Proteomic Analysis Of Trichuris Trichiura Egg Extract Reveals Potential Immunomodulators And Diagnostic Targets

Background: Trichuris trichiura embryonated eggs are the infectious developmental stage and the rst signal to the immune system of the denitive host. Each infective T. trichiura egg carries the antigens needed to challenge the immune system with a wide variety of proteins present in the shell, larvae’s surface, and the accompanying uid that contains their excretions/secretions. The parasite eggs constitute the rst antigenic stimuli to evoke the host response to this intestinal parasite with direct life cycle and enteric development. Methods: The soluble egg extract of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus) was investigated using a proteomic approach by mass spectrometry. The antigenic prole of the egg soluble proteins against sera IgG from C. sabaeus with trichuriasis was also investigated by Western blot and LC-MS/MS from the corresponding SDS-PAGE gel. Results: A total of 231 proteins were accurately identied, 168 with known molecular functions. The proteome of the egg lysate revealed common protein families including energy and metabolism; cytoskeleton, motility and muscle; proteolysis; signaling; stress and detoxication; transcription and translation and; lipid binding and transport. Vitellogenin N and VWD and DUF1943 domain containing protein, Poly-cysteine and histidine tailed protein isoform 2, Heat shock protein 70, Glyceraldehyde-3-phosphate dehydrogenase, Actin and Enolase, were among the potential immunoactive proteins. Conclusions: To our knowledge, this study represents the rst attempt to identify the proteome of the T. trichiura egg extract as a novel source of immunomodulators and targets for immunodiagnosis able to contribute to the treatment of human autoimmune diseases and to the control of this neglected disease. bands at a ow rate of 300 nL/min. The eluted peptides were analyzed with a nanoESI-Q-TOF mass spectrometer (5600 TripleTOF, AB SCIEX © ) in an information dependent acquisition mode (IDA). The eluted sample was ionized applying 2.8 kV to the spray emitter and survey MS1 scans were acquired from 350 to 1250 m/z for 250 ms. The quadruple resolution was set to ‘UNIT’ for MS2 experiments, which were acquired from 100 to 1,500 m/z for 50 ms in ‘high sensitivity’ mode. Adenosylhomocysteinase, Tubulointerstitial nephritis antigen, Calponin domain 3 ketoacyl coenzyme domain

Each infective T. trichiura egg carries the antigens needed to challenge the immune system of the host after the larvae and the accompanying uids are released. Herein, we carried out the analysis of the soluble egg extract as a stage-speci c proteomic approach and the identi cation of immunomoactive molecules. Detailed information about the speci c protein patterns and functional analysis of the molecules provides a rst insight into their intricate role within the life cycle and the interactions with the host. The antigenic identi cation and characterization of these egg-derived proteins will complement the current efforts with the T. suis and T. muris models focused on the prevention of autoimmune diseases and the development of new immunodiagnostic techniques.

Sera samples
Sera samples were collected from 10 African green monkeys (AGMs) naturally infected with T. trichiura as part of other studies and transferred to 5 mL vacutainers (Covidien Monoject ™ , Massachusetts, USA). Serum was isolated immediately by centrifugation at 2,000 g for 15 min at 4˚C and stored at -80˚C until analysis.
T. trichiura adults Adult worms were obtained at necropsy from the large intestine of naturally infected animals. The large intestine was placed in 0.9% saline solution for approximately 2 h at room temperature (30-32°C). During this time the nematodes were released from the mucosa.
Thereafter, the large intestine was opened and washed over a 100 µm sieve, and the sieved content was examined under a stereomicroscope (7x -10x magni cation) for the presence of T. trichiura adults, which were isolated, sexed and preserved at -80˚C.
T. trichiura egg extract Uteri were removed from T. trichiura females using a 30G ½" needle (BD Microlance™, Fraga, Huesca, Spain) (10x -30x magni cation) and placed on phosphate buffered saline (PBS; pH 7.4). The uteri were opened with a longitudinal incision to facilitate the release of eggs which were pooled. The pooled eggs were washed ve times in PBS (10,000 g; 1 min), after the nal wash the supernatant was removed and PBS containing 1% protease inhibitors cocktail (Complete mini EDTA-free™, Roche, Berlin, Germany) and 1% Triton™ X-100 (Sigma-Aldrich, Steinhiem, Germany) was added to prepare homogenates as described elsewhere [26]. To ensure disruption of Trichuris eggshells, the homogenate was frozen at -20˚C and sonicated while frozen using ten cycles of 10x 1second pulses at maximum intensity with a Microson Ultrasonic Cell Disruptor XL™ (Misonix, Farmingdale, NY, USA). Homogenates were checked under a stereomicroscope, centrifuged (10,000 g; 10 min at 4˚C) and the supernatant containing the soluble egg proteins was recovered as the T. trichiura egg extract (EE). The total protein concentration of the extract was determined by a commercial Protein Assay (Bio-Rad®, Hercules, USA), which is based on the Bradford method of quanti cation of soluble proteins [27]. The EE was stored frozen at -20˚C until further analysis.
T. trichiura female extract The T. trichiura female extract (FE) was analyzed in parallel to make a comparative study of the immunogenic capacity of the egg proteins with respect to those of the whole females. Female adults were obtained from the intestine of infected AGMs. The collected worms were washed several times with PBS and homogenized with a Te on homogenizer in PBS containing 1% protease inhibitors cocktail (Complete mini EDTA-free™, Roche). After initial centrifugation at a low speed to remove larger particles, the homogenate was centrifuged again (15,000 g; 30 min at 4ºC) and the supernatant collected and stored frozen at -20ºC until further analysis. The protein content was measured in the same way as the EE.
To analyze the protein patterns obtained after electrophoresis, the gels were stained with Coomassie brilliant blue, which allowed identifying and excising the most prominent bands for proteomic analysis. The procedure for staining was as follows: Fixing solution (50% methanol and 10% glacial acetic acid) overnight with gentle agitation (change solution once at rst 1 h). Gels were soaked in staining solution (0.1% Coomassie brilliant blue R-250, 50% methanol and 10% glacial acetic acid) for 20 min, being gently shaken.
Destaining solution was used (40% methanol and 10% glacial acetic acid) and the solution was replenished several times until the gel background was fully removed. Finally, gels were stored at 4ºC in 5% glacial acetic acid.

Western Blot
For immunoblotting, following one dimensional electrophoresis, proteins were transferred onto nitrocellulose paper using a Trans-Blot® Turbo™ transfer system (Bio-Rad®) for 7 min. The blotted membrane was blocked with 5% skimmed milk in 0.05% PBS-Tween 20® (PBST) for 2 h at room temperature and, after successive washes in PBST, they were incubated overnight at 4˚C with a pool of serum samples diluted 1:500 in PBST. After three washes for 30 min in PBST, the membranes were incubated for 4 h at room temperature with the secondary antibody (peroxidase-labeled goat anti-primate IgG (Novusbio™, Colorado, USA) (1:5,000 in PBST)). Finally, membranes were washed three times in PBST, for 30 min each and the assay developed using Clarity™ Western ECL substrate (Bio-Rad®) mixed in a 1:1 ratio. The positive reactions were determined by the appearance of clearly de ned protein bands detected by chemiluminescence with an Amersham™ Imager 600 (GE Healthcare, New Jersey, USA). The relative molecular masses of the recognized protein fractions were determined by comparison with molecular weight markers (kDa) and data analysis was completed as previously described [29].
Proteomic analysis of the T. trichiura egg extract (EE)

Sample preparation
Following electrophoresis and staining, a complete gel strip of egg extract was cut and digested with 500 ng of sequencing grade trypsin (Promega, Wisconsin, USA) in 200 µL of ammonium bicarbonate solution as described elsewhere [30]. The selected bands from other gels (egg and female extracts) were manually excised and digested with 100 ng of sequencing grade trypsin (Promega) in 100 µL of ammonium bicarbonate as described elsewhere [30]. Digestion was stopped with 1% tri uoracetic acid (TFA) and a double extraction with acetonitrile (ACN) was performed. The nal peptide solution was vacuum-dried and resuspended with 25 µL of 2% ACN and 0.1% TFA (pH 2.0) for the EE and 9 µL of 2% ACN and 0.1% TFA (pH 2.0) for the individual bands as previously described [28].
Liquid chromatography and tandem mass spectrometry (LC-MS/MS) Liquid chromatography and tandem mass spectrometry were performed at the Proteomics facility of "Servei Central de Suport a la Investigació Experimental (SCSIE)" of Universitat de València (Burjassot, Spain).
To initiate the elution process, 5 µL of the nal peptide solution was loaded onto a trap column (Nano-LC Column, 3 µm C18-CL, 350 m x 0.5 mm, Eksigen®, AB SCIEX © , California, USA) and desalted with 0.1% TFA at 3 µL / min for 5 min. The peptides were loaded onto an analytical column (LC Column, 3 µm C18-CL, 75 µm x 12 cm, Nikkyo, Nikkyo Technos Co., Ltd. Tokyo, Japan) equilibrated in 5% acetonitrile, 0.1% formic acid (FA) and eluted using a linear gradient (5-35%) of solvent B (0.1% FA in ACN) in A (0.1% FA) for 120 min for the EE and 30 min for the individual bands at a ow rate of 300 nL/min. The eluted peptides were analyzed with a nanoESI-Q-TOF mass spectrometer (5600 TripleTOF, AB SCIEX © ) in an information dependent acquisition mode (IDA). The eluted sample was ionized applying 2.8 kV to the spray emitter and survey MS1 scans were acquired from 350 to 1250 m/z for 250 ms. The quadruple resolution was set to 'UNIT' for MS2 experiments, which were acquired from 100 to 1,500 m/z for 50 ms in 'high sensitivity' mode.
The following switch criterion was used: charge 2+ to 5+, minimum intensity, 70 counts per second (cps). Up to 50 ions were selected for fragmentation after each survey scan. Dynamic exclusion was set to 15 s. The system sensitivity was controlled with 2 fmol of 6 proteins (LC Packings, A Dionex Company, Amsterdam, Netherlands).

Bioinformatics
ProteinPilot (version 4.5.1, revision 2768; Paragon™ Algorithm 4.5.1.0, 2765; SCIEX © ) Applied Biosystems® / MDS SCIEX © ) with default parameters was used to generate a peak list directly from 5600 TripleTof wiff les. All wiff. les from the samples were combined in a single search. The Paragon™ Algorithm included in ProteinPilot™ software was used for searching the NCBI protein database (version 01-2016) with the following parameters: tryptic speci city, cys-alkylation, Metazoa, Nematoda and Trichuris trichiura protein taxonomy restrictions.
Protein grouping was done by Pro Group™ algorithm (a set of proteins that share physical evidence guided by observed peptides only) and identi cation was considered accurate when the ProteinPilot™ unused score was > 1.3 corresponding to a 96% con dence according to the following equation: ProtScore= -log (1-(percent con dence/100)).
Protein identi cation was conducted against the T. trichiura adult proteome from the Parasite WormBase (version of 2017-262 05 -WormBase -www.parasite.wormbase.org). All identi ed proteins were subsequently assigned to the UniProt database and classi ed in Gene Ontology (GO) (https://www.uniprot.org) in accordance to their molecular function and biological process.

Results
In this study we describe for the rst time the proteome of the egg extract of T. trichiura from AGMs (C. sabaeus) and the potential antigens recognized by sera of naturally infected animals.
Proteomic characterization of the T. trichiura egg extract (EE) In a rst analysis of the EE, the spectrometric data using ProteinPilot™ software v4.5 identi ed 246 proteins from which 212 showed signi cant homologies with known T. trichiura adult stage proteins, in addition to 19 novel or uncharacterized proteins with unknown ontology. A total of 231 proteins were accurately identi ed by ProteinPilot™ and accession numbers from Parasite WormBase. These proteins were categorized by their molecular function according to information obtained from the Gene Ontology (GO); 168 presented known molecular functions (Additional le 1).

Gene ontology (GO)
The different functional groups and biological processes of the most representative proteins of our analysis (with 10 or more distinct peptides) are shown in Table 1 and Figure 1. Only a single annotation was assigned to a given protein. Functional annotation of the identi ed proteins was assigned using GO, which revealed functionally diverse molecules of the common protein families or groups: energy and metabolism; cytoskeleton, motility and muscle; proteolysis; signaling; stress and detoxi cation; transcription and translation; and lipid binding and transport (Table 1). Their speci c molecular functions range from molecules involved in ATP, actin, carbohydrate, chitin, lipid and magnesium ion binding, as well as molecules that take part in oxidoreductase, aminopeptidase, glycogen phosphorylase and metallopeptidase activity (Table 1). Others include lipid transporter, motor and protein disul de isomerase activity, together with structural constituents of the ribosome or proteins associated with the elongation phase of protein synthesis. Proteins with kinase and intracellular cholesterol transport functions where also identi ed ( Table 1). The most abundant category for the biological process assigned to the egg proteins were protein folding, translation, gluconeogenesis and glycolytic process all equally represented (13%), followed by cell redox homeostasis (12%) and chitin metabolic function (12%), and to a lesser extent: metabolic process, carbohydrate metabolic process, protein biosynthesis and stress response ( Figure 1).

1-DE and immunoblot analysis of Egg and female adult proteins T. trichiura
To identify the species-speci c parasite antigens, the 1-DE SDS-PAGE and Western blot were performed to investigate the antigenic pro le of the EE and FE, and analyze which peptides were recognized by serum IgG from naturally infected AGMs.
The immune-complexes identi ed by Western blot for the EE, were in the range of 37 and 200 kDa, with two marked bands including the most immunogenic ones, band 1W (≈ 170 kDa) and band 2W (≈ 37 kDa) ( Figure 2, lane 1). The possible identity of the proteins in those bands was investigated by matching the predicted molecular weight from the EE proteome (Table 2). In addition, the same regions of interest were excised from the SDS-PAGE corresponding gel (labeled as 1G and 2G, Figure 2, lane 2), for new con rmatory proteomic analysis (Table 3).
Regarding the identi cation of antigens in the gel bands, our proteomic analysis revealed Vitellogenin N and VWD and DUF1943 domain containing protein (VgNVD) and Heat shock protein 70 (HSP-70) among the potential egg immunodominant proteins in band 1W based on the EE proteome ( Table 2). The subsequent mass spectrometry of the Coomassie-stained band con rmed that VgNVD is the most representative protein within this area with 241 distinct peptides ( Table 3).
In both samples a highly reactive band, with a mobility corresponding to 37 kDa was detected (2W and 4W, Figure 2). It corresponded to material detected by Coomassie staining, more abundant in the FE; while other two bands (1W and 3W) seemed to be stagespeci c ( Figure 2). As shown in the Figure 2, the band labeled 1W in EE was barely detected in the stained gel, indicating its low abundance, while band 3W corresponded to a prominent band when stained with Coomassie (3G) (Figure 2).
Regarding the proteomic analysis of reactive areas displayed in FE Western blot, the analysis of the band 3G (≈ 60-70 kDa) (Figure 2, lane 4), which corresponded to band 3W, revealed again PCHTP-2 as one of the proteins identi ed with the highest number of matching peptides. This protein was also identi ed in bands, 4.1G and 4.2G ( Table 4). The proteomic results of both sections of the band 4W showed some proteins shared between the egg and female extracts, such as PCHTP-2, Actin and GAPDH, suggesting them likely to be the major ones in both samples. This is not surprising, since most of the EE antigens are also present in FE (eggs contained in the uterus).

Discussion
Foth and collaborators [15] described the whole-genome sequences of the human-infective T. trichiura, as well as the wholetranscriptome in a mouse laboratory model T. muris and identi ed numerous genes that are differentially expressed in a sex-or stage-speci c manner. The most abundant transcripts found in this extensive study, included proteins we have also identi ed in the EE proteome such as two WAP domain containing SLP-like proteins, protease inhibitors such as Cystatin-domain containing protein and nematode cuticle collagen N-terminal domain containing proteins and Chitin binding domain containing proteins such as CBM14 domain containing proteins. Furthermore, with more or less representation, but of particular interest within the context of the present work, we have found trichuris egg proteins with known immunomodulatory properties such as Macrophage migration inhibitory factor homolog (MIF), previously identi ed in T. trichiura adult [14], and 14-3-3 protein which has also been identi ed in several developmental stages of other nematodes, Trichinella britovi [31] and Trichinella spiralis [32] and trematodes, Schistosoma japonicum [33]. Both proteins are considered as enhancers of humoral and cellular immune responses [34].
Interestingly, two of the proteins identi ed with the largest numbers of distinct peptides in the EE proteome presented in this study, Vitellogenin N and VWD and DUF1943 domain containing protein (VgNVD) and Poly-cysteine and histidine tailed protein isoform 2 (PCHTP-2), were also found among the top 25 most abundant transcripts found by Foth and collaborators [15]. Vitellogenins are a lipid transfer proteins present in the eggs of most oviparous animals as the major component of yolk. They play a signi cant role in embryonic development and are extensively conserved amongst insects, nematode and vertebrates [35]. They are produced by extraovarian tissues, secreted into the circulatory system and then taken up by the developing oocytes through receptor mediated endocytosis to provide the growing embryo with amino acids [36]. On the other hand, the detection of PCHTP-2 as the second most frequently detected protein is in accordance to Shears and collaborators [37] who found it to be the most abundant protein in the T. muris adult secretome; even though a speci c function has not been assigned yet. Likewise, Bancroft and collaborators [38] identi ed PCHTP-2 as the most abundant protein in cecal mucus from chronically infected mice with T. muris and con rmed its expression in all developmental stages.
One of the most represented groups of proteins is that of energy and metabolism including proteins related to glycolysis (Enolase and Glyceraldehyde-3-phospate dehydrogenase (GADPH)) and gluconeogenesis (Triosephosphate isomerase and Phoshoenolpyruvate carboxykinase GTP) and other metabolic enzymes such as Alpha-1,4 glucan phosphorylase and Malic enzyme. This fact is consistent with previous studies in which those metabolic enzymes were described in the surface of the helminths, nematodes and trematodes, participating in parasite invasion and migration processes within the host and in oxidative processes [28,29,[39][40][41][42].
The ensuing functional group with the largest number of representatives is the cytoskeleton and motility and muscle proteins. Actin, Tropomyosin, Paramyosin, Intermediate lament protein IFA 1 and Epididymal secretory protein E1 were found with a high number of distinct peptides. These proteins are essential to enhance the motility of the nematodes and have also been recorded in many helminthic proteomes: somatic extract of adults of T. spiralis [43], T. britovi [31], Syphacia muris [42] and Echinostoma caproni [44]; and in egg secretions of Schistosoma mansoni [17]. Speci cally, Intermediate lament protein IFA1 has been studied in Caenorhabditis elegans demonstrating that in nematodes they allow epidermal elongation in the larval stages to grow into adults [45].
In addition to the proteins already mentioned, another group essential for the survival of the nematode within its host is that of the stress and detoxi cation, including antioxidants and chaperones. The Cu/Zn superoxide dismutase (Cu/Zn-SOD) was found in the EE and it has also been identi ed on the adult surface and larval extracts (secreted and somatic) of Toxocara canis [34], in somatic extract of adults of Fasciola hepatica, and in S. mansoni egg secretome [17,46]. This essential enzyme antagonizes the in ammatory responses in the host by regulating the free radical balance and reactive oxygen species in cells protecting helminths against cell death [47]. Heat shock proteins (HSP90, HSP70, HSP60) are inducible conserved proteins widely described in parasite proteomes and secretomes, acting as molecular chaperones which fold, assemble and translocate other proteins to ensure the survival of the parasite by defending it against stressful situations being important in stress tolerance [48]. Small heat shock proteins HSP-20 and HSP-20 domain containing protein were also identi ed in EE, which are known to aid parasite survival under hostile conditions such as heat or nutritional stress [49].
Within the proteins implicated in signaling pathways, we identi ed galectin, a type of lectin found in different extracts of nematodes such as adults and larvae of T. canis [34] and extract of infective larvae (L3) of Haemonchus contortus [50] with a role in immune signaling pathways. Nematode galectins are believed to be immunological mediators with implications in survival and interaction with the host [51] and modulate a range of immune responses including the cellular immune response, in ammatory processes and immune regulation [52].

Antigenic pro le of T. trichiura EE and FE extracts and identi cation of immunodominant proteins
This type of immunoproteomic approach has been was applied in previous studies to determine both the antigenic proteins of different helminths developmental stages (larvae and adults), and evaluate the serological response to the soluble protein extracts of Ascaris lumbricoides [53], T. britovi [31], Schistosoma japonicum [54] and Taenia solium [55].
Parasitic worms have a remarkable ability to modulate the host immune response through several mechanisms; speci c parasitederived proteins can modulate immune functions playing an important role in the parasite-host interaction. Excretion/secretion proteins from larvae and adults of the porcine whipworm, T. suis, closely related to the human T. trichiura, were investigated by Leroux et al. [21], who identi ed a subset of proteins that promote speci c anti-in ammatory functions and immunomodulatory properties.
HSP-70 and heat shock proteins in general, have caught the attention of researchers for acting typically as immunodominant antigens eliciting strong humoral responses as major targets of host immune responses, suggesting them out as possible candidates for antiparasitic, allergic and autoimmune diseases treatments [61,62]. The HSP70 is amongst the most highly abundant protein identi ed in egg secretions of S. mansoni and H. polygyrus [17,60], and also heavily represented in E. caproni, F. hepatica, H. polygyrus, Schistosoma bovis, T. trichiura, T. britovi and Zygocotyle lunata adult worms extract [14,31,41,44,63,64]. Others have reported on their immunogenicity linked to stimulation of IgG and IgM responses [39,65,66] and they have been suggested as possible vaccine targets [67].
PCHTP-2 was identi ed as a strong immunogen of Trichinella pseudospiralis adult secretome [68]. Another protein of the same family, Poly-cysteine and histidine-tailed metalloprotein, implicated in metal storage and/or transport, was the rst member of the nematode poly-cysteine protein family described in T. spiralis. Since these proteins are unique for parasites of the Superfamily Trichinelloidea their potential applications in diagnostics and treatment could be exploited in the future [69]. Bancroft [38] hypothesized that the unique structural features of this protein allow binding to IL-13 which is considered the key effector cytokine responsible for T. muris expulsion, able to inhibit IL-13 function both in vitro and in vivo.
Certain glycolytic enzymes, Enolase and GAPDH, have been identi ed as inmunoactive components of the Trichuris egg proteome. Both of them are present in the surface of helminths interacting with the host surface. Furthermore, Enolase plays an important role in brinolysis and degradation of the intracellular matrix through the activation of plasminogen, which may induce plasmin-mediated proteolysis and facilitate the invasion, migration and xation in the host [15,17,29,42]. In T. spiralis [39] and T. britovi [31] this enzyme has been con rmed as immunodominant suggesting that it may assist in tissue migration of the larvae. Enolase and heat shock proteins have also been classi ed as exosome markers [37,70]. Likewise, GAPDH has been previously linked to bronectin, laminin, entactin and collagen binding [71]. Cass and collaborators [17] suggested that in the case of S. mansoni this protein could be involved in the attachment of the eggs to host tissues or aid the passage of live eggs across host tissues to the external environment.
The present study seeks to identify and characterize the soluble extracts of T. trichiura eggs by proteomic and immunoproteomic approaches. The T. trichiura life cycle inside the host starts with the egg hatching and the release of the larva, this period of time remains as an undiagnosed stage, while the proteins described here are directly exposed to the immune system, and as we demonstrate herein, can elicit anti-Trichuris antibodies by the host.

Conclusions
This is the rst attempt to identify the proteome of the T. trichiura eggs as a novel source of potential targets that can be used to develop better strategies for improving diagnosis, treatment and control of this neglected disease.
Eggs as the infective developmental stage of the nematode will signal the host interface with its shell surface antigens and with the

Consent for publication
Not applicable

Availability of data and materials
The data supporting the conclusions of this study are included within the article and the datasets generated during the present study are included as supplementary information les (Additional le 1).

Competing interests
The authors declare that they have no competing interests.

Funding
The proteomic analysis was supported by the project GVPROMETEO16/156 of Generalitat Valenciana (Spain) (AM and MT). The work was carried out while the rst author (KC) was funded by Ross University School of Veterinary Medicine under a collaborative with Universitat of València.
Author's contributions KC performed the experiments, the data curation and analysis, and drafted the manuscript. AM contributed with the conceptualization, methodology, validation, writing review, editing, and funding acquisition. PK and MV contributed with writing review, editing, supervision and resource acquisition. AO contributed with conceptualization, methodology, validation, writing review and editing. MT contributed with the design, conceptualization, methodology and validation of the experiments as well as the writing review and editing, supervision, funding and resource acquisition. Table 1 Main proteins identi ed in the EE (10 or more distinct peptides) organized by functional annotation. Only a single annotation was assigned to a given protein.  Figure 1 Biological process of the identi ed proteins in the egg extract (EE) of T. trichiura according to information obtained from the Gene Ontology (GO) database https://www.uniprot.org.