Lipid profile of Trichinella papuae muscle-stage larvae

Outbreaks of trichinellosis caused by Trichinella papuae have been reported in South-East Asia. Mebendazole and thiabendazole are the treatments of choice for trichinellosis; however, both drugs result in significant side effects and are less effective for muscle-stage larvae (L1). An alternative therapeutic agent is needed to improve treatment. Information on lipid composition and metabolic pathways may bridge gaps in our knowledge and lead to new antiparasitics. The T. papuae L1 lipidome was analysed using a mass spectrometry-based approach, and 403 lipid components were identified. Eight lipid classes were found and glycerophospholipids were dominant, corresponding to 63% of total lipids, of which the glycerolipid DG (20:1[11Z]/22:4[7Z,10Z,13Z,16Z]/0:0) (iso2) was the most abundant. Overall, 57% of T. papuae lipids were absent in humans; therefore, lipid metabolism may be dissimilar in the two species. Proteins involved T. papuae lipid metabolism were explored using bioinformatics. We found that 4-hydroxybutyrate coenzyme A transferase, uncharacterized protein (A0A0V1MCB5) and ML-domain-containing protein are not present in humans. T. papuae glycerophospholipid metabolic and phosphatidylinositol dephosphorylation processes contain several proteins that are dissimilar to those in humans. These findings provide insights into T. papuae lipid composition and metabolism, which may facilitate the development of novel trichinellosis treatments.

www.nature.com/scientificreports www.nature.com/scientificreports/ pathways has led to several antiparasitic designs. Glycosylphosphatidylinositols of T. gondii and Plasmodium falciparum were found to be candidates for immunotherapeutic strategies 16,17 . Another phosphatidylcholine metabolism in plasmodium was demonstrated to be a novel drug target 18 . Amphotericin B is an antiparasitic drug which binds to ergosterol, which is present in leishmania membranes and absent in mammals. The amphotericin B-induced membrane pore is responsible for ion leakage, contributing to parasite death 19 . Miltefosine 20 , edelfosine 21 and sitamaquine 22 are lipid-like drugs used for leishmaniasis treatment; they affect membrane lipid rafts and accumulate in L. donovani through passive diffusion.
In this research, we aimed to profile the lipid components of T. papuae larvae using MS-based lipidomics. In addition, proteins relating to lipid metabolism in T. papuae were compared with those in humans to explore potential new drug targets. The study was designed to identify drug target candidates and their related pathways.

Comparison of
Quantification of lipid components. The quantities of each lipid species can be estimated through the ion intensities of LC-MS/MS analysis. The top-10 most abundant lipids are shown in Fig. 3 ,16Z]/0:0) in T. papuae L1. While, sterol lipids, e.g., alloavicholic acid and 27-norcholestanehexol, were also highly abundant. However, the total ion abundance of a specific lipid species doesn't necessarily indicate this lipid amount since ion abundance was affected by both ionization efficiency and lipid concentration. The different lipid class has quite different ionization efficiency. Therefore, relative ion abundance of lipids from the same class were more appropriate to consider. The top-10 most abundant lipids in each class in are provided in Supplementary Table 1   www.nature.com/scientificreports www.nature.com/scientificreports/ T. papuae lipid mechanisms. Because of the distinct structures of T. papuae L1 lipids, their metabolism and synthesis mechanisms may also differ from those of humans. To investigate the proteins involved in the lipid metabolic processes in T. papuae, a bioinformatics pipeline was designed, which is shown in Fig. 4. Multiple gene ontology (GO) can be used to annotate the function of single genes; therefore, all genes involved in T. papuae lipid metabolism were collated using QuickGO, a web-based tool for GO searching. This annotation program provides GO annotations to proteins in the UniProt Knowledgebase (UniProtKB), RNA molecules from RNACentral and protein complexes from the Complex Portal. The GO:0006629 pathway was used to search for lipid metabolism process genes and 193 gene products were found that belonged to T. papuae. All protein sequences involved in T. papuae lipid metabolic processes were subjected to the Basic Local Alignment Search Tool (BLAST) to compare their similarity to T. spiralis, T. britovi, T. native, T. pseudospiralis, C. elegans and human protein sequences. We found 41 T. papuae proteins containing less than 50% sequence similarity to human proteins (Supplementary Table 2 and Supplementary Dataset 2), providing evidence that some cellular lipid processes of T. papuae may be dissimilar to those in human. T. spiralis, T. britovi, T. native and T. pseudospiralis demonstrated high similarity to T. papuae proteins. Whereas, C. elegans protein sequences were slightly different from Trichinella species. Compared with human proteins, T. papuae 4-hydroxybutyrate coenzyme A transferase (A0A0V1M4U9), uncharacterized protein (A0A0V1MCB5) and ML domain-containing protein (A0A0V1MEM4) had the lowest sequence similarity. T. papuae 4-hydroxybutyrate coenzyme A transferase is involved in the acetate metabolic process (GO:0006083), propionate metabolic process and methylcitrate cycle (GO:0019679). Whereas, the uncharacterized protein and ML domain-containing protein are associated with the ganglioside catabolic process (GO:0006689) and sphingolipid metabolic process (GO:0006665), respectively. These three proteins are putative candidates for future anti-trichinella drug development. The entire 41 T. papuae proteins with low sequence similarity to human proteins involved in lipid metabolism were classified into the 16 lipid pathways shown in Table 2. The major pathways were the glycerophospholipid metabolic process (GO:0006650, 6 proteins), phosphatidylinositol dephosphorylation (GO:0046856, 6 proteins), acetate metabolic process (GO:0006083, 3 proteins), ganglioside catabolic process (GO:0006689, 3 proteins), glycosylphosphatidylinositol (GPI)-anchor  www.nature.com/scientificreports www.nature.com/scientificreports/ biosynthetic process (GO:0006506, 3 proteins) and sphingolipid metabolic process (GO:0006665, 3 proteins). T. papuae glycerophospholipid metabolic and phosphatidylinositol dephosphorylation processes present the most attractive targets for drug design because they contain several proteins that are dissimilar to equivalent proteins in humans. These include phospholipase B-like (PLB) and 85/88 kDa calcium-independent phospholipase A2 (PLA2) of the glycerophospholipid metabolic process (GO:0006650, 6 proteins) (Fig. 5A) and type I inositol 1,4,5-trisphosphate 5-phosphatase (I145P), inositol polyphosphate 5-phosphatase K (INPP5) and 72 kDa inositol polyphosphate 5-phosphatase of the phosphatidylinositol dephosphorylation process (Fig. 6A). In addition, a total 15,304 protein sequences of T. papuae was collected from UniProt database and analyzed by blast2GO software to match the T. papuae proteins into the glycerophospholipid metabolism process and phosphatidylinositol dephosphorylation process as shown in Figs 5A and 6A, respectively. Moreover, the T. spiralis, C. elegans and human glycerophospholipid metabolism process and phosphatidylinositol dephosphorylation process are also demonstrated for comparison in Figs 5 and 6, respectively. The green and white boxes are proteins which present and absent in each organism. Due to these finding, proteins in both processes are similar between T. papuae and T. spiralis. They are slightly different between C. elegans and Trichinella. While, the proteins in the pathways are distinguishable between nematode and human.

Discussion
According to our LC-MS/MS lipid analyses, both the negative and positive ESI ionization modes increased the number of identified lipids. Some sodium adducts were observed on T. papuae polar and non-polar lipids. Cation adducts form easily on the dipoles of lipids in the positive ionization mode 24 . In the negative mode, T. papuae PE, PI, PS, sulphatide and non-esterified free fatty acids were detected. These lipid species are acidic lipids containing ionic bonds and were simply deprotonated and ionized as negative ion species. T. papuae PC and SM were also detected in the negative ionization mode as a result of their strong zwitterionic structures which can form anionic adducts with small anions 25 .
In the lipid classification experiment, glycerophospholipids (also called phosphoglycerides) were identified as the major lipid class of T. papuae. Glycerophospholipids are fatty acid diglycerides with a phosphatidyl ester attached to the terminal carbon. Their abundance is likely to be related to the fact that glycerophospholipids, sphingolipids and sterols are the major constituents of membrane bilayers in eukaryotic cells, with glycerophospholipids being the most abundant 26 . A high percentage of glycerophospholipids has been observed in other parasites, such as Trypanosoma brucei 27 and S. mansoni 28 . The T. papuae glycerophospholipids were sub-classified into PC (39%), PE (31%), PS (13%), PI (6%), PG (6%) and glycerophosphate (5%). Onchcerca ochengi, a filarial nematode, was found to contain PC (57.6%), PE (25.2%), PI (8.5%) and PS (7.8%) 29 . The glycerophospholipids of the two nematodes appear to have similar concentration trends. A study of S. mansoni glycerophospholipids reported contents of PC (28%), PE (25%), PS (15%), and PG (8%) 30 . The percentages of PC in T. papuae and O. ochengia are higher than that in S. mansoni, which may be related to their different body walls structures. The outer body wall of S. mansoni is a tegument of carbohydrate-containing macromolecules known as the glycocalyx 31 . In contrast, the body wall surface of T. papuae is coated with an extracuticular layer of lipids comprised of an abundance of PC 32 . PC is composed of a choline head group and glycerophosphoric acid with a variety of fatty acids, and it has roles not only in biological membranes but also in strategies by which parasitic helminths evade host immune responses. PC attached to a secreted glycoprotein ES-62 of the rodent filarial nematode Acanthocheilonema viteae is thought to be responsible for modulating host cytokine production 33   www.nature.com/scientificreports www.nature.com/scientificreports/ MyD88 protein expression leading to suppression of toll-like receptor 4 (TLR4) and interleukin-33 (IL-33) signalling 34 . Synthetic PC analogues of A. viteae reproduce many anti-inflammatory effects and have potential uses as anti-inflammatory drugs 35 . Furthermore, the PC components of T. papuae membranes may also play roles in host immune modulation. PE and PS contain phosphorylethanolamine and phosphoserine, respectively, at glycerol substitution sites and many different combinations of fatty acids can attach at the C-1 and C-2 positions of the glycerol. In addition, PEs and PSs are involved in cell membrane metabolism 36 . Oxidized PE and PS promote or suppress human inflammatory phenotypes in monocytes and myeloid dendritic cells, and they promote an inflammatory response by increasing tumour necrosis factor alpha (TNF-α) expression. However, stimulation of monocytes with oxidized PE and PS in the presence of lipopolysaccharides (LPS) results in a decrease in TNF-α and IL-1β 37 . Oxidized PEs and PSs have also been observed in T. papuae, and they may be involved in modulating the host inflammatory response. Furthermore, they are possible drug development candidates for human diseases related to oxidative stress and inflammation. One such glycerophospholipid class molecule in T. papuae is PI; it contains a glycerol molecule with phosphoinositide and various fatty acids and is a core component of GPI anchors. The GPI consists of PI, glycans and a terminal phosphoethanolamine linked to the C-terminus of the protein. The GPIs of Plasmodium, Trypanosoma, Leishmania and Toxoplasma are distinguishable from mammalian GPIs 38 . The GPI-anchoring surface antigens are involved in the pathogenicity of parasitic diseases by causing inflammatory responses and other symptoms, for example hypoglycaemia, acidosis and anaemia 39 . GPI-based vaccines have been demonstrated to reduce parasitaemia and malaria severity 40 . In addition, GPI-related pathways have been previously studied for antiparasitic purposes, e.g., for immune therapeutics and novel drug design [41][42][43] . There are differences in the structural details of PIs between T. papuae and humans, such as oxidization and fatty acid composition. Therefore, T. papuae PIs and related biological mechanisms might be useful areas for anti-trichinella research.
With regards to glycerolipids, their structures are composed of a glycerol condensed with one, two, or three fatty acids, yielding MG, DG and TG, respectively. In most eukaryotes, glycerolipids are mainly involved in lipid storage. In Toxoplasma, TG is de-novo synthesized through an acyl-CoA:diacylglycerol acyltransferase-mediated pathway localized to the parasite endoplasmic reticulum 44 . DGAT (acyl-CoA: diacylglycerol acyltransferase) is a transmembrane enzyme that is thought be the rate-limiting enzyme for glycolipid synthesis. DGAT also presents in T. papuae as a hypothetical protein T10_12081 (GenBank: KRZ77819.1). The T. papuae protein showed 72% sequence similarity to the diacylglycerol O-acyltransferase of Brachionus plicatilis (data not shown). For that reason, T. papuae may synthesize glycerolipids through a similar pathway to B. plicatilis, and T. papuae DGAT may be a good candidate for drug development. Galactolipids, such as MGDG and DGDG, are also members of the www.nature.com/scientificreports www.nature.com/scientificreports/ glycerolipid class. Interestingly, the MGDGs and DGDGs identified in T. papuae were completely distinct from the human galactolipids. DGDGs in P. falciparum and T. gondii are also different from those in humans, but they show similar properties to plant DGDGs analysed by chromatographic lipid separation 45 . A thorough examination of the galactolipid biosynthesis pathway of T. papuae may provide further insights into lipid metabolism in this parasite.
The backbone of sphingolipid is a sphingoid (long-chain) base, which is usually linked to a fatty acid via an amide bond. Sphingolipids are important for cell division, differentiation, cell death, and cell recognition and signalling, and sphingolipid biosynthesis pathways have been proposed as promising targets for therapeutic intervention 46 . A key step in the synthesis of the complex sphingolipids is the glucosylation of ceramide, which is mediated by ceramide glucosyltransferase (CGT). CGT of C. elegans has been reported to have roles in oocyte formation and early embryonic cell division 47 . A CGT inhibitor, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, was able to reduce cyst transformation of Giardia lamblia by 90% 48 . In T. papuae, CGT also presents as a ceramide glucosyltransferase-B (GenBank: KRZ80132.1), and it could represent a drug target for trichinellosis treatment. The schistosomal sphingolipids induce T helper 2 cell responses in helminth infections through T cell stimulation 49 . A structural analogue of sphingosine, 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol (fingolimod), is a new generation of immunomodulator for several autoimmune diseases and has recently completed a phase II clinical trial for sclerosis 50 . Sphingolipids may be involved in T. papuae cellular processes and may also act as anti-inflammatory molecules during host immune evasion.
Sterols are necessary for cell membrane structure and function and act as precursors for several steroid hormones. Sterols are generated from acetyl-CoA via a multistep metabolic pathway that includes the key steps acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and HMG-CoA reductase. These three enzymes are expressed in T. papuae as 3-ketoacyl-CoA thiolase (GenBank: KRZ75105.1), hydroxymethylglutaryl-CoA synthase 1 (GenBank: KRZ69053.1) and HMG-CoA reductase (GenBank: KRZ76401.1). Statin is one of the main classes of sterol biosynthesis inhibitors, which inhibit HMG-CoA reductase and have been widely used for cholesterol reduction in humans 51 . In T. cruzi, inhibitors of HMG-CoA reductase, such as mevinolin (lovastatin), reduced the growth rate of amastigotes. Furthermore, a combination of mevinolin, terbinafine and ketoconazole showed a synergistic effect on amastigotes 52 . Therefore, interference of the T. papuae sterol biosynthesis pathway is a potential approach for anti-trichinella development.
Neutral-lipid and glycolipid uptake have been observed in parasitic nematodes, and, although the actual mechanism of lipid absorption has not been investigated, it is likely to be a form of diffusion 53 . Because of the www.nature.com/scientificreports www.nature.com/scientificreports/ chemical properties of lipids, they must be transported through aqueous environments by other molecules. There are two main types of lipid binding proteins (LBPs) found in nematodes; one carries lipids through aqueous compartments and the other transports lipids across cell membranes. LBPs may also be involved in cellular functions, nutrient acquisition, host-parasite interactions and pathogenesis 54 . Long-chain fatty acid transport protein 1 (GenBank: KRZ68592.1) and fatty acid-binding-like protein 6 (GenBank: KRZ72130.1) were found in T. papuae and may play roles in lipid uptake or transportation. In addition, lipid metabolic processes are important for parasitic nematodes. In our study, three T. papuae lipid metabolic proteins, 4-hydroxybutyrate coenzyme A transferase, uncharacterized protein (A0A0V1MCB5) and ML domain-containing protein, are conserved among Trichinella species and not found in humans. In T. papuae, 4-hydroxybutyrate coenzyme A transferase takes part in the fermentation of 4-aminobutyrate to ammonia, acetate and butyrate and has been found in anaerobic bacteria such as Clostridium aminobutyricum and Porphyromonas gingivalis 55 . T. papuae juveniles within nurse cells use facultative anaerobic metabolism 56 , thus, they may use this enzyme for the same purpose as anaerobic bacteria. There is no published information on uncharacterized protein (A0A0V1MCB5) and ML domain-containing protein in T. papuae. Therefore, it would be useful to characterize these proteins to understand their functions, as they might be potential drug targets for further anti-trichinella development.
The glycerophospholipid metabolic and phosphatidylinositol dephosphorylation processes could be adopted as pathways for drug design because they contain several proteins that are dissimilar to the equivalent proteins in humans. Two phospholipases, PLB and PLA2, are involved in glycerophospholipid metabolism. Three phosphatases, I145P, INPP5 and 72 kDa inositol polyphosphate 5-phosphatase, play roles in the T. papuae phosphatidylinositol dephosphorylation process. PLB has structural features similar to Ntn-hydrolases and it could catalyze the hydrolysis of specific amide bonds of many substrates such as antibiotics and proteins 57 . The PLB contributes to the virulence of many pathogens, for example Candida albicans, Cryptococcus neoformans, Aspergillus spp., Legionella pneumophila and Pseudomonas aeruginosa, through host membrane lysis 58,59 ,. When triazole, a synthetic phospholipase inhibitor, was administered to mice infected with C. albicans, parasite tissue penetration was blocked and host death prevented 60 . In case of PLA2, it is an important energy source enzyme and crucial in the development of Clonorchis sinensis mediated hepatic fibrosis 61 . PLA2 has been reported its role on membrane remodelling and vesicle secretion in several organisms for example P. falciparum 62 and Leishmania amazonensis 63 . Plasmodial PLA2 involves the egress of merozoites from liver-stage schizonts 64 and merozoite invasion of red blood cells 58 . According to phospholipase roles in other organisms, T. papuae phospholipases are potential targets for further drug development. I145P, INPP5 and 72 kDa inositol polyphosphate 5-phosphatase are related to secondary-messenger-mediating cell responses to various stimulations and regulate Golgi-vesicular trafficking 65 . No studies have been carried out on T. papuae I145P, INPP5 or 72 kDa inositol polyphosphate 5-phosphatase in term of their roles and functions. Therefore, it would be useful to characterize these proteins for a better understanding of T. papuae lipid metabolic processes.
Our lipidomics study of T. papuae larvae provided insights into the lipids and lipid metabolism in the parasites, revealing several potential drug targets which may facilitate the development of innovative parasite control mechanisms. The glycerophospholipid metabolism process and phosphatidylinositol dephosphorylation process are different between T. papuae and human. However, the lipidome of T. spiralis, an encapsulated Trichinella species, will need to be studied and comparatively analysed with that of T. papuae. These studies may lead to the development of effective prevention and control strategies against trichinosis caused by both Trichinella clades.

Methods
T. papuae preparation. The T. papuae used in this study were laboratory strains maintained in the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Thailand. Six-week-old female ICR mice were orally infected with 100 larvae. After 2 months of infection, muscle stage larvae (L1) were obtained from the muscle tissue by pepsin digestion (0.7% pepsin [BDH, UK], 0.7% HCl). All procedures performed on animals in this study were approved by the Faculty of Tropical Medicine Animal Care and Use Committee (FTM-ACUC), Mahidol University. The approval number was FTM-ACUC No. 009/2555. All experiments were performed in accordance with relevant guidelines and regulations Lipid extraction. T. papuae L1 were quickly frozen in liquid nitrogen and then finely ground in a pestle and mortar. Liquid/liquid extraction was then performed following the Bligh and Dyer method 66 . Briefly, the lipids were extracted from the finely ground L1 by adding 1:1 (v/v) chloroform/methanol and mixed using a vortex. The mixture was then centrifuged at 3,000 × g for 10 min to achieve phase separation. The lower chloroform phase was carefully transferred to a separate 1.5-ml tube and dried in a fume hood.

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Lipid
profile analysis was conducted using ACQUITY UPLC with a 2777 C autosampler (Waters Corp, USA) coupled to a Xevo G2-XS QTof mass spectrometer (Waters, Manchester, UK) with an ESI source 67 . For each sample, triplicate injections were made for each ionization mode. Intact molecular masses and associated fragmentation products were recorded for all compounds using MS, during which retention-time aligned data were collected in two channels simultaneously, i.e., low collision energy for precursor ions and high collision energy for daughter ions, with no precursor ion selection in the quadrupole. A mass range of m/z 100 to 2000 was acquired for lipids. High mass accuracy was maintained by lock mass correction using leucine enkephalin as a reference mass (m/z 556.2771 in ESI+ and m/z 554.2615 in ESI-).
For data analysis and identification, the retention time alignment, peak picking and database searching were accomplished using Progenesis QI (version 2.1, Nonlinear Dynamics, Newcastle, UK). Lipids were identified using (i) MetaScope by searching compound features against LIPID MAPS (http://www.lipidmaps.org/) and (ii) LipidBlast (http://fiehnlab.ucdavis.edu/). The search parameter used for precursor ion mass accuracy was set at 5 ppm and the theoretical fragmentation ion mass accuracy was set at 10 ppm. Reported compound identifications were scored and filtered by isotope similarity ≥90%. Identified compounds with abundance >100 and abundance variation among three replicates with <30% coefficient of variation were investigated. Bioinformatics analysis. All genes involved in lipid metabolism were gathered using QuickGO, a web-based tool for gene ontology searching. The term GO:0006629 assigns lipid metabolic processes. Gene products belonging to T. papuae were collated. The obtained protein sequences were subjected to the Basic Local Alignment Search Tool (BLAST) and used to search the human non-redundant protein, Trichinella and C. elegans sequence databases. Any proteins with less than a 50% sequence similarity to human proteins were reported as being distinctive T. papuae proteins.