Mass Spectrometry and Metabolomics—New Approaches for Helminth Biochemical Studies

1.1. Metabolomics and mass spectrometry in biomedical research

The composition of the intracellular environment depends on harmonic and orchestrated synthesis and interaction of molecules that are essential for cellular survival and healthy life maintenance [1], and it is true for both eukaryotic and prokaryotic organisms. Eukaryotic cells present a higher degree of intricacy compared with prokaryotic cells because of the huge biochemical complexity they present [2]. Helminths represent a pluricellular eukaryotic group of worms that establish parasitic relationships with humans, which has been registered since ancient Greek writings [3]. Structurally, helminths are completely different from Homo sapiens, and it is assured that peculiar metabolic differences exist between these organisms. These differences enable understanding and studying helminth-human interaction, transmission routes, and life cycles through molecular studies, the cornerstone in all fields of biomedical research.

The search for faster, more accurate methodologies that describe the biochemical/pathophysiological status of an organism of interest has driven major efforts in biomedical research in the late years in order to meet the clinical needs for a health community [4]. Bioanalytical methodologies have then emerged as suitable choices for expeditious approaches that exhibit great resolution and sensitivity, thus generating the development of more accurate and precise methodologies. In instrumental bioanalysis, several compounds of interest can be measured and/or monitored depending on their molecular nature. As there is no “one-size-fits-all” approach, different molecular classes have demanded the creation of different analytical strategies in which both comprehensive and specific analyses can be performed. This, therefore, has elicited the development of the “omics” strategies, which were subdivided according to the needs of the researchers. For example, for large and bulky molecules such as proteins, a wide array of analytical methods compose what is called the proteomics approach, which is dedicated to study each and every aspect of proteins and their levels, interactions, and structure in biological systems [5]. The same rationale was then applied for other biological components, such as genes (genomics) [6], lipids (lipidomics) [7], and small molecules/metabolites (metabolomics) [8].

Metabolomics can be understood in more detail as the systematic study of small, nonprotein, both endogenous and exogenous molecules, which represent the end of the entire biological chain started by gene expression (i.e., phenotype) [9]. Metabolomics studies usually measure compound classes such as lipids, nucleic acids, amino acids, and glycans, composing a useful biochemical platform for studying mechanisms of interactions, signalling synthetic pathways of systems and molecules under different metabolic conditions (e.g., diseases), providing specific molecules or molecular groups that characterize such conditions: the biomarkers. This assists in understanding, diagnosing, preventing, and predicting conditions, as well as helps assessing risks and benefits of pharmacological interventions [10].

Cell metabolism is one of the challenges that have been the driving force of metabolomics over the years [11]. The efforts on development were to obtain increasingly accurate and well-resolved methods, introducing more sophisticated and specific methods such as nuclear magnetic resonance (NMR) and mass spectrometry (MS) [12]. This made room for a number of improvements also in data processing and bioinformatics, where multivariate analyses and big data analytics found a whole new application in resolving and integrating complex biological matrices and systems [13], giving rise to the four main metabolomics strategies: metabolic profiling, the identification and quantification of known metabolites that are related to a specific number of metabolic pathways [14]; metabolic fingerprinting, which comprises fast-paced analysis to classify a determined sample during high-throughput screenings (positive/negative or disease/health) [15]; metabolic footprint, which analyses the extracellular metabolites (i.e., excretion/secretion products) of a given organism [16]; and targeted analysis, a series of quali-quantitative analyses of any given known metabolite in an orga-nism or a metabolic pathway [17].

The technical aspects of both NMR and MS place them both as the main tools for metabolomics studies, especially for their complementary nature. NMR is a spectroscopy-based technique that relies on the incidence of a magnetic field over the nuclei of atoms with nonzero spin counts, such as ¹H, ¹³C, and ¹⁵N, thus providing accurate structural and quantitative information of the metabolic profile under study [18]. There are several advantages associated with the use of NMR as an analytical tool for metabolomics; as it is not a destructive analysis, i.e., it is possible to recover the sample after the analysis, which is useful especially for target analyses. Moreover, NMR requires little sample preparation and shows great reproducibility, two desirable features for bioanalyses [19]. In contrast, some disadvantages hamper the wide distribution of NMR as a routine approach; the high costs associated with instrument maintenance and the need for relatively high amounts of the analyte usually turn down NMR as a high-throughput choice, which finds its best use as a research tool.

Mass spectrometry, on the other hand, despite being sample-destructive, shows great potential for routine analyses. MS-based bioanalytical systems show superior performance in detection and quantification limits, easily reaching a working range of nano to picomoles [20]. Relying on a basic structure of ionization source-mass analyser, MS brings a wide array of combinations of both, which comprehends virtually any type of sample for metabolomics studies [21], in addition to the possibility of coupling with chromatographic approaches such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) [22]. Moreover, structural elucidation strategies with MS are diverse and can be suited according to the needs and to the instrument available, either with mass accuracy on high-resolution instruments or with fragmentation profile (MS/MS) in standard devices—or even a combination of the two [23]. These factors contributed to the popularization of MS among researchers and clinical experts and have, therefore, provided the basis for great development in this area.

Hence, this chapter will provide a comprehensive overview on the application of metabolomics analysis and strategies developed for the analyses of helminths, with a particular focus on MS, reinforcing the great importance of these organisms for general public health. The main aim is to address recent analytical chemistry methodologies, challenges to overcome and promising results, expanding possibilities at helminthiasis research, and thus contributing with advances in the area.

2.1. Schistosoma mansonibiochemical characterization

S. mansonimetabolomics studies are conducted to improve the knowledge of disease patho-logy, to evaluate the behaviour and infectivity of different strains, to elucidate mechanisms of action, and to evaluate the efficacy of available drugs in addiction to discover biomarkers that can be applied as diagnostic and drug targets [24].

The main analytical metabolomics approaches are MS and NMR [25], and both of them allow performing target or untargeted analysis, which are mostly focused on two classes of metabo-lites: lipids and oligosaccharides. In case of living organisms, such as parasites, lipids are necessary for cell-cell signalling, for protection from host immunity, for maintenance of integrity and parasite structure during the life cycle and are also involved in the mechanisms of sexual development, egg production, and cercaria penetration [26–28]. Concerning oligosaccharides, these molecules are precursors of glycoconjugates such as glycoproteins and glycolipids that are mainly involved in development of innate and adaptive immune response in the host, parasite survival, and the chronic infection establishment [29, 30]. Lipids as well glycans may change as long as the parasite develops, thus it is crucial to understand metabolomics patterns according to the different lifecycle stages for each parasite studied.

2.1.1. Metabolomics approach for S. mansonilifecycle stages

2.1.1.1. Mature and immature eggs

The main class of lipids present at S. mansonieggs is glycosphingolipids [31], which are involved in interactions with the different affected organs, such as gut, lungs, or liver, facilitating its passage through the tissues [32]. Glycosphingolipids are also reported to be associated with egg’s high immunogenicity and pathogenicity in different strains [33]. For example, the granuloma formation occurs by the reaction of host immune response mediated by parasite glycoconjugates; therefore, the greater abundance of glucosylceramides (GlcCer) in one strain compared to another, the higher pathogenicity [32, 34]. The same study using high-resolution mass spectrometry (HRMS) showed that phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), triacylglycerol (TG), and diacylglycerol (DG) are involved in the differentiation of S. mansonistrains [34]. In respect to maturation, immature and mature eggs present also been differentiated using Matrix-Assisted Laser Desorption/Ionization—Time of Flight Mass Spectrometry (MALDI-TOF-MS). This research showed that immature eggs have a common N-glycan profile and are formed predominantly of nonspecific oligomanoses, while mature eggs have highly complex and fucosylated N-glycans [30]. Therefore, lipid molecules in eggs show differences according to maturation phases and diverge between distinct strains.

2.1.1.2. Miracidium

This is about a free-living and motile form that penetrates in Biomphalaria sp.snails, the intermediate host of the S. masonilifecycle. Different strains were analyzed by HRMS, and the results showed the possibility to differentiate two strains of miracidium by identifying different lipids: triacylglycerol (TG), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol-ceramide (PI-Cer), and dodecanoid acid (DA). The most interesting lipid among them is the last compound, reported to be responsible for facilitating the penetration of miracidium in snails [27]. Considering glycolipid studies through MALDI-TOF-MS analysis, miracidium glycolipids have been almost devoid of LeX- or LN-motifs, what is coherent with previous data that showed snail infection is apparently independent of LeX-motifs, but LDN-motifs are fairly present in miracidium and snail. Observing that the parasite presented the same spectra compared to the intermediate host, miracidium appear to develop a snail-compatible glycosylation pattern [30]. These molecular studies show lipid variability and/or similarity among different lifecycle stages, opening opportunities to better understand interactions between parasite and host/intermediate host or to interfere at metabolic pathways, as long as we know where and how [30, 35].

2.1.1.3. Cercaria

Cercaria, the final larval stage of S. mansonilifecycle, produces secretions that facilitate skin penetration. These fluids are reported to be weak immunogenic and composed of glucoproteins and glycolipids. Mass spectrometry approaches demonstrate the predominance of ceramides-linked glucose and/or galactose with fucose and xylose cores at cercaria secretions [30, 33, 36]. Another study also determined the cercaria differentiation between two S. mansonistrains by HRMS. The lipids TG, DG, PE, PA, and phosphatidylglycerol (PG) were reported to be responsible for this discrimination [27].

2.1.1.4. Adults

The tegument surface structure of S. mansoniadult worms is crucial for parasite survival and modulation of host immune response. This structure is syncytial and is covered by two closely apposed lipid bilayers, one of them is an inner plasma membrane and the other an outer membranocalyx complex [28, 37]. Mass spectrometry studies conducted with tegument and whole worm demonstrate an enrichment of the phosphatidylcholine (PC) and PS of tegumental compositions, but this phenomena does not occur with PE and PI, which presents the same composition for the two worm fractions. The tegumental membranes are also enriched with lysophospholipids, mainly lyso-PS and lyso-PE containing eicosanoic acid (20:1) [28].

As regards glycans, their composition dramatically changes during the development stages. Comparing with early life-stages, adult worms present smaller ratio between complex type-glycans and oligomannosidic glycans and the antigenic core composed by xylose is undetectable [30].

Mass spectrometry imaging (MSI) enables the study of spatial distribution of biomarkers. Ferreira and co-workers (2014) used MALDI-MSI to image two different S. mansonistrains. Figure 1 shows the sum of all ions obtained by the imaging analysis where it is possible to detect both male and female worms’ bodies. The spectra obtained from the single worms were compared with the pair and enabled the election of specific ions for male and female worms [34].

The same study determined the ions responsible for the differentiation of male and female worms of two different strains and their localization in a worm body. The fingerprint of each worm was submitted to Principal Component Analysis (PCA), and the groups of strains are discriminated, enabling the selection of chemical markers for each group. New MS/MS imaging was performed and the distribution of the parental ions selected by PCA can be seen in Figures 2 and 3.

Figure 2 shows the male and female image of BH strain obtained by MALDI-MSI analysis. Female worm presents the predominance of PC (823, 665 and 761 m/z) and PG (764 m/z) distributed to reproductive and digestive systems. Male worm presents diacylcglycerol (DAG) (635 and 651 m/z) in ventral sucker and gynaecophoric channel, triacylglycerol (TAG) (846 and 886 m/z) in ventral sucker and reproductive system, phosphatidic acid (PA) (675 m/z) in ventral sucker and gynaecophoric channel, PI (862 m/z) in reproductive and digestive systems and ventral sucker, and finally, PC (887 m/z) in reproductive system and ventral sucker.

Figure 3 shows male and female image of SE strain obtained by MALDI-MSI analysis. Female worm presents the predominance of PI (623 m/z) and DAG (606 m/z) distributed mainly in tegument while male worm presents TAG (786 and 850 m/z) and PC (825 m/z) in ventral and oral suckers and reproductive system.

Ferreira and coworkers (2014) demonstrate that it is possible to determine spatial distribution of biomarkers for parasites and also show that more studies are necessary to understand the function of each chemical markers according to the organs they are located, searching for its influence through pathogenicity of different strains. Furthermore, a new platform of study putting together metabolomic and mass spectrometry techniques was proposed and was named as Parasitomics.

2.2. Ascaris lumbricoidesand chemical markers at moultingprocess

According to Pullan and coworkers [38] and CDC [39], it is estimated that 800–1200 million people are infected with Ascaris lumbricoidesworldwide and more than 60,000 deaths occur annually because of Ascariasis(spectrum of disease caused by Ascaris lumbricoidesinfection). World Health Organization has applied some strategies for disease burden control, such as deworming programmes [40]. In case of soil-transmitted helminthiasis, the main public health strategy is the mass drug administration (MDA), which consists of anthelmintic drug treatment regularly provided to risk population, mainly school-aged children [40, 41]. Despite the great efforts, approximately 30% of children are really receiving the anthelmintic treatment in the whole world [42]. Besides the absence of total coverage of MDA worldwide, there is still another issue to pay attention, the development of anthelmintic drug resistance [43–45].

Taking into account all of these issues cited above, beyond the disability-adjusted life years (DALYs) in consequence of morbidity and mortality indexes, it is necessary to better comprehend and to deal with helminthic parasites, such as Ascaris lumbricoides, in a different point of view. It is urgent to evaluate and discover different and better ways to control and eliminate it. Therefore, to outline strategies that are suitable for its life cycle could bring light to mana-gement of helminthiasis. Since part of the development of A. lumbricoideshappens in the soil [46, 47], it is therefore reasonable to consider that breaking the life cycle during the phases in the environment would be a valuable attempt in containing the infection in endemic areas [48].

Molecular detailed analysis of the diverse A. lumbricoidesforms may elucidate new targets for parasite control. Metabolic fingerprinting using mass spectrometry has been widely employed, targeting parasite-related metabolites [34, 49, 50]. The trend of establishing target molecules and linking them with important metabolic pathways in the parasite life cycle broadens the knowledge in the field, generating a platform that will assist in better knowledge on their biology, elucidation of survival mechanisms, pharmacodynamics, diagnostic methodologies, development of pharmaceutical products to eradicate these diseases, whether in soil or living hosts, etc. [50, 51].

Melo and coworkers aimed to characterize, in a simple way, the larval development stages of A. lumbricoides(the traditional human ascariasis agent) by metabolic fingerprinting using HRMS from stool samples [52]. The molecular elucidation in different life stages presents a huge potential for application in parasite infection control, breaking the life cycle and reducing infection and re-infection of exposed human beings, mainly at endemic zones.

From Ascaris lumbricoidesspecimens, it is possible to separate different development larval stages using optical microscopy. Melo’s researchers group analyzed stool samples collected from school-aged children at Brazil. Nonembryonated and embryonated eggs, containing L1 and L3 larvae, could be separated from stool pool. L2 stage larvae were excluded from samples because it is an overlapping period between the end of L1 and the beginning of L3, what makes it difficult to identify and isolate. Considering the high sensitivity of mass spectrometer analyser, it was necessary to clean as much as possible this complex sample, what favors the real identification of significant molecules [52].

For molecular screening like this, starting from a complex sample, it is necessary to do a multivariate statistical analysis for correct identification of targets. For the present study, Principal Component Analysis (PCA) was chosen to statistically determine relevant groups of ions, what led to identification of the correspondent molecular biomarkers for each development stage under analysis. From the elected ions by PCA, it was possible to achieve the most suitable chemical markers through metabolome databases analysis, along with a good literature review. ESI-HRMS analysis of Ascaris lumbricoidesspecimens showed different spectral signals among the egg, L1 and L3, and PCA statistical analysis elected distinct chemical markers for each structure/larval stage.