Foth and collaborators [15] described the whole-genome sequences of the human-infective T. trichiura, as well as the whole-transcriptome in a mouse laboratory model T. muris and identified numerous genes that are differentially expressed in a sex- or stage–specific manner. The most abundant transcripts found in this extensive study, included proteins we have also identified 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 identified in T. trichiura adult [14], and 14-3-3 protein which has also been identified 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 identified 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 significant role in embryonic development and are extensively conserved amongst insects, nematode and vertebrates [35]. They are produced by extra-ovarian 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 specific function has not been assigned yet. Likewise, Bancroft and collaborators [38] identified PCHTP-2 as the most abundant protein in cecal mucus from chronically infected mice with T. muris and confirmed 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 [28,29,39-42].
The ensuing functional group with the largest number of representatives is the cytoskeleton and motility and muscle proteins. Actin, Tropomyosin, Paramyosin, Intermediate filament 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]. Specifically, Intermediate filament 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 detoxification, including antioxidants and chaperones. The Cu/Zn superoxide dismutase (Cu/Zn-SOD) was found in the EE and it has also been identified 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 inflammatory 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 identified 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 identified 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, inflammatory processes and immune regulation [52].
Antigenic profile of T. trichiura EE and FE extracts and identification 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; specific parasite-derived 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 identified a subset of proteins that promote specific anti-inflammatory functions and immunomodulatory properties.
Shears and collaborators [37] identified VgNVD in extracellular vesicles (EVs) of T. muris as a potential immunogenic candidate. Antigenic homologs have been identified in both free-living nematodes such as C. elegans, and adult parasites secretomes of Ascaris suum, Nippostrongylus brasiliensis, Heligmosomoides polygyrus and Litomosoides sigmodontis [56-59] and also in H. polygyrus eggs [60].
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 identified 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 identified 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 first 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 identified 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 fibrinolysis and degradation of the intracellular matrix through the activation of plasminogen, which may induce plasmin-mediated proteolysis and facilitate the invasion, migration and fixation in the host [15,17,29,42]. In T. spiralis [39] and T. britovi [31] this enzyme has been confirmed as immunodominant suggesting that it may assist in tissue migration of the larvae. Enolase and heat shock proteins have also been classified as exosome markers [37,70]. Likewise, GAPDH has been previously linked to fibronectin, 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.