haem biosynthesis inhibitor, iii) S-methyl-L-thiocitrulline, a nitric oxide synthase inhibitor, and iv) O-arachidonoyl glycidol, a fatty acid amide hydrolase inhibitor, The highest concentrations of 3-methylindole and succinylacetone were 178 ± 18.7 pmoles at 36 hours and 157±30.5 pmoles at 48 hours respectively in parasite infected supernatant.ConclusionHRM with bioinformatics identified four potential parasite-specific metabolite biomarkers using in vitro culture supernatants. Further study in malaria infected human is needed to determine presence of the molecules and its relationship with parasite densities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0651-1) contains supplementary material, which is available to authorized users.

Metabolome-wide association study of peripheral parasitemia in Plasmodium vivax malaria

“…Metabolomics represents a powerful analytical approach to uncover the activity of physiological and pathological processes (Li et al, 2016) for the discovery of biomarkers of infectious diseases, including malaria (Park et al, 2015; Salinas et al, 2014). Nuclear magnetic resonance or gas/liquid chromatography coupled with mass spectrometry (GC/LC-MS) have been applied to understand the metabolic changes in models of host- Plasmodium interactions in vitro (Lakshmanan et al, 2012; MacRae et al, 2013; O’Hara et al, 2014; Olszewski et al, 2009; Park et al, 2015; Sana et al, 2013) and in vivo (Basant et al, 2010; Ghosh et al, 2012, 2013; Olszewski et al, 2009; Sengupta et al, 2013; Tritten et al, 2013).…”

“…Nuclear magnetic resonance or gas/liquid chromatography coupled with mass spectrometry (GC/LC-MS) have been applied to understand the metabolic changes in models of host- Plasmodium interactions in vitro (Lakshmanan et al, 2012; MacRae et al, 2013; O’Hara et al, 2014; Olszewski et al, 2009; Park et al, 2015; Sana et al, 2013) and in vivo (Basant et al, 2010; Ghosh et al, 2012, 2013; Olszewski et al, 2009; Sengupta et al, 2013; Tritten et al, 2013). However, studies of human malaria are limited to evaluation of plasma from patients infected with P. falciparum (Lakshmanan et al, 2012; Surowiec et al, 2015; Sengupta et al, 2016) or urine from patients infected with P. vivax (Sengupta et al, 2011).…”

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

“…Recent developments in high-resolution mass spectrometry and the associated data analysis tools have enabled the application of untargeted metabolomics to cell culture systems, including P. falciparum (24,25). These untargeted metabolomics techniques have already been used to identify mechanisms of action for novel drug candidates in other protozoan pathogens (26)(27)(28), providing an unbiased, hypothesis-free approach to reveal the actions of novel antiparasitic compounds with no known target, such as those found in the Malaria Box.…”

Cholesterol-dependent enrichment of understudied erythrocytic stages of humanPlasmodiumparasites

“…The P. falciparum genome is poorly annotated (40% of genes) compared to many model organisms (85% of genes for S. cerevisiae), making metabolomics an attractive method for unbiased study of parasite biology (7,8). Indeed, Plasmodium metabolomics has become increasingly popular in recent years for investigation of parasite biology and antimalarial drug action (9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”