Background
Among four species of human malaria parasites,
Plasmodium falciparum is responsible for most malaria-attributed morbidity and mortality. Over the past decade, successful scale-up of malaria control has resulted in substantial reductions in malaria cases and deaths [
1,
2]. As malaria transmission decreases due to control efforts, the epidemiology of malaria may change; that is, an increasing proportion of infections at the community level may be asymptomatic and of low parasite density [
3,
4]. Current malaria diagnostic tools include: 1) parasite detection by microscopic examination of blood smears, 2) antigen-based rapid diagnostic tests (RDTs), and 3) sensitive DNA-based assays. All these diagnostic methods require blood sampling by finger-prick and their implementation has been limited by either their labour/time intensive nature and requirement for specialized training and skills (microscopic method), moderate sensitivity (RDTs, microscopy), or high cost of sample preparation and supporting infrastructure needed (DNA-based methods). For programmes aiming to reduce transmission by further decreasing the parasite reservoir in humans through large scale screening approaches to detect and then to radically cure asymptomatic low-density malaria infections in real time, a field-deployable non-invasive, sensitive, low-cost, simple diagnostic tool would be very useful at the community level. Currently, available diagnostic tools cannot meet this challenge. Therefore, it was proposed to identify malaria parasite-specific low molecular-weight metabolites that could potentially be used for future development of such diagnostic tools.
As the first step for proof of concept, this study was designed to identify parasite specific low molecular-weight metabolites in an
in vitro 48-hour time-course culture system of
P. falciparum using high-resolution metabolomics (HRM) [
5-
7]. Earlier metabolomic studies on malaria have mainly focused on metabolic pathways and enzymes for the development of therapeutic strategies and interpretation of malaria pathogenesis. For example, using metabolomics with LC-MS/MS, Olszewski
et al. identified the potential mechanism of cerebral malaria pathogenesis which was associated with the depletion of arginine [
8]. More recently, NMR techniques in malaria metabolite profiling has been applied to identify biomarkers for infections. Tritten
et al. identified two urinary metabolites in
Plasmodium berghei infected mice, while a study by Sengupta et al. suggested urinary ornithine seems to have the potential as biomarkers of
Plasmodium vivax malaria [
9,
10]. Although the two studies were conducted in rodent malaria and
P. vivax malaria respectively, their analytical approaches could be used in mining for biomarkers of
P. falciparum malaria infection. A study by Teng
et al. showed strain-specific differences in a range of metabolites in erythrocytes infected with
P. falciparum from
in vitro culture and further highlighted the variation in levels of choline and phosphocholine among the strains [
11]. In addition, Sana
et al. investigated global mass spectrometry-based metabolomic profiling between
in vitro P. falciparum infected and uninfected erythrocytes [
12]. They demonstrated the alteration of metabolic profiling including the glycolysis pathway and tricarboxylic acid (TCA) cycle elucidating the mechanism of host-parasite interactions.
In contrast to the above reports, this study was designed to explore
P. falciparum specific waste products, low molecular-weight metabolites, in the supernatant from the erythrocyte culture system. The rationale for profiling low molecular-weight metabolites in culture supernatants, first, was based on the hypothesis that parasite-specific small molecular wastes could be secreted into urine, saliva or sweat at high concentrations in malaria infected human and the ultimate goal is to use the small molecules as potential biomarkers for development of non-invasive and sensitive malaria diagnostic tools. This study demonstrated HRM could achieve a relatively comprehensive and quantitative analysis of
Plasmodium-specific metabolites in supernatant from parasite infected culture system, and explores low molecular-weight biomarkers [
6,
13] associated with
Plasmodium.
Discussion
The primary objective of this study was to explore P. falciparum-specific low molecular-weight metabolites that can be used as biomarkers for future development of non-invasive malaria diagnostic tools. Using the supernatant samples from in vitro erythrocytic stage asynchronized cultures, four molecules, 3-methylindole, succinylacetone, S-methyl-L-thiocitrulline and O-arachidonoyl glycidol, were identified as potential biomarkers.
Use of HRM is a uniquely good approach to identify putative biomarkers for a complex disease like malaria since
Plasmodium parasites divert nutrients toward proliferating parasite cells while the host cells try to maintain homeostasis and deal with metabolic changes during the parasites’ intraerythrocytic life cycle [
33,
34]. This study demonstrated the strength of HRM in measuring
P. falciparum specific waste products and toxins which were expected to increase in concentration during the infection. Therefore, this approach allows for the identification of potential biomarkers associated with
Plasmodium-specific products. Previous studies also showed that the analytic capabilities of HRM could measure the relative levels of all metabolites simultaneously in
in vitro and
in vivo malaria infection systems [
12]. Furthermore, using the outcomes of HRM, KEGG mapping was performed in this study. Significant features (n=1025) were identified in both human and
Plasmodium metabolic pathways to distinguish which metabolic compounds are being utilized by both. Surprisingly, 439 metabolites were found to be used in both human and plasmodium metabolic pathways. However, despite the similarities in this large number of metabolites, the pathways in which these metabolites are mapped are likely to be different due to the absence of certain metabolic pathways in plasmodium compared to human metabolic pathways. Meanwhile, of the 586 unmatched features, four (4) were found to be potential biomarkers from the parasite during the erythrocytic stage culture system. This was based on the fact that the ion intensities of the four molecules were increased with culture time, suggesting a positive association between relative quantity of these molecules and level of parasitaemia. In addition, 3-methylindole and succinylacetone molecules in the supernatant samples from 3% haematocrit 48 hour time-course cultures were further quantified. The concentration of succinylacetone peaked at 48 hours compared to 36 hours for 3-methylindole. As 3-methylindole is volatile molecule, it is possible that 3-methylindole was evaporated due to extended culture to 48 hours. Importantly, 3-methylindole has been shown to stimulate an odorant receptor to attract malaria mosquito vector [
26], potentially playing a role in enhancing the probability of transmission for the parasite. Practically, 3-methylindole could be used in traps for mosquito research purposes. Although 3-methylindole is found in large intestine of humans via ingestion of tryptophan, this molecule is well-known as a highly selective pulmonary toxicant for ruminants [
35]. A study showed that metabolism and bioactivation of 3-methylindole in human is mediated by human liver microsomes [
36]. In spite of the fact that this molecule is found in human gastrointestinal system, it is probable that the elevation of this molecule due to malaria infection in human could still be used as a potential biomarker when urine or sweat samples are tested.
Another potential biomarker identified was succinylacetone which is known to inhibit haem biosynthesis (delta-aminolevulinate dehydrolase inhibitor) [
37]. Succinylacetone either makes the haem synthesizing system nonfunctional or decreases its functionality [
37]. Although this molecule has been shown to be a biomarker for Tyrosinaemia Type 1 (Tyr I), the genetic disorder is rare and worldwide prevalence is very low. Therefore, utilizing this compound as a potential biomarker in malaria-endemic areas could account for the malaria infection rather than the Tyr I. The production range of succinylacetone suggests it may be useful as a basis for developing a biosensor for non-invasive diagnosis of malaria. Two other potential metabolites were also identified in this study: S-methyl-L-thiocitrulline is a potent NOS inhibitor to reduce nitric oxide production and endothelial dysfunction [
38] while O-arachidonoyl glycidol was reported to be an inhibitor of fatty acid amide hydrolase [
30]. The increase in ion intensities during culture period for these two molecules indicated a potential as biomarkers for malaria diagnosis. Currently, a metabolomics study using urine, saliva, sweat and blood samples collected from
P. falciparum infected people from Africa is underway. The ongoing study will provide information on concentration and stability of the molecules in malaria infected people and their association with parasite densities.
An apparent disappearance of two amino acids, arginine and isoleucine, was observed in the 48 hr culture system. Several important host-parasite interaction studies elucidated a mechanism that isoleucine was taken by
Plasmodium sp. to develop blood stage parasites [
25,
39]. Malaria parasite utilizes amino acids largely through the degradation of host erythrocyte haemoglobin [
40]. Isoleucine is the only amino acid not present in human mature haemoglobin, resulting in utilization of this amino acid from other sources by parasites. The untargeted HRM confirmed previous finding regarding the depletion of isoleucine in culture supernatant in time dependent manner, supporting the reliability of experimental system and analytical approach in identifying the above four parasite-specific waste metabolites and toxins which were released into culture supernatant and were shown increase in concentration during 48 hr culture period.
In summary, HRM coupled with network and pathway analysis using the significant metabolites from culture supernatants of infected erythrocytes and incorporating the broader human and malaria parasite metabolomic knowledge identified four potential parasite-specific biomarkers. The findings from the current study may provide improved opportunities for innovative prevention and management programmes such as development of new malaria diagnostic tools.
Acknowledgements
This research was supported in part by the Center for Health Discovery and Well-being of Emory University, grant # NRF-2014R1A4A1007304 (Y.H.J) and grant# NRF-2014R1A1A2053787 of Korea University, and by Malaria Branch, Division of Parasitic Diseases and Malaria (DPDM), Center for Global Health, Centers for Disease Control and Prevention (CDC). DPJ is supported in part by NIH grants HL113451, ES009047, ES019776 and AG038746 and NIAID Contract HHSN272201200031C. The authors acknowledged Jae Ho Cho, Korea University in making figures. We also thank Dr. S. Patrick Kachur, Malaria Branch, DPDM, CGH, CDC for his critical review of this manuscript and valuable suggestions. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
Competing interests
The findings from the current study were filed for Emory Intellectual Property Disclosure and for CDC employee Discovery and Invention Report (EIR) respectively. Emory University was included in the CDC EIR.
Authors’ contributions
YP and DJ designed the current study and YP carried out the metabolomics experiments and comprehensive data analysis. YPS and LS conceived the concept of identification of parasite-specific low molecular-weight metabolites for future development of non-invasive, low cost, sensitive and simple malaria diagnostic tool and participated in the current study design. ETL conducted parasite cultures, and BL and KU assisted in metabolomics experiments. CM and YHJ assisted in generating additional files for this manuscript. YP, YPS and DJ wrote the paper. All authors read and approved the final manuscript.