Abstract

   Soil-transmitted helminths, including hookworms and whipworms, infect
   billions of people worldwide. Their capacity to penetrate and migrate
   through their hosts’ tissues is influenced by the suite of molecules
   produced by the infective developmental stages. To facilitate a better
   understanding of the immunobiology and pathogenicity of human hookworms
   and whipworms, we investigated the metabolomes of the infective stage
   of Nippostrongylus brasiliensis third-stage larvae (L3) which penetrate
   the skin and Trichuris muris eggs which are orally ingested, using
   untargeted liquid chromatography-mass spectrometry (LC-MS). We
   identified 55 polar metabolites through Metabolomics Standard
   Initiative level-1 (MSI-I) identification from N. brasiliensis and T.
   muris infective stages, out of which seven were unique to
   excretory/secretory products (ESPs) of N. brasiliensis L3. Amino acids
   were a principal constituent (33 amino acids). Additionally, we
   identified 350 putative lipids, out of which 28 (all known lipids) were
   unique to N. brasiliensis L3 somatic extract and four to T. muris
   embryonated egg somatic extract. Glycerophospholipids and glycerolipids
   were the major lipid groups. The catalogue of metabolites identified in
   this study shed light on the biology, and possible therapeutic and
   diagnostic targets for the treatment of these critical infectious
   pathogens. Moreover, with the growing body of literature on the
   therapeutic utility of helminth ESPs for treating inflammatory
   diseases, a role for metabolites is likely but has received little
   attention thus far.

   Keywords: metabolites, infective stage, Nippostrongylus brasiliensis,
   Trichuris muris, parasites, LC-MS, metabolomic, lipidomic

1. Introduction

   Infection with parasitic helminths is one of the most common and
   detrimental of the neglected tropical diseases [[36]1]. Indeed, eight
   out of the 17 recognised neglected tropical diseases are caused by
   parasitic helminths [[37]2]. More than 1.5 billion people
   (approximately 24% of the world’s population) are infected with
   soil-transmitted helminth infections (STHIs) [[38]3] and contribute to
   a substantial burden of human disease and disability worldwide. The
   roundworm (Ascaris lumbricoides), the whipworm (Trichuris trichiura),
   and hookworms (Necator americanus and Ancylostoma duodenale) are the
   major Soil-transmitted helminths (STHs) that infect humans. Unlike T.
   trichiura and A. lumbricoides, which are prevalent among young
   children, N. americanus and A. duodenale tend to infect older children
   and adults [[39]4,[40]5]. Soil-transmitted helminths are ubiquitous in
   tropical climates and rural temperate areas with inadequate sanitation
   facilities—that is, mostly poverty-stricken areas across the world.
   There is no vaccine for any human helminth infections, and current
   control efforts focus on mass drug administration, which is only
   partially effective [[41]6]. Currently, the World Health Organization
   recommends that anthelmintic drugs such as mebendazole and albendazole
   are commonly used for mass administration to control STHIs [[42]7], but
   they do not give lasting protection against re-infection. Meanwhile,
   other treatment drugs such as niclosamide (for Taenia saginata),
   piperazine (for A. lumbricoides), and ivermectin (for Strongyloides
   stercoralis) [[43]8] are not suitable for mass control.

   Eradication of helminths is challenging, mainly due to their complex
   life-cycles. Hookworms enter the human hosts when their infective stage
   3 larvae (L3) penetrate the skin. Initially, L3 migrates through
   subcutaneous venules and lymphatics and then subsequently enters the
   afferent circulation to reach inside the lungs [[44]4]. From there, L3
   break through the alveolar spaces and are coughed up and swallowed into
   the gastrointestinal tract as they mature into L4 and adulthood.
   Maturation proceeds in the small intestine, where L3 moult twice to
   become adult male and female hookworms. After mating, female worms
   produce thousands of eggs that exit the body in the faeces. Whipworms,
   on the other hand, enter the human host through the ingestion of
   embryonated eggs from the environment. First-stage (L1) larvae hatch
   from the eggs and penetrate the epithelial cells at the crypt base to
   undertake an intracellular existence, where larvae grow and moult
   through the larval (L2, L3 and L4) and adult stages [[45]9]. Examining
   helminth eggs in stool samples by Kato-Katz thick-smear technique is
   the only widely used diagnostic tool in helminth epidemiology
   [[46]10,[47]11], but this technique is less sensitive in the case of
   low-intensity helminth infections. Alternative sensitive diagnostic
   tools such as FLOTAC (a multivalent faecal egg count technique) and
   McMaster are available [[48]7,[49]12], but they are not adequate. It
   is, therefore, essential to understand these helminths holistically and
   devise precise diagnostic tools and effective treatment regimens that
   would provide long-term protection against diseases caused by STHs.

   While there are numerous published studies on STH genomes
   [[50]13,[51]14,[52]15,[53]16,[54]17] and proteomes
   [[55]18,[56]19,[57]20], less attention has been afforded to their
   metabolomes [[58]21,[59]22] and, specifically, the lipidome [[60]23]. A
   number of studies [[61]24,[62]25] have shown the importance of STH
   excretory/secretory products (ESPs) in host–parasite communication,
   including parasite survival and host protection against
   immunopathology. For example, ESPs such as TT47 and TM43 produced by
   whipworms are important for invasion of the gut wall by forming a pore
   in the epithelial cell membrane [[63]26], and the major ESP, p43m,
   suppresses secretion of IL-13, a cytokine with anti-helminth properties
   [[64]27]. Adult Necator and Ancylostoma hookworms release proteases
   [[65]28] and anti-coagulant peptides [[66]29] into the infection site
   to digest the host’s mucosal tissues and secrete abundant netrin-like
   proteins to suppress inflammatory responses by inducing regulatory T
   cells [[67]30]. Moreover, a small-molecule linoleic acid, used by the
   cercariae of Schistosoma mansoni to produce prostaglandin, PGE2, helps
   them to successfully migrate inside their host [[68]31]. However, very
   little is known about the small-molecule complement of STHs,
   particularly of their infective stages.

   Since it is challenging to obtain parasite material from humans, in
   this study, we used the rodent model STH parasites, N. brasiliensis
   (model organism for the human hookworms Ancylostoma and Necator spp.)
   and T. muris (model organism for whipworm T. trichiura) to characterise
   and identify small-molecule components of their infective stages.
   Previously, we reported the ESP metabolomes of their adult
   developmental stages, both of which reside in the gut [[69]21]. In this
   study, we hypothesised that the capacity of hookworms and whipworms to
   establish infection successfully might be related to the types of
   metabolites produced by their infective stages in the lung and gut,
   respectively. These findings would shed light on whether the
   metabolomes of infective-stage hookworms and whipworms are conserved or
   display stage-, species- or niche-specific molecules. Moreover, some of
   the metabolites discovered herein might be useful as potential
   diagnostic biomarkers.

2. Results

2.1. MSI Level-1 Identification of Polar Metabolites Present in the Infective
Stages of N. brasiliensis and T. muris

   Using an untargeted LC-MS protocol, we analysed the metabolome
   composition of the infective stages of N. brasiliensis and T. muris as
   outlined in the Methods ([70]Figure 1). From the infective L3 stage of
   N. brasiliensis, we prepared two biological samples—somatic tissue
   extract and ESP—and each biological sample had five replicates. The
   replicates of the L3 somatic tissue extract were named NB_SE_1 through
   NB_SE_5; ESP replicates were named NB_ESP_1 through NB_ESP_5. From the
   infective embryonated eggs of T. muris, we prepared five replicates of
   the somatic tissue extract, which were named TM_SE_1 through TM_SE_5.
   For the quality control, we prepared a pooled quality control from the
   samples (QC_P_1–QC_P_3) and media control (QC_Media_1–QC_Media_3) from
   the same media used for the experimental cultures. Using Metabolomics
   Standards Initiative level-2 (MSI-II) identification based on accurate
   mass and predicted retention time, using the open-source software IDEOM
   (an Excel interface for analysis of LC-MS-based metabolomic data), we
   identified a total of 164 putative polar metabolites altogether (i.e.,
   after subtracting media peak area values from each metabolite)
   ([71]Supplementary Table S1 from three different samples (NB_SE, NB_ESP
   and TM_SE). Most of the polar metabolites were the products of amino
   acid metabolism, followed by carbohydrate metabolism and nucleotide
   metabolism ([72]Figure 2A–C).

Figure 1.

   [73]Figure 1
   [74]Open in a new tab

   Schematic flowchart of the metabolomic and lipidomic profiling strategy
   applied for the infective stages of the N. brasiliensis (L3) and T.
   muris (embryonated eggs).

Figure 2.

   [75]Figure 2
   [76]Open in a new tab

   Distribution of total putative metabolites by different metabolite
   classes (A) in the somatic extract of infective third-stage larvae (L3)
   of N. brasiliensis (NB_SE); (B) in the excretory/secretory products of
   N. brasiliensis L3 (NB_ESP); and (C) in the somatic extract of T. muris
   embryonated eggs (TM_SE).

   Of these 164 putative polar metabolites, authentic chemical standards
   were available for 55 metabolites, allowing confident identification of
   55 polar metabolites by accurate mass and retention time (MSI-I
   identification) ([77]Table 1). Of the 55 metabolites, 21 were common to
   all three samples (NB_SE, NB_ESP, and TM_SE), comprising mostly
   products of amino acid metabolism, followed by carbohydrate and
   nucleotide metabolism. Based on the intensity of peak areas, adenosine,
   betaine, adenine, lactose, and choline were the top five compounds
   present in NB_SE. In NB_ESP, betaine, (S)-malate, l-glutamine,
   isocitrate, and 5-oxoproline were present as the major compounds. TM_SE
   contained l-leucine, lactose, adenosine, l-proline, and urocanate as
   the major metabolites ([78]Table 1).

Table 1.

   Polar metabolites (MSI-I identified) of infective stages of N.
   brasiliensis (L3) and T. muris (embryonated eggs).
   Polar Metabolites Formula ^a Mass (m/z) Rt (min) ^c KEGG ID ^d
   Log[2](FC) ^e Chemical Taxonomy ^b Reported Pharmacological Activities
   Average Peak Area (mz/rt)
   NB_SE NB_ESP TM_SE
   Adenine C[5]H[5]N[5] 135.054 8.32 [79]C00147 5.23 6-aminopurines
   Anti-inflammatory [[80]32] 103,160,746 309,024 101,274
   Adenosine C[10]H[13]N[5]O[4] 267.096 8.23 [81]C00212 5.21 Purine
   nucleosides Anti-inflammatory [[82]33] 327,057,005 0 347,270
   Inosine C[10]H[12]N[4]O[5] 268.080 10.32 [83]C00294 4.77 Purine
   nucleosides Anti-inflammatory [[84]34,[85]35] 14,631,676 0 18,472
   l-Carnitine C[7]H[15]NO[3] 161.105 12.32 [86]C00487 4.56 Carnitines
   Anti-inflammatory [[87]36] and anti-oxidant [[88]37] 81,471,762
   1,911,162 125,132
   Choline C[5]H[13]NO 103.099 20.72 [89]C00114 4.53 Cholines
   Anti-inflammatory [[90]38] 82,418,790 0 129,808
   N(pi)-Methyl-l-histidine C[7]H[11]N[3]O[2] 169.085 11.58 [91]C01152
   3.86 Histidine and derivatives N/A 5,268,444 2,170,489 13,260
   Xanthine C[5]H[4]N[4]O[2] 152.033 11.08 [92]C00385 3.62 Xanthines
   Proinflammatory [[93]39] 37,256,722 3,609,773 103,264
   l-Aspartate C[4]H[7]NO[4] 133.037 14.57 [94]C00402 3.52 Aspartic acid
   and derivatives Anti-inflammatory and neuroprotective [[95]40,[96]41]
   3,886,437 402,353 9321
   Succinate C4H6O4 118.026 15.15 [97]C00042 3.14 Dicarboxylic acids and
   derivatives Activate inflammatory pathways [[98]42,[99]43] 29,148,318
   5,130,431 99,396
   5-Aminolevulinate C[5]H[9]NO[3] 131.058 13.74 [100]C00430 3.11 Delta
   amino acids and derivatives Anti-inflammatory [[101]44] 8,231,888 0
   33,627
   Adenosine 5′-monophosphate C[10]H[14]N[5]O[7]P 347.063 13.03
   [102]C00020 3.05 Purine ribonucleoside monophosphates Anti-inflammatory
   [[103]45,[104]46] 9,485,566 0 43,938
   Hypoxanthine C[5]H[4]N[4]O 136.038 9.56 [105]C00262 2.84 Hypoxanthines
   Anti-inflammatory and wound healing [[106]47] 48,567,229 425,871
   230,726
   Lactose C[12]H[22]O[11] 342.116 15.25 [107]C00243 2.61 O-glycosyl
   compounds N/A 94,498,840 420,550 552,215
   l-Glutamate C[5]H[9]NO[4] 147.053 14.24 [108]C00025 2.30 Glutamic acid
   and derivatives Antioxidant [[109]48] 16,940,920 4,993,538 104,717
   l-Methionine C[5]H[11]NO[2]S 149.051 10.81 [110]C00073 2.06 Methionine
   and derivatives Anti-inflammatory [[111]49] and antioxidant [[112]50]
   2,078,174 890,947 15,017
   l-Histidine C[6]H[9]N[3]O[2] 155.069 14.25 [113]C00135 1.82 Histidine
   and derivatives Anti-inflammatory [[114]51] 14,964,799 1,089,738
   135,916
   4-Hydroxybenzoate C[7]H[6]O[3] 138.031 10.37 [115]C00156 1.73
   Hydroxybenzoic acid derivatives Neuroprotective [[116]52] 1,566,927
   1,019,203 13,620
   l-Tyrosine C[9]H[11]NO[3] 181.074 12.44 [117]C00082 1.09 Tyrosine and
   derivatives N/A 3,708,634 6,686,668 58,822
   Deoxyadenosine C[10]H[13]N[5]O[3] 251.101 7.48 [118]C00559 −1.03 Purine
   2′-deoxyribonucleosides Cell growth inhibitor and cytotoxic [[119]53]
   628,485 0 39,096
   (S)-Malate C[4]H[6]O[5] 134.021 16.18 [120]C00149 −1.28 Beta hydroxy
   acids and derivatives N/A 51,344,999 43,880,026 0
   D-Glucose 6-phosphate C[6]H[13]O[9]P 260.029 15.83 [121]C00092 −1.66
   Hexose phosphates N/A 402,720 0 42,982
   2-Oxoglutarate C[5]H[6]O[5] 146.021 15.77 [122]C00026 −1.80 Gamma-keto
   acids and derivatives Anti-inflammatory [[123]54] and antioxidant
   [[124]55] 249,067 2,843,652 0
   l-Pipecolate C[6]H[11]NO[2] 129.079 11.42 [125]C00408 −1.94
   l-alpha-amino acids N/A 139,920 0 16,319
   Mannitol C[6]H[14]O[6] 182.079 13.30 [126]C00392 −2.08 Sugar alcohol
   Anti-edema [[127]56] 299,808 0 37,781
   l-Alanine C[3]H[7]NO[2] 89.047 14.12 [128]C00041 −2.27 l-alpha-amino
   acids Anti-inflammatory [[129]57,[130]58,[131]59] 3,102,359 3,529,005 0
   Betaine C[5]H[11]NO[2] 117.079 10.39 [132]C00719 −2.39 Alpha amino
   acids Neuroprotective [[133]60]; improves intestinal barrier function
   [[134]61]; hepatoprotective [[135]62]; anti-inflammatory [[136]38]
   261,777,360 56,072,188 0
   l-Lysine C[6]H[14]N[2]O[2] 146.105 22.50 [137]C00047 −2.45
   l-alpha-amino acids Anti-inflammatory [[138]63,[139]64] 2,260,735
   1,941,102 0
   l-Arginine C[6]H[14]N[4]O[2] 174.111 24.05 [140]C00062 −2.47
   l-alpha-amino acids Anti-inflammatory [[141]65,[142]66,[143]67]
   30,934,546 3,589,173 0
   l-Glutamine C[5]H[10]N[2]O[3] 146.069 14.44 [144]C00064 −2.48
   l-alpha-amino acids Anti-inflammatory [[145]57,[146]58,[147]59]
   45,487,251 39,780,748 0
   l-2-Aminoadipate C[6]H[11]NO[4] 161.068 14.47 [148]C00956 −2.49
   l-alpha-amino acids N/A 137,122 0 22,389
   l-Phenylalanine C[9]H[11]NO[2] 165.079 9.35 [149]C00079 −2.50
   l-alpha-amino acids Anti-diabetic [[150]68] 1,534,294 18,355,000 0
   3’,5’-Cyclic AMP C[10]H[12]N[5]O[6]P 329.052 8.72 [151]C00575 −2.54
   3′,5′-cyclic purine nucleotides Anti-inflammatory [[152]69] 281,873 0
   57,179
   N-Acetylputrescine C[6]H[14]N[2]O 130.110 21.02 [153]C02714 −2.58
   Carboximidic acids Lung cancer biomarker [[154]70] 125,036 0 22,355
   Thymine C[5]H[6]N[2]O[2] 126.043 6.96 [155]C00178 −3.62
   Hydroxypyrimidines N/A 169,388 0 64,297
   l-Leucine C[6]H[13]NO[2] 131.094 9.97 [156]C00123 −4.14 Leucine and
   derivatives Analgesic and anti-inflammatory [[157]71,[158]72] 1,593,314
   10,121,672 735,972
   Urocanate C[6]H[6]N[2]O[2] 138.043 10.49 [159]C00785 −4.16 Imidazolyl
   carboxylic acids and derivatives Chemoattractant [[160]73] 452,897
   169,049 232,883
   Azelaic acid C[9]H[16]O[4] 188.105 10.69 [161]C08261 −4.89 Medium-chain
   fatty acids Anti-inflammatory [[162]74] 131,028 874,645 116,635
   Maleic acid C[4]H[4]O[4] 116.011 12.18 [163]C01384 −5.67 Dicarboxylic
   acids and derivatives Inflammatory/Cytotoxic [[164]75] 98,623 0 154,549
   D-Glycerate C[3]H[6]O[4] 106.026 11.85 [165]C00258 ns Sugar acids and
   derivatives N/A 343,824 0 8833
   Homogentisate C[8]H[8]O[4] 168.042 9.42 [166]C00544 ns
   2(hydroxyphenyl)acetic acids Pro-inflammatory [[167]76,[168]77] 0
   640,416 0
   Isocitrate C[6]H[8]O[7] 192.027 19.04 [169]C00311 ns Tricarboxylic
   acids and derivatives N/A 0 37,798,044 0
   l-Citrulline C[6]H[13]N[3]O[3] 175.095 14.84 [170]C00327 ns
   l-alpha-amino acids Anti-inflammatory and antioxidant
   [[171]78,[172]79,[173]80] 672,469 0 21,477
   l-Proline C[5]H[9]NO[2] 115.063 11.96 [174]C00148 ns Proline and
   derivatives Anti-inflammatory [[175]81] 9,760,345 1,106,588 269,125
   l-Serine C[3]H[7]NO[3] 105.042 15.56 [176]C00065 ns Serine and
   derivatives Modulates adaptive immunity by controlling T cell
   proliferative capacity [[177]82]; colon protection and mucosal healing
   [[178]81] 337,344 45,878 6178
   l-Threonine C[4]H[9]NO[3] 119.058 14.14 [179]C00188 ns l-alpha-amino
   acids Anti-inflammatory [[180]83,[181]84] 425,688 0 14,375
   l-Tryptophan C[11]H[12]N[2]O[2] 204.090 11.03 [182]C00078 ns Indolyl
   carboxylic acids and derivatives Anti-inflammatory
   [[183]85,[184]86,[185]87] 2,002,647 1,435,161 127,849
   l-Valine C[5]H[11]NO[2] 117.079 11.76 [186]C00183 ns Valine and
   derivatives Anti-inflammatory [[187]85] 474,387 9,518,904 25,791
   Ll-2,6-Diaminoheptanedioate C[7]H[14]N[2]O[4] 190.095 17.63 [188]C00666
   ns Amino acid N/A 0 115,608 0
   N6,N6,N6-Trimethyl-l-lysine C[9]H[20]N[2]O[2] 188.152 21.12 [189]C03793
   ns l-alpha-amino acids Cardiovascular disease biomarker [[190]88]
   462,527 208,659 12,965
   Orotate C[5]H[4]N[2]O[4] 156.017 10.27 [191]C00295 ns
   Pyrimidinecarboxylic acids N/A 0 437,266 0
   Pterin C[6]H[5]N[5]O 163.049 10.30 [192]C00715 ns Pterins and
   derivatives Biomarker of exercise-induced stress [[193]89] 0 460,935 0
   Pyridoxal C[8]H[9]NO[3] 167.058 7.46 [194]C00250 ns Pyridoxals and
   derivatives N/A 323,537 0 10,226
   5-Oxoproline C[5]H[7]NO[3] 129.042 9.82 [195]C01879 ns Alpha amino
   acids and derivatives Promotes oxidative stress in neuropathology
   [[196]90] 0 32,495,730 0
   2,5-Dihydroxybenzoate C[7]H[6]O[4] 154.026 8.30 [197]C00628 ns
   Hydroxybenzoic acid derivatives Anti-cancer activity [[198]91] 0
   1,377,617 0
   4-Trimethylammoniobutanoate C[7]H[15]NO[2] 145.110 12.25 [199]C01181 ns
   Straight chain fatty acids N/A 217,954 0 2264
   [200]Open in a new tab

   ^a Formula; ^b Chemical taxonomy = Formula and chemical taxonomy for
   compounds were taken from human metabolome database (HMDB,
   [201]http://www.hmdb.ca); ^c Rt = retention time in minutes; ^d KEGG ID
   ([202]http://www.genome.jp/kegg/) contains information on biosynthetic
   and metabolic pathways of identified compounds; ^e log 2(FC) is a fold
   change between NB_SE and TM_SE; Abbreviations: ns = not significant; ID
   = identity; NB_SE = the somatic extract of infective third-stage larvae
   (L3) of N. brasiliensis; NB_ESP = the excretory/secretory products
   (ESP) of N. brasiliensis L3; TM_SE = the somatic extract of T. muris
   embryonated eggs. Note: peak areas values of media were subtracted from
   the samples.

2.2. Metabolic Pathways of Putatively Identified Polar Metabolites

   Based on the 164 putative polar metabolites identified from the three
   biological samples (114 metabolites from NB_SE, 99 from NB_ESP, 70 from
   TM_SE), we deduced metabolic pathways (MAPs) by mapping all the
   compounds against known metabolite pathways for helminths in the Kyoto
   Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Database
   (HMDB), and MetaCyc Metabolic Pathway database. In all three sample
   groups, the highest number of identified metabolites/compounds were
   products of amino acid metabolism, followed by carbohydrate metabolism
   in NB_SE and NB_ESP, and nucleotide metabolism in TM_SE, as shown in
   [203]Figure 2A–C. For example, of 114 metabolites identified from the
   NB_SE, 57 metabolites were produced through amino acid metabolism, 28
   by carbohydrate metabolism, and 14 by nucleotide metabolism. A similar
   pattern can be seen in its ESP. Of 70 polar metabolites identified from
   TM_SE, 36 of them were the products of amino acid metabolism, 13 were
   from nucleotide metabolism, and 12 were from carbohydrate metabolism.

2.3. Chemometric Analysis of the Polar Metabolites of N. brasiliensis L3 and
T. muris Embryonated Eggs

   We performed statistical analysis on the 55 polar metabolites
   identified in [204]Table 1 (MSI-I identification) using MetaboAnalysis
   4 ([205]http://www.metaboanalyst.ca) to determine metabolite
   differences. First, the differences in the abundance of polar
   metabolites between NB_SE and TM_SE were evaluated by univariate
   analysis (volcano plot analysis). NB_ESP was excluded from the
   univariate analysis since there was no ESP from the infective stage of
   T. muris. Volcano plot analysis identified differential metabolites
   using the t-test and fold-change (FC) methods, and we plotted log[2]
   (fold-change > 2) on the X-axis against -log[10] (p-value) from the
   t-test on the Y-axis. Benjamini–Hochberg correction or false discovery
   rate was applied to compute the number of false positives out of
   significantly varied metabolic features. [206]Table 1 shows the fold
   change and the p-values of these significantly different metabolites.
   When we compared NB_SE metabolites against TM_SE, 38 of 49
   (approximately 78%) metabolites showed significant differences (p <
   0.05), where both the samples (i.e., NB_SE and TM_SE) had 19
   metabolites each with an absolute log2 fold change > 2 and an absolute
   p-value < 0.05 (represented as pink dots in [207]Figure 3).

Figure 3.

   [208]Figure 3
   [209]Open in a new tab

   Volcano plot of untargeted metabolomics analysis of differential
   features (i.e., 49 MSI-I (Metabolomics Standard Initiative level-1)
   identified polar metabolites from somatic extracts) between the somatic
   extract of infective third-stage larvae (L3) of N. brasiliensis (NB_SE)
   and the somatic extract of T. muris embryonated eggs (TM_SE). The
   volcano plot displays log2 fold changes versus Benjamini–Hochberg
   adjusted p-values (−log10 transformed). Features that exhibited an
   absolute log2 fold change > 2 and an absolute p-value < 0.05 are
   coloured in pink. Most significant metabolites are labelled with the
   corresponding name.

   We next performed multivariate analysis by a principal component
   analysis (PCA) and hierarchical clustering analyses (with heat-map
   analyses) to determine the overall metabolic similarities and
   differences between three sample groups (i.e., NB_SE, NB_ESP, and
   TM_SE). As expected, the PCA plot ([210]Figure 4) showed a clear
   separation among the three biological groups, indicating that the
   infective stages of the two STHs (N. brasiliensis and T. muris) produce
   distinct metabolite profiles. The plot shows that the variation of
   metabolites between the different helminth species and the difference
   shown on PC1 was greater than the difference between somatic extract
   and excretory/secretory product.

Figure 4.

   [211]Figure 4
   [212]Open in a new tab

   Principal component analysis scores plot for three sample groups, i.e.,
   the somatic extract of infective third-stage larvae (L3) of N.
   brasiliensis (NB_SE) (green), the excretory/secretory products (ESP) of
   N. brasiliensis L3 (NB_ESP) (red), and the somatic extract of T. muris
   embryonated eggs (TM_SE) (blue) with five replicates each. Parentheses
   on each axis explain the amount of variance. Media (QC_Media) and
   pooled quality control (QC_P) values were excluded from the analysis,
   as they were subtracted from the samples.

   Additionally, we used hierarchical clustering heat-map analysis
   (distance measured by Euclidean, and clustering algorithm using ward.D)
   to evaluate the difference in the concentration of each metabolite
   between sample groups. Clustered heat-maps allow easy visualisation of
   changing patterns in metabolite concentrations across sample groups and
   experimental conditions. The metabolite pattern of each biological
   replicate from three sample groups clustered, and individual clusters,
   were distinct, indicating the uniqueness of the metabolome profiles of
   the two helminths. Unlike score plots, heat-maps display the actual
   data values using carefully chosen colour gradients, as shown in
   [213]Figure 5, where blue bars indicate a low concentration and red bar
   denotes a high concentration. Based on the colour intensity pattern in
   the heat-map, adenine (purine nucleobase), adenosine (purine
   nucleoside), choline (amino acid), and hypoxanthine were notable
   features in the NB_SE. Amino acids such as l-tyrosine, l-valine,
   2-oxoglutarate, l-phenylalanine, and 4-hydroxybenzoate were prominent
   in NB_ESP. In TM_SE, l-2-aminoadepate, l-pipecolate, and
   N-acetylputrescine (amino acids), thymine (nucleic acid), and maleic
   acid (organic acid) were prominent.

Figure 5.

   [214]Figure 5
   [215]Open in a new tab

   Hierarchical clustering analysis (HCA) of the three samples (the
   somatic extract of infective third-stage larvae (L3) of N. brasiliensis
   (NB_SE), the excretory/secretory products (ESP) of N. brasiliensis L3
   (NB_ESP), and the somatic extract of T. muris embryonated eggs (TM_SE))
   showing discrimination between the sample types and differential
   abundances of 55 polar metabolites. Each column represents sample
   groups with their replicates, and each row represents the expression
   profile of a metabolite across sample groups. The scale bar represents
   the normalised intensity of metabolites, where blue indicates a
   decrease/low and red an increase/high. Media (QC_Media) and pooled
   quality control (QC_P) values were excluded from the analysis, as they
   were subtracted from the samples.

2.4. Lipidomics Analysis of the N. brasiliensis and T. muris Infective Stages

   After acquiring LC-MS data (mass and retention time) using open source
   software IDEOM, lipids were putatively identified by accurate mass
   within 3 ppm. We putatively identified (MSI level-2 identification) a
   total of 350 lipids ([216]Supplementary Table S2) in all three samples
   (332 in NB_SE, 256 in NB_ESP, and 283 in TM_SE), out of which 203
   lipids were common in all three sample groups. Of the vast array of
   lipids identified, glycerophospholipids such as phosphatidylcholine
   (PC) and phosphatidylethanolamine (PE) were predominant in NB_SE. In
   NB_ESP and TM_SE, glycerolipid triglycerides (TG) were present in
   higher intensities. Based on their intensity of peak areas,
   glycerophospholipid species phosphatidylcholine (PC) such as PC(38:6),
   PC(40:5), PC(40:7), PC(40:8), and PC(40:9) were the top five lipids in
   NB_SE. In NB_ESP and TM_SE, glycerolipid species such as triglyceride
   (TG) were dominant. TG(48:1), TG(45:0), TG(50:2), TG(47:1), and
   TG(52:2) were the top five lipids in NB_ESP and TG(30:0), TG(38:0),
   TG(38:1), TG(40:0), and TG(42:1) in TM_SE.

2.5. Lipidomic Pathways of Identified Lipids

   We mapped lipidomic pathways for putative lipids against known lipid
   pathways in lipid databases such as LIPID MAPS Lipidomics Gateway
   ([217]http://www.lipidmaps.org) and LipidBank
   ([218]http://www.lipidbank.jp). Most lipids in all three sample groups
   were the glycerophospholipid metabolism pathway products, followed by
   glycerolipid and fatty acyl metabolism ([219]Figure 6). Elements of
   glycerophospholipid metabolism such as phosphatidic acid (PA),
   phosphatidylethanolamine (PE), phosphatidylcholine (PC),
   phosphatidylserine (PS), and phosphatidylinositol (PI) were major lipid
   constituents in all three sample groups (NB_SE, NB_ESP, and TM_SE),
   followed by triacyl-glycerides (TG), the product of the glycerolipid
   pathway ([220]Supplementary Table S2).

Figure 6.

   [221]Figure 6
   [222]Open in a new tab

   Distribution of putative lipids by lipid classes: (A) in the somatic
   extract of infective third-stage larvae (L3) of N. brasiliensis
   (NB_SE); (B) in the excretory/secretory products (ESP) of N.
   brasiliensis L3 (NB_ESP); (C) in the somatic extract of T. muris
   embryonated eggs (TM_SE).

2.6. Chemometric Analysis of Putatively Identified Lipids

   We performed both univariate and multivariate chemometric analyses
   using Metaboanalyst 4.0 software, as described above for polar
   metabolites. For comparison of lipid profiles in the somatic extracts
   of the infective stages of the two helminths, univariate analysis
   (volcano plot analysis) between NB_SE and TM_SE yielded 204 significant
   features out of top 350 lipids (represented by pink dots in [223]Figure
   7). All features presented possessed values above a given count
   threshold (>75% of pairs/variable) and had a fold change of >2 and
   p-value of <0.05 ([224]Supplementary Table S3). When the NB_SE lipid
   profile was compared against TM_SE, 10 lipids in NB_SE and five lipids
   in TM_SE (labelled pink dots in [225]Figure 7) showed the most
   significant differences in their peak intensities (p < 0.05).

Figure 7.

   [226]Figure 7
   [227]Open in a new tab

   Volcano plot of the untargeted lipidomics analysis of differentially
   regulated features between the somatic extract of infective third-stage
   larvae (L3) of N. brasiliensis (NB_SE) and the somatic extract of T.
   muris eggs (TM_SE). The volcano plot displays log2 fold changes versus
   Benjamini–Hochberg adjusted p-values (-log10 transformed). Features
   that exhibited an absolute log[2] fold change > 2 and an absolute
   p-value < 0.05 are coloured in pink. Most significant features which
   could be structurally annotated are labelled with the corresponding
   name. Gray dots represent features that were not significantly altered
   (having fold change less than 2). The further its position away from
   the (0,0), the more significant the lipid is.

   A principal component analysis of the 350 putative lipids (listed in
   [228]Supplementary Table S2) showed clear segregation of the clusters
   among all three sample groups (NB_SE, NB_ESP, and TM_SE) as presented
   in [229]Figure 8. These results indicate a high level of heterogeneity
   in the lipidomes of the two helminths.

Figure 8.

   [230]Figure 8
   [231]Open in a new tab

   Principal component analysis scores plot comparing LC-MS metabolomic
   profiles for the lipids among three sample groups: the somatic extract
   of infective third-stage larvae (L3) of N. brasiliensis (NB_SE)
   (green), the excretory/secretory products of N. brasiliensis L3
   (NB_ESP) (red), and the somatic extract of T. muris embryonated eggs
   (TM_SE) (blue) with five replicates each. The amount of variance
   explained is shown in parentheses on each axis. Media (QC_Media) and
   pooled quality control (QC_P) values were excluded from the analysis,
   as they were subtracted from the samples.

   We evaluated the diversity of lipidomic patterns across the three
   samples by cluster heat-map analysis using Metaboanalyst 4.0
   statistical analysis package. The variation in the intensities of 350
   putative lipids between groups is shown in [232]Figure 9. The somatic
   extract of N. brasiliensis L3 contained a very high intensity of
   glycerophospholipids such as phosphatidylcholine (PC) and
   phosphatidylethanolamine (PE) species. Meanwhile, NB_ESP and TM_SE both
   contained glycerolipids such as triglycerides (TG) species in higher
   intensities. The [233]Supplementary Table S2 contains the individual
   intensity of all 350 lipids in each group.

Figure 9.

   [234]Figure 9
   [235]Open in a new tab

   Hierarchical clustering analysis (HCA) of the three samples (the
   somatic extract of infective third-stage larvae (L3) of N. brasiliensis
   (NB_SE), the excretory/secretory products (ESP) of N. brasiliensis L3
   (NB_ESP), and the somatic extract of T. muris embryonated eggs
   (TM_SE)), showing discrimination between the sample types and
   differential abundances of 350 putative lipids. Each row represents
   putative lipids, and each column represents the biological replicates
   of an individual sample group. The scale bar represents the normalised
   intensity of metabolites, where blue colour indicates low/decrease, and
   red colour indicates high/increase. Media (QC_Media) and pooled quality
   control (QC_P) values were excluded from the analysis, as they were
   subtracted from the samples. Note: Heat-map was purposefully generated
   on all 350 putative lipids to show the overall pattern between the
   sample groups. For specific lipid details with peak areas, refer to
   [236]Supplementary Table S2.

2.7. Common and Unique Metabolites in N. brasiliensis and T. muris Infective
Stages

   Out of 55 polar metabolites identified in this study, 21 were common to
   all three sample groups, mostly amino acids. Seven (7) polar
   metabolites were unique to the ESP of the N. brasiliensis infective
   stage ([237]Table 1 and [238]Figure 10A), and interestingly, two of
   them, namely isocitrate and 5-oxoproline/pyroglutamic acid, were also
   detected in their adult ESP [[239]21]. The remaining five, namely
   homogentisate, orotate, ll-2,6-Diaminoheptanedioate, pterin, and
   2,5-dihydroxybenzoate, were absent in the ESP as well as somatic
   extracts of both helminths. Meanwhile, out of 350 putative lipids
   identified from all three samples (NB_SE, NB_ESP, and TM_SE), 203
   lipids were common to all three sample groups (NB_SE, NB_ESP, and
   TM_SE). A total of 62 lipids were common between NB_SE and TM_SE, 39
   lipids between NB_SE and NB_ESP, and 14 lipids between NB_ESP and
   TM_SE. Twenty-eight (28) lipids were unique to NB_SE and four lipids to
   TM_SE ([240]Table 2 and [241]Figure 10B).

Figure 10.

   [242]Figure 10
   [243]Open in a new tab

   Distribution of common and unique metabolites among the three sample
   groups: the somatic extract of infective third-stage larvae (L3) of N.
   brasiliensis (NB_SE), the excretory/secretory products (ESP) of N.
   brasiliensis L3 (NB_ESP), and the somatic extract of T. muris
   embryonated eggs (TM_SE). (A) MSI-I identified polar metabolites; (B)
   MSI-II identified lipids.

Table 2.

   Putative lipids unique to infective stages of N. brasiliensis (L3) and
   T. muris (embryonated eggs).
   Putative Lipids Formula ^a Mass (m/z) Rt (min) ^c Chemical Taxonomy ^b
   LipidMAPS ID ^d Peak Areas (mz/rt)
   NB_SE NB_ESP TM_SE
   DG(41:7) C[44]H[72]O[5] 680.536 16.05 Glycerolipids LMGL02010545
   124,404 0 0
   FA hydroxy(12:0) dodecanoic acid C[12]H[24]O[3] 238.155 2.19 Fatty
   Acyls NA 0 0 51,014
   LacCer(d38:0) C[50]H[97]NO[13] 919.696 18.98 Sphingolipids LMSP05010122
   139,285 0 0
   LacCer(d40:0) C[52]H[101]NO[13] 947.727 20.22 Sphingolipids
   LMSP05010124 182,175 0 0
   LysoPE(22:2) C[27]H[52]NO[7]P 533.350 4.84 Glycerophospholipids
   LMGP02050024 136,295 0 0
   PE-Cer(d40:1) C[42]H[85]N[2]O[7]P 760.610 13.49 Sphingolipids
   LMSP03020086 117,114 0 0
   PE-Cer(d38:1) C[40]H[81]N[2]O[7]P 732.579 12.26 Sphingolipids
   LMSP03020064 112,168 0 0
   PA(25:0) C[28]H[55]O[8]P 550.364 12.05 Glycerophospholipids
   LMGP10010001 0 0 374,621
   PA(26:0) C[29]H[57]O[8]P 564.379 12.58 Glycerophospholipids
   LMGP10010980 0 0 1,921,006
   PC(36:7) C[44]H[74]NO[8]P 775.514 10.73 Glycerophospholipids
   LMGP01012100 105,372 0 0
   PC(P-32:2) C[40]H[76]NO[7]P 713.536 13.07 Glycerophospholipids NA
   657,868 0 0
   PC(P-36:2) C[44]H[84]NO[7]P 769.599 14.95 Glycerophospholipids
   LMGP01030137 138,297 0 0
   PE(28:2) C[33]H[62]NO[8]P 631.422 7.68 Glycerophospholipids
   LMGP02011238 127,324 0 0
   PE(48:2) C[53]H[102]NO[8]P 911.734 20.75 Glycerophospholipids
   LMGP02010893 466,471 0 0
   PE(40:5) C[45]H[80]NO[8]P 815.542 12.76 Glycerophospholipids
   LMGP02010893 483,243 0 0
   PE(48:1) C[53]H[104]NO[8]P 913.751 21.37 Glycerophospholipids NA
   1,154,195 0 0
   PE(O-20:0) C[25]H[54]NO[6]P 495.370 7.71 Glycerophospholipids
   LMGP02060005 108,008 0 0
   PE(P-20:0) C[25]H[52]NO[6]P 493.354 7.86 Glycerophospholipids
   LMGP02070004 375,557 0 0
   PE(P-36:4) C[41]H[74]NO[7]P 723.521 12.51 Glycerophospholipids
   LMGP02030093 195,178 0 0
   PE(P-36:5) C[41]H[72]NO[7]P 721.504 11.93 Glycerophospholipids
   LMGP02030028 87,036 0 0
   PE(P-38:6) C[43]H[74]NO[7]P 747.519 12.36 Glycerophospholipids
   LMGP02030001 308,137 0 0
   PG(36:1) C[42]H[81]O[10]P 776.557 11.98 Glycerophospholipids
   LMGP04010037 699,995 0 0
   PI(37:6) C[46]H[77]O[13]P 868.512 9.51 Glycerophospholipids
   LMGP06010790 119,218 0 0
   PI(38:7) C[47]H[77]O[13]P 880.512 9.44 Glycerophospholipids
   LMGP06010792 168,877 0 0
   PI(P-37:2) C[46]H[85]O[12]P 860.576 11.78 Glycerophospholipids
   LMGP06030067 94,153 0 0
   PS(28:2) C[34]H[62]NO[10]P 675.412 6.97 Glycerophospholipids
   LMGP03010919 253,811 0 0
   PS(36:4) C[42]H[74]NO[10]P 783.506 10.48 Glycerophospholipids
   LMGP03010038 141,937 0 0
   PS(36:5) C[42]H[72]NO[10]P 781.491 10.06 Glycerophospholipids
   LMGP03010654 115,385 0 0
   PS(O-38:0) C[44]H[88]NO[9]P 805.621 15.40 Glycerophospholipids
   LMGP03020051 86,582 0 0
   PS(O-34:0) C[40]H[80]NO[9]P 749.558 12.46 Glycerophospholipids
   LMGP03020043 0 0 1883
   SM(d41:2) C[46]H[91]N[2]O[6]P 844.667 14.55 Sphingolipids LMSP03010074
   29,023 0 0
   SM(d42:2) C[47]H[93]N[2]O[6]P 812.676 15.24 Sphingolipids LMSP03010007
   78,211 0 0
   [244]Open in a new tab

   ^a Formula; ^b Chemical taxonomy = Formula and chemical taxonomy for
   compounds were taken from LipidMAPS database
   ([245]https://www.LipidMAPS.org); ^c Rt = retention time in minutes; ^d
   LIPIDMAPS ID ([246]https://www.LipidMAPS.org) contains information on
   biosynthetic and metabolic pathways of identified lipids;
   Abbreviations: ID = identity; NB_SE = the somatic extract of infective
   third-stage larvae (L3) of N. brasiliensis; NB_ESP = the
   excretory/secretory products (ESP) of N. brasiliensis L3; TM_SE = the
   somatic extract of T. muris embryonated eggs.

2.8. Reported Pharmacological Activities of Identified Compounds

   A comprehensive literature search revealed that 42 out of the 55 polar
   metabolites were associated with various pharmacological activities. Of
   the 42 polar metabolites, 31 metabolites possess anti-inflammatory or
   antioxidant properties ([247]Table 1). For example, polar metabolites
   such as l-valine (prominent in N. brasiliensis ESP), adenine, choline,
   inosine, adenosine, hypoxanthine (prominent features in N. brasiliensis
   somatic extract), and l-tryptophan (prominent feature in T. muris eggs
   somatic extract) possess anti-inflammatory activity. A few of the
   detected metabolites were unique to the extracts, such as homogentisate
   in the N. brasiliensis ESP and maleic acid in T. muris eggs
   (embryonated) somatic extract, both (compounds) known for their
   inflammatory and cytotoxic properties, respectively. Unlike polar
   metabolites, non-polar metabolites or lipids in helminths are the least
   studied for biological activities. There is either limited or no record
   of studies for all putative lipids identified in this study.

3. Discussion

   Parasites have co-evolved with humans for millennia and produce ESPs
   that allow them to navigate through circuitous pathways (for hookworm
   at least) to reach the gut, where they survive for a prolonged period.
   STHs are masterful at modulating the host’s immune response to avoid
   elimination from the body and facilitate the establishment of chronic
   infection [[248]92]. Their ESP contain a plethora of biomolecules,
   including proteins, peptides, lipids, and other small molecules
   [[249]21]. However, small molecules remain less-studied, especially
   those produced by the infective stages. In this study, we show the
   metabolomes of the infective stages of N. brasiliensis and T. muris
   using untargeted LC-MS for the first time. Our results also provide new
   insights into comparative metabolome profiles of different
   developmental stages of the helminths. LC-MS is a preferred analytical
   technique for metabolome profiling [[250]93,[251]94,[252]95],
   especially when employing high-resolution accurate mass (HRAM)
   detection. In this study, we applied HRAM mass spectrometer-Q-Exactive
   (Thermo Scientific) to detect both polar metabolites and lipids.

   Helminths are known to produce stage-specific metabolites [[253]21].
   Although both adult and infective stages of N. brasiliensis had many
   fatty acids in common, interestingly, 44 of the polar metabolites
   detected in the infective stage of N. brasiliensis were not detected in
   the ESP of their adult stage [[254]21]. Such differences in the
   metabolic profiles could have resulted partially due to the different
   experimental conditions and analytical techniques used in two separate
   studies. Nevertheless, such marked differences in the metabolic
   profiles of two different life-cycle stages of N. brasiliensis suggest
   that there could be major metabolic changes accompanying the transition
   from one life-cycle stage to the next in parasitic helminths. Moreover,
   according to Barrett [[255]96], there are variations in the
   distribution pattern and activity of enzymes and metabolite levels
   among the different developmental stages of helminths. For instance,
   adult and larval stages of A. lumbricoides and S. mansoni possess a
   marked difference in the isoenzyme patterns. When we compared
   metabolites identified from the somatic extracts of N. brasiliensis and
   T. muris infective stages, the percentage similarity for both polar and
   non-polar metabolites was quite low (34.5% and 18%, respectively)
   despite sharing some common metabolites such as l-citrulline,
   l-threonine, deoxyadenosine, and pyridoxal. Metabolites such as
   inosine, hypoxanthine, and l-pipecolate were comparatively higher in
   NB_SE and were also reported significantly in high levels in the blood
   plasma samples of patients suffering from onchocerciasis (caused by
   nematode Onchocerca volvulus) [[256]97]. The lower percentage
   similarity of metabolites in the somatic extracts of the two helminths
   could have resulted from the different conditions they were exposed to,
   as N. brasiliensis L3 were incubated at 26 °C with activated charcoal
   and later at 37 °C, 5% CO[2] in glutamax/Phosphate-buffered saline
   (PBS) media. At the same time, T. muris eggs were kept in PBS at room
   temperature. Thus, suggesting that the different environmental niches
   in which they survive—for instance, the L3 of human hookworms N.
   americanus and A. duodenale remain outdoors, while their adult stage
   dwells inside the host gut [[257]4]—could potentially influence the
   types as well as the level of metabolites produced. However, the
   effects of CO[2] tensions and temperature on the overall metabolic
   pathways are either complex or difficult to predict [[258]96].

   More than half of the metabolites identified in all sample groups were
   amino acids. l-citrulline, l-methionine, N(pi)-Methyl-l-histidine,
   succinate, and 4-hydroxybenzoate were the top five most abundant
   metabolites (based on LC-MS peak intensity) in the somatic extract and
   excretory/secretory products of N. brasiliensis L3. Succinate, one of
   the amino acids detected in the current study samples, is known to be
   produced by the intestinal microbiota to induce intestinal tuft cells
   to trigger T-helper cell type 2 (T[H]2) responses [[259]98]. Somatic
   and ESP of the adult stage of N. brasiliensis also contained succinate
   [[260]21]. In the T. muris embryonated eggs, l-2-aminoadepate,
   N-acetylputrescine, l-pipecolate, thymine, and maleic acid were present
   in the highest intensities. Betaine is another amino acid detected in
   the infective stage of N. brasiliensis but not in their adult stage
   [[261]21]. Betaine (a derivative of amino acid glycine with three
   methyl groups), present in microorganisms, plants and animals, is known
   to function as an osmolyte in their cells [[262]99]. The plasma and
   spleen of rats infected with the liver fluke Fasciola hepatica also
   contained a high level of betaine [[263]100]. Another metabolite that
   was prominent in both N. brasiliensis L3 ESP and T. muris embryonated
   eggs was l-glutamate. The dormant infective eggs of other helminths
   such as A. lumbricoides also contained l-glutamate [[264]101]. The
   relevance of betaine and l-glutamate (and glutamine) in this study
   should be interpreted with caution, as it is possible that samples were
   contaminated by the sulfobetaine polymer (PSB) used for the somatic
   extract preparation and the glutamax used in the media. Pyroglutamic
   acid (also called 5-oxoproline), reported as one of the major
   metabolites in the somatic extracts of the adult stage of N.
   brasiliensis and T. muris [[265]21], was a common metabolite in the
   infective stages of two helminths. Somatic extract of adult Ancylostoma
   caninum (dog hookworm) also contained pyroglutamic acid as one of the
   major metabolite constituents [[266]102]. Pyroglutamic acid is formed
   as an intermediate product by the enzyme γ-glutamylcyclotransferase in
   glutathione metabolism, and it is ultimately converted into l-glutamic
   acid by 5-oxoprolinase [[267]103]. It is also known to be produced due
   to disordered glutathione metabolism [[268]104] and usually tends to
   accumulate abnormally in the case of metabolic acidosis.

   Another exciting difference among the samples from two helminths is the
   presence of unique metabolites. Seven polar metabolites, namely
   orotate, pterin, 2,5-dihydroxybenzoate, ll-2,6-Diaminoheptanedioate,
   isocitrate, 5-oxoproline, and homogentisate, were found unique to the
   ESP of N. brasiliensis. Out of these seven, only isocitrate and
   5-oxoproline were reported in their adult ESP [[269]21]. In mammals,
   orotate (orotic acid) is released by mitochondrial dihydroorotate
   dehydrogenase for conversion to uridine monophosphate during pyrimidine
   metabolism [[270]105]. Moreover, five main enzymes involved in
   pyrimidine metabolism are present in many helminths, including N.
   brasiliensis and T. muris [[271]106], indicating that de novo
   pyrimidine biosynthesis could be the main source of orotate in
   helminths. Pterins are found in all living organisms starting from tiny
   bacteria to mammals and serve as a urine biomarker for
   hyperphenylalaninaemia [[272]107]. Homogentisate is the central
   intermediate product in the catabolism of phenylalanine and tyrosine
   [[273]108]. Whilst we have not ruled out the possibility that this
   feature could be another isomer of dihydroxyphenylacetate, it is most
   likely an intermediate in tyrosine/phenylalanine metabolism. Tyrosine
   catabolism is considered as a critical metabolic pathway in Rhodnius
   prolixus, a blood-sucking insect vector of Trypanosoma cruzi, because
   R. prolixus dies after a blood meal if the pathway is disturbed by
   silencing two critical enzymes, tyrosine aminotransferase and
   4-hydroxyphenylpyruvate dioxygenase [[274]109]. Thus, the presence of
   phenylalanine and tyrosine in N. brasiliensis L3 ESP could mean that
   tyrosine catabolism may be important for the survival of N.
   brasiliensis L3 when establishing a successful infection. Homogentisate
   also possesses antioxidant activity higher than α-tocopherol and
   moderate anti-inflammatory activity [[275]110]. Thus, the capacity of
   ESP from many helminths, including N. brasiliensis, to reduce T-cell
   proliferation [[276]111] and confer protection against T-cell mediated
   immunopathology in a mouse model of colitis [[277]102], are
   attributable to the presence of such metabolites.

   Identifying different metabolic pathways within a species is considered
   necessary, mainly to understand any malfunctions or alterations that
   may occur during disease state [[278]112]. Similarly, identifying
   metabolic pathways in soil-transmitted helminths might reveal a unique
   metabolic pathway(s) critical in the infection process and, therefore,
   present as drug targets. The metabolic pathway analysis revealed that
   the majority of the metabolic pathways are associated with amino acid
   metabolism, a finding that aligns with earlier metabolic profiling of
   the ESP from adult stage STHs [[279]21], suggesting that both the
   infective and adult stages share similar (amino acid and carbohydrate)
   metabolic pathways. Polar metabolites identified from the infective
   stages of both helminths mostly belonged to common amino acid pathways
   such as aminoacyl-tRNA biosynthesis, arginine biosynthesis, lysine
   degradation, alanine, aspartate, and glutamate metabolism. Amino acids,
   such as leucine, lysine, and phenylalanine, are detected significantly
   in high concentrations in the herbivorous youngstock acutely infected
   with helminths [[280]113]. l-arginine is known to enhance intestinal
   mucosa function by reducing tissue damage in intestinal ischemia of
   animal models [[281]114,[282]115]. We also found purine, glyoxylate,
   and dicarboxylate metabolism as major pathways following amino acid
   pathways. Glyoxylate metabolism, which is characteristic of free-living
   parasitic nematodes (but not in the adult) and catalyses the conversion
   of lipids into carbohydrates [[283]96], could be the source of
   isocitrate (organic acid), which was one of the unique metabolites in
   the N. brasiliensis ESP.

   Lipids and fatty acids are also known to play a crucial role in the
   maturation and completion of different life-cycle stages of helminths
   and host–parasite interaction [[284]23]. Generally, the parasitic stage
   of helminths utilise lipids for the long-term adaptation inside their
   host and completion of the life-cycle [[285]116]. Lipids are also
   involved in essential biological processes such as apoptosis, cell
   proliferation, angiogenesis, immunity, and inflammation [[286]117].
   Fatty acids, including the cis-form of octadecenoic acid and other
   branched-chain and monoenoic acids (oleic acids and vaccenic acid), are
   known to play a vital role in helminth infections by altering the
   physical properties of the host cell membrane and ultimately causing it
   to rupture [[287]118]. Thus, we presume that fatty acids, such as
   octadecanoic acid detected in the ESP of N. brasiliensis L3, might be
   playing a key role during the process of host invasion and infection.
   Fatty acids were also reported in other nematodes such as Haemonchus
   contortus (in all stages of life-cycle) [[288]119] and adult
   Caenorhabditis elegans [[289]120], and in both studies, fatty acids
   with 18 carbons (i.e., 18:1, 18:2, and 18:3) were commonest. We also
   obtained a similar result, where ~7% of total lipids were fatty acids,
   and the above-mentioned fatty acids with 18 carbons were present in all
   three samples (i.e., NB_SE, NB_ESP, and TM_SE). Nematodes regulate the
   saturation levels of fatty acids while adapting to the changing
   environmental temperature [[290]121], but the saturation level varies
   among different nematodes. For example, in C. elegans, the saturated
   fatty acid level increases with increasing temperature [[291]122]. In
   contrast, Wang et al. [[292]119] observed the opposite in H. contortus,
   where the level of fatty acid saturation tended to decrease as they
   transitioned from their free-living stage to the parasitic stage.

   Glycerophospholipids and glycerolipids were the major lipid groups in
   all three sample groups (NB_SE, NB_ESP, and TM_SE), constituting
   approximately 83% of the total lipids. This is in congruence with
   lipidomic studies in the muscle-stage larvae of Trichinella papuae
   (~63% glycerophospholipids) [[293]123] and in the H. contortus (all
   life-cycle stages), where more than 90% of the total lipids were
   glycerophospholipids and glycerolipids [[294]119]. The somatic extract
   of N. brasiliensis L3 contained elements of glycerophospholipids such
   as phosphatidylcholine, PC(38:6), PC(40:8), PC(40:7), PC(40:9), and
   PC(46:5). Wewer et al. [[295]124] also reported PC as dominant lipid
   constituents in filarial nematodes Onchocerca volvulus, O. ochengi, and
   Litomosoides sigmodontis but they have studied only their adult stage.
   Meanwhile, glycerolpids such as TG(45:0), TG(48:1), TG(50:2), TG(30:0),
   TG(38:0), and TG(38:1) were dominant in the excretory/secretory product
   of N. brasiliensis L3 and somatic extract of T. muris embryonated eggs.
   TG constituted major lipids (80.9% of total 327 lipids) identified from
   the somatic extract of the L3 stage of H. contortus and it decreases as
   they mature into L4 stage [[296]119]. Lee [[297]125] also reported
   triglycerides as a major lipid constituent in the adult tissue of N.
   brasiliensis, which was presumed to be due to reduced lipid metabolism
   under anaerobic conditions inside their host. Triglyceride is the major
   neutral lipid in the majority of helminths [[298]126]. According to
   Ward [[299]118], unlike mammals, helminths are capable of storing a
   large amount of energy in the form of glycerolipids as triglycerides or
   triacylglycerides have more energy content (9 kcal/g) compared to
   carbohydrates (4 kcal/g). Thus, it is likely that infective stages of
   both helminths N. brasiliensis L3 and T. muris embryonated eggs store
   energy in the form of triglycerides for their later developmental
   stages of the life-cycle. Our findings complement a similar study where
   the Schistosoma mansoni infective stage (cercariae) had a unique lipid
   profile compared to other stages of the life-cycle [[300]23].
   Interestingly, although S. mansoni belongs to a different phylum
   (Platyhelminthes), it shares some behavioural aspects with N.
   brasiliensis—notably, the penetration of host skin by the infective
   stage larvae. Although we could not retrieve literature on
   pharmacological activities of specific triglycerides identified in this
   study, a high level of triglycerides was associated with inflammation
   and inflammation-related disorders [[301]127]. T. muris embryonated
   eggs showed enrichment of species of phosphatidic acids (PA), out of
   which PA(25:0) and PA(26:0) were completely absent in the infective
   stage L3 of N. brasiliensis. Phosphatidic acid is a lipid of interest
   as a vital signalling molecule and a central intermediate in the
   synthesis of membrane lipids and storage lipids [[302]128,[303]129].
   Phosphatidic acid, such as lysoPA (C14:2), was also detected in the
   surface coat of the infective stage of the parasitic nematode
   Trichinella spiralis [[304]130]. Other species of helminths such as
   Hymenolepis diminuta, A. lumbricoides, Dirofilaria immitis, and Setaria
   cervi also contained phosphatidic acids [[305]126].
   Phosphatidylcholines (PC) reported to be produced by all life-cycle
   stages of parasitic trematode [[306]23] were also detected in both
   helminths studied here. PC lipids are associated with the maintenance
   of gastrointestinal mucus barrier function [[307]131], besides their
   good anti-inflammatory properties [[308]132,[309]133]. PC lipids such
   as PC(36:7), PC(P-32:2), and PC(P-36:2) were unique to N. brasiliensis
   L3 somatic extract.

4. Materials and Methods

4.1. Ethics, Source, and Housing of Mice and Rats

   Mice strain B10.Br (5 weeks old, 5 mice per cage) and rat strain
   Sprague–Dawley (2 weeks old, 2 rats per cage) were purchased from
   Animal Resources Centre (Perth, Australia). All experiments using these
   animals were approved by the animal ethics committee of James Cook
   University (JCU), Cairns, Australia (animal ethics number: A2647). Mice
   and rats were kept in the JCU animal facility centre in compliance with
   JCU approved protocols, Australian Code of Practice for the Care and
   Use of Animals for Scientific Purposes, (7th edition, 2007), and the
   Queensland Animal Care and Protection Act 2001. The animal facility
   room had an ambient temperature (20–22 °C) and humidity (60%), and
   animals were exposed to a 12 h day/night cycle and fed irradiated
   mouse/rat chow (Specialty Feeds, Glen Forrest, Western Australia) and
   autoclaved tap water ad libitum.

4.2. Collection of N. brasiliensis L3 and Its ESP

   Sprague–Dawley rats were infected with N. brasiliensis L3 (~3000
   larvae) by subcutaneous injection and sacrificed on day seven
   post-infection [[310]134]. We collected faecal pellets on day five and
   six post-infection. Subsequently, faecal pellets were cultured with
   activated charcoal—untreated, granular, 8–20 mesh (Sigma-Aldrich, New
   South Wales, Australia). The culture plates were sealed inside an
   airtight plastic container in an incubator (Binder, model: BD 115
   #02-040007) at 26 °C for one week. After one-week incubation, L3 were
   harvested, washed with warmed 5× pen/strep PBS, and then transferred to
   a 12-well culture plate (1500 worms per well) containing warmed 2 mL 5×
   glutamax, 2× pen/strep PBS media. Plates were incubated in a CO[2]
   incubator (Sanyo MCO-18AIC CO[2] incubator, SANYO Electric Co., Ltd.,
   Moriguchi, Japan) at 37 °C supplied with 5% CO[2]. The supernatant
   (ESP) was collected and replaced with fresh media twice daily (09:00
   and 17:00) for four consecutive days. The supernatants were centrifuged
   at 3000× g for 30 min, and then aliquot was transferred to Amicon^®
   Ultra-15, centrifugal 10 kDa filters (Merck Millipore, Victoria,
   Australia), and centrifuged at 4000× g for 20 min. Concentrated ESP
   filtrate containing small molecules (<10 kDa) was collected and stored
   at −80 °C until further analysis.

4.3. Collection of Eggs (Infective Stage) from T. muris Adult Worms

   B10.Br mice were infected with T. muris embryonated eggs (200 μL of PBS
   containing ~200 eggs) through oral gavage. We euthanised mice with
   CO[2]on day 30–33 post-infection to harvest adult T. muris for egg
   collection. Caecum was collected, split longitudinally, and washed in
   warmed 5× pen/strep in PBS. Adult worms were carefully pulled from the
   caecum of mice with fine forceps and washed with 5x pen/strep in PBS,
   and then transferred to 6-well culture plates (~100 worms per well)
   containing 4 mL of warmed 5× glutamax, 2× pen/strep in PBS media. Worms
   were incubated in a CO[2] incubator at 37 °C in 5% CO[2]. For egg
   collection, we replaced culture media with fresh media twice daily as
   described above for three consecutive days. The supernatant was
   collected twice daily and centrifuged at 3000× g for 30 min. Eggs from
   adult T. muris worms were resuspended in 40 mL milli-Q water and
   filtered through a 100 μm nylon sieve before transferring to a fresh
   cell culture tube. Finally, for eggs to become embryonated, eggs were
   kept in the sterile PBS and stored in the dark at room temperature for
   approximately six weeks, and then stored at 4 °C.

4.4. Somatic Extract Preparation

   Somatic extracts of N. brasiliensis L3 (five biological replicates
   named as NB_SE_1, NB_SE_2, NB_SE_3, NB_SE_4, and NB_SE_5, each
   replicate containing ~17000 L3) and T. muris embryonated eggs (five
   biological replicates named as TM_SE_1, TM_SE_2, TM_SE_3, TM_SE_4, and
   TM_SE_5, each replicate containing ~17000 eggs) were suspended in 1 mL
   chilled sulfobetaine polymer (PSB) and centrifuged at 1000× g for 5 min
   at 4 °C. The supernatant was discarded, and the remaining solid was
   resuspended in 250 µL of chilled extraction solvent
   (CHCl[3]:MeOH:H[2]O, 1:3:1, v/v) containing 1 µM of internal standards
   3-(cyclohexyl amino)-1-propane sulfonic acid (CAPS),
   3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS),
   piperazine-N,N’-bis(2-ethane sulfonic acid (PIPES), and
   Tris(hydroxylmethyl)amino-methane (Tris). A blank sample containing
   water instead of a tissue pellet was extracted simultaneously as a
   control. After three freeze-thaw cycles, the samples were mixed
   thoroughly for 30 min at 4 °C. The samples were centrifuged at 14,800×
   g for 10 min at 4 °C. Supernatant (100 µL) was transferred to the vials
   for metabolomics analysis and analysed on the same day. Another 100 µL
   was transferred to microfuge tubes for lipidomics analysis. The solvent
   was evaporated using a centrifugal evaporator at 55 °C for 50 min.
   Dried extracts were frozen at −80 °C until LC-MS analysis was
   performed. On the day of analysis, the samples were dissolved in 80 µL
   of BuOH:MeOH:H[2]O (4.5:4.5:1, v/v). The samples were shaken for 30 min
   at room temperature and sonicated for 1 h while maintaining the
   temperature below 25 °C. The samples were centrifuged at 14,800× g for
   10 min at 20 °C, and then 70 µL was transferred to LC-MS vials.

4.5. ESP Extract Preparation

   ESP from N. brasiliensis L3 was thawed on ice and 100 µL aliquots of
   ESP (five biological replicates named as NB_ESP_1, NB_ESP_2, NB_ESP_3,
   NB_ESP_4, and NB_ESP_5) were transferred to microfuge tubes kept on
   ice. Subsequently, 400 µL of extraction solvent (CHCl[3]:MeOH, 1:3,
   v/v) containing 1 µM of internal standards CAPS, CHAPS, and PIPES was
   added to each replicate. The mixture was shaken at 4 °C for 30 min and
   centrifuged at 14,800× g for 10 min at 4 °C. Then, 100 µL of
   supernatant was transferred to the vials for metabolomic analysis and
   10 µL was combined to make a pooled quality control (QC) sample.

   For the lipidomics analysis, 240 µL of supernatant was transferred to
   microfuge tubes and evaporated at 20 °C under a stream of nitrogen and
   stored at −80 °C. On the day of analysis, the samples were dissolved in
   80 µL of BuOH/MeOH/H[2]O (4.5:4.5:1, v/v), shaken for 30 min at room
   temperature, and sonicated for 1 h, keeping the temperature below 25
   °C. The samples were centrifuged at 14,800× g for 10 min at 20 °C and
   then 70 µL of the sample was transferred to LC-MS vials and the
   leftover was combined to make a pooled QC sample.

4.6. Metabolomics LC-MS Data Acquisition

   LC-MS data were acquired on a Q-Exactive Orbitrap mass spectrometer
   (Thermo Fischer Scientific, Waltham, MA, USA) coupled with
   high-performance liquid chromatography (HPLC) system Dionex Ultimate^®
   3000 RS (Thermo Scientific, Waltham, MA, USA) [[311]135] as outlined in
   the [312]Figure 1. The samples were analysed as a single batch to avoid
   batch-to-batch variation and randomised to account for the LC-MS system
   drift over time. Chromatographic separation was performed on ZIC-pHILIC
   column (5 µm, 4.6 × 150 mm, SeQuant^®, Merck) (Merck Millipore,
   Victoria, Australia) equipped with a guard (ZIC-pHILIC). The mobile
   phase (A) was 20 mM ammonium carbonate (Sigma Aldrich, New South Wales,
   Australia), and (B) acetonitrile (Thermo Fischer Scientific, Melbourne,
   Australia). The needle wash solution was 50% isopropanol. The gradient
   program started at 80% B and was decreased to 50% B over 15 min, then
   to 5% B until 18 min, kept at 5% B until 21 min, returned to 80% B by
   24 min, and equilibrated at 80% B to 32 min. The flow rate was 0.3
   mL.min^−1 and column compartment temperature was 25 °C. The total run
   time was 32 min, with an injection volume of 10 µL. The mass
   spectrometer was operated in full scan mode with positive and negative
   polarity switching at 35 k resolution at 200 m/z with a detection range
   of 85 to 1275 m/z, AGC target 1 × 10^6 ions, maximum injection time 50
   ms. Heated electro-spray ionisation source (HESI) was set to 4.0 kV
   voltage for positive and negative mode, and sheath gas was set to 50,
   aux gas to 20, and sweep gas to 2 arbitrary units (AU), capillary
   temperature was 300 °C, and probe heater temperature was 120 °C.

4.7. Lipidomics LC-MS Data Acquisition

   Chromatographic separation was performed on an Agilent Zorbax C8 (1.8
   µm, 2.1 × 100 mm, Agilent Technologies, Victoria, Australia) equipped
   with a guard column (C8, 2 × 2 mm, Phenomenex, New South Wales,
   Australia) [[313]136]. The mobile phase (A) was 40% isopropanol, 8 mM
   ammonium formate, and 2 mM formic acid, and (B) 98% isopropanol, 8 mM
   ammonium formate and 2 mM formic acid, and needle wash solution was 50%
   isopropanol. The gradient program started at 0% B and was increased
   stepwise to 20% B over 1.5 min, to 28% B over 5.5 min, to 35% B over 1
   min, to 65% B over 16 min, and 100% B over 1 min. Wash at 100% B was
   continued for 2 min before decreasing to 0% B over the next 2 min,
   followed by equilibration at 0% B for 1 min. The flow rate was 0.2
   mL.min^−1 and column compartment temperature was 40 °C. The total run
   time was 30 min, with an injection volume of 10 µL. The mass
   spectrometer was operated in full scan mode with positive and negative
   polarity switching at 70 k resolution at 200 m/z with a detection range
   of 140 to 1300 m/z, AGC target 1 × 10^6 ions, maximum injection time 50
   ms. Heated electro-spray ionisation source (HESI) was set to 3.5 kV for
   both positive mode and negative modes, and sheath gas was set to 34 AU,
   auxiliary gas to 13 AU, and sweep gas to 1 AU. Capillary and probe
   heater temperatures were set to 250 and 190 °C, respectively.

4.8. Data Processing Using IDEOM

   The acquired LC-MS data were processed in untargeted fashion using open
   source software IDEOM [[314]137], which was initially used ProteoWizard
   to convert raw LC-MS files to mzXML format and XCMS to pick peaks.
   Mzmatch.R was used to convert to peakML files [[315]138], and for
   sample alignment and the filtering of peaks using a minimum detectable
   intensity of 100,000, relative standard deviation (RSD) of <0.5
   (reproducibility), and peak shape (codadw) of >0.8. Mzmatch was also
   used to retrieve missing peaks and annotate related peaks. Default
   IDEOM parameters were used to eliminate unwanted noise and artefact
   peaks. The loss or gain of a proton was corrected in negative and
   positive ESI mode, respectively, followed by putative identification of
   metabolites by accurate mass within 3 ppm mass error by searching
   against the Kyoto Encyclopedia of Genes and Genomes (KEGG), MetaCyc,
   and LIPIDMAPS databases. Additional manual curation was performed to
   remove putative lipids that did not elute at the expected retention
   time.

4.9. Data Analyses and Statistical Interpretation

   We performed chemometric univariate and multivariate statistical
   analyses using the Metaboanalyst website
   ([316]http://www.metaboanalyst.ca) [[317]139]. Before chemometric
   univariate and multivariate statistical analyses, data integrity was
   checked and filtered to ensure all data had been included. The sample
   data (spectral data) were normalised, log transformed, and auto-scaled
   before analysis. For univariate analysis, volcano plot analysis was
   performed to identify differential metabolites using the t-test and
   fold-change (FC) methods, and plots log2 (fold-change > 2) on the
   X-axis against −log10 (p-value) from the t-test on the Y-axis.
   Benjamini–Hochberg correction or false discovery rate (FDR) was applied
   to compute the number of false positives out of significantly varied
   metabolic features.

   In multivariate analyses, we performed principal component analysis
   (PCA) unsupervised method and hierarchical clustering analysis (HCA)
   with Euclidean measured distance, and the ward.D clustering algorithm
   was used to evaluate the difference in the concentration of each
   metabolite between sample groups.

   Before the pathway analysis, IDs for the metabolites were obtained from
   the KEGG, LipidMAPS, PubChem Compound ID (PubChem CID), Human
   Metabolome Database (HMDB), and the Chemical Translation service (CTS;
   [318]https://cts.fiehnlab.ucdavis.edu). Subsequently, we performed
   pathway enrichment analysis using the Metaboanalyst website
   ([319]http://www.metaboanalyst.ca) [[320]139].

4.10. Literature Review on Pharmacological Properties of Identified
Metabolites

   We conducted a comprehensive literature search for the pharmacological
   properties of metabolites identified in this study using various search
   engines, including PubMed, Medline, Google Scholar, and SciFinder
   Scholar. Keywords such as “anti-inflammatory,” “bioactivity,”
   “pharmacological activity,” and “anti-oxidant activity” were used to
   identify reported pharmacological activities of metabolites.

5. Conclusions

   In summary, we show that the infective stages of two different STHs
   produce characteristic metabolites. The current study identified many
   unique metabolites (both polar as well as non-polar metabolites)
   present in the infective stages of N. brasiliensis (seven unique polar
   metabolites and 28 lipids in the somatic extract) and T. muris eggs
   (four unique lipids). Future studies should further characterise their
   identity and bioactivity in more detail. The vast array of metabolites
   identified from these two helminths’ infective stages could potentially
   serve as a database for the in-depth understanding of helminth
   biochemistry, which is currently lacking. Moreover, the suite of
   metabolites identified in this study presents a potential avenue for
   future research, particularly for the development of metabolite-based
   diagnosis tools and the identification of novel targets for
   anthelmintic drugs.

Supplementary Materials

   The following are available online at
   [321]https://www.mdpi.com/2218-1989/10/11/446/s1, Table S1: Total
   putative polar metabolites identified in the infective stages of
   Nippostrongylus brasiliensis and Trichuris muris, Table S2: Total
   putative lipids identified in the infective stages of Nippostrongylus
   brasiliensis and Trichuris muris, Table S3: Fold change analysis of
   putative lipids between the infective stage of N. brasiliensis somatic
   extract (NB_SE) and T. muris embryonated egg extract (TM_SE).
   [322]Click here for additional data file.^ (13.7MB, zip)

Author Contributions

   Conceptualisation, P.W. and A.L.; methodology, P.W., L.B. and K.Y.;
   software, D.A., K.Y. and P.W.; validation, D.J.C. and D.A.; formal
   analysis, D.A., D.J.C., K.Y. and P.W.; investigation, D.A. and D.J.C.;
   resources, A.L., and P.W.; data curation, D.A., and D.J.C.;
   writing—original draft preparation, K.Y.; writing—review and editing,
   P.W., A.L., L.B., E.R. and D.J.C.; visualisation, P.W.; supervision,
   P.W. and A.L.; project administration, P.W.; funding acquisition, A.L.
   and P.W. All authors have read and agreed to the published version of
   the manuscript.

Funding

   This work was funded by a James Cook University Postgraduate Research
   Scholarship (JCUPRS) to K.Y.; a National Health and Medical Research
   Council (NHMRC) Ideas Grant (APP1183323) to P.W., an Australian
   Institute of Tropical Health and Medicine Capacity Development Grant to
   P.W.; an NHMRC Program Grant (APP1132975) to A.L., and a Senior
   Principal Research Fellowship (APP1117504) to A.L.

Conflicts of Interest

   All authors declare that there are no conflicts of interest.

Footnotes

   Publisher’s Note: MDPI stays neutral with regard to jurisdictional
   claims in published maps and institutional affiliations.

References