Identification of somatic proteins in Haemonchus contortus infective larvae (L₃) and adults

Gastrointestinal (GI) parasites are one of the main causes of diseases, death and economic loss in small ruminant production. Haemonchus contortus has been considered the most pathogenic nematode in production systems based on grazing (Bordoloi et al., 2012; Arsenopoulos et al., 2021).

Since anthelmintic resistance has been described in several species of parasites (Pandey and Sivaraj, 1994; De Albuquerque et al., 2017), new strategies to control the infections and their adverse effects have been developed. Among these, the selection of animals with resistance to GI parasites has been in the spotlight (Palomo-Couoh et al., 2017; Aguerre et al., 2018; Estrada-Reyes et al., 2019; Zaragoza-Vera et al., 2019 ). Thus, the study of protein molecules (involved mainly in secretion-excretion) focused on the parasite-host interactions and the identification of targets to elaborate new drugs or vaccines (Gadahi et al., 2016; Wang et al., 2019a). Some studies concerning the genome, transcriptome and secretome of H. contortus have been conducted (Yatsuda et al., 2003; Laing et al., 2013; Gadahi et al., 2016; Lu et al., 2020).

The somatic proteins of H. contortus were recently reported in a study (Wang et al., 2019b), while, their biological implications and particular functions have been scarcely discussed. In this line, comparing infective larvae (L₃) with adult parasites can lead to the identification of proteins that play an important role in diverse parasite-host interactions, such as the biological mechanisms involved in the establishment of the infection and the survival of the parasite, evasion of the immune response, immunogenic protein recognition by the host, metabolic functions, and other interactions that could improve the understanding of this parasitic infection.

Proteomics studies have contributed to elucidate the role of somatic proteins in the modulation of cellular immune response against H. contortus (Laing et al., 2013; Gadahi et al., 2016; Naqvi et al., 2020; Tian et al., 2020). Somatic proteins such as HC23 and their recombinant version (rHC23), rHcp26/23 and recombinant galectin (rHco-gal-m and rHco-gal-f) have been used for inducing partial protection against of H. contortus infection (Yanming et al., 2007; García-Coiradas et al., 2010; González-Sánchez et al., 2018). In the same way, a potential use of H. contortus somatic proteins could be to generate immunodiagnostic antigens to identify early and late H. contortus infections in sheep, similar to the reported in goats (Naqvi et al., 2020), or as an alternative method to identify specific antibodies of resistant sheep that contribute with the traditional method of faecal egg count (FEC) to identify resistant animals (Palomo-Couoh et al., 2016; Palomo-Couoh et al., 2017; Cruz-Tamayo et al., 2020).

In this study, we report a list of somatic proteins of H. contortus and a comparative analysis between somatic proteins from L₃ and adult parasites recovered from an infected lamb.

This study was approved by the Ethical Committee of University in State of Yucatan, Mexico (Approval ID: CB-CCBA-D-2019-004). The animal used in this study, biological samples and residues were treated and handled in accordance with federal regulations in Mexico (NOM-046-zoo-1995, NOM-087-ECOLSSA1-2002) and following the recommendations emitted by Ethical Committee.

The lamb infected with H. contortus (L₃) presented an FEC of 7, 850 EPG at 35 days after inoculation. The necropsy revealed 634 adult specimens of H. contortus that were directly collected from the abomasum of the infected lamb and used for total protein extraction.

A comparative analysis between L₃ and adult worms was performed after protein identification. L₃ showed a protein profile different from the adult parasites. Data analysis and search on SEQUEST and MASCOT against H. contortus from the WormBase ParaSite database resulted in the identification of 775 unique peptide sequences corresponding to 227 proteins at 1 % FDR (Supplementary Table S1). From these, 18 proteins were specific to L₃ and 63 to adult parasites.

We performed gene ontology (GO) enrichment analysis of the proteins specific to L₃ and adult worms to gain insight into cellular components, molecular functions and biological processes that affect the parasite-host interaction. In the L₃ stage, the GO category of cellular components included the following: transcription complex, intracellular and extracellular region, intracellular organelle and non-membrane bound organelle (Fig. 1). The molecular functions category included binding (e.g., heterocyclic binding, organic cyclic compound binding, carbohydrate derivative binding) and catalytic activity (hydrolase, transferase and catalytic activity acting on a protein). The biological functions category included metabolic processes (primary and organic and nitrogen compound metabolic process) and protein folding.

Fig. 1

In the adult worm stage, the GO category of cellular components included intracellular organelle, non-membrane bound organelle, the endomembrane system, the nuclear outer membrane-endoplasmic reticulum membrane network, and protein complexes such as myosin complex, catalytic complex and proteasome core complex (Fig. 1). As for the molecular functions category, it included catalytic activity (transferase, hydrolase, lyase, oxidoreductase, and catalytic activity that acts on protein), binding (ion, organic cyclic, heterocyclic compound binding, carbohydrate derivative and drug binding), and structural constituents of the cytoskeleton and ribosome. The biological processes category included active metabolic processes linking organic substances, primary metabolic processes, nitrogen metabolic processes, oxidoreduction processes, and cellular processes such as microtubule-based processes and regulation of cellular processes. The comparison between the GO categories of adult worms and L₃ suggests that the adult stage implicates an array of enzymatic activities, structural changes and degradation of proteins.

To identify significant differences in protein abundance between L₃ and adult parasites, the altered proteins were defined with spectral counts that were normalised across L₃ and adult parasites by calculating Z-score values for each protein and with a significance level of p < 0.001. As result, 52 from the 219 proteins showed different abundance between L₃ and adult parasites (Fig. 2). Proteins accumulated in adults include vitellogenin (CDJ88321.1), proteases (AAC03561.1, CDJ97529.1, CDJ87267.1 and AAV68383.1), ubiquitination component (CDJ87593.1), heat shock proteins such as HSP20 (CDJ94402.1), HSP70 (AEO14648.1 and CDJ90614.1) and HSP90 (ACU00668.1), key metabolic proteins such as phosphoenolpyruvate carboxykinase (CDJ86804.1), isocitrate lyase phosphoryl mutase and malate synthase (CDJ95172.1), 6-phosphogluconate dehydrogenase (CDJ88445.1), glutamate dehydrogenase (ACT34055.1), acetyl-CoA hydrolase transferase (CDJ95142.1), thioredoxin, and structural proteins such as actin (CDJ93106.1) and myosin (CDJ95284.1). Collectively, the above-identified proteins suggest that scavenging of reactive oxygen species (ROS), protein folding and degradation, and metabolic changes are implicated in the parasite-host interaction.

Fig. 2

The immune response against GI parasites is considered the main factor associated with the resistance level of the sheep (Aguerre et al., 2018; Estrada-Reyes et al., 2019 ). This response is toward different parasite proteins, either secretory-excretory products or structural compounds (Gadahi et al., 2016; Sakthivel et al., 2018; Lu et al., 2020). The secretory-excretory products have been widely studied due to functions such as tissue penetration and their capability to degrade host proteins (Cox et al., 1990; Yatsuda et al., 2003; Wang et al., 2019a). Moreover, some secretory-excretory products also establish the infection because they regulate the immune response of the host, facilitating the immune evasion of the parasite that induces chronicity related to a T cell-mediated hyporesponsiveness (Lu et al., 2020).

Somatic proteins also play an important role in the immune response against GI nematodes due to which some of them have been evaluated as vaccine candidates (Sakthivel et al., 2018; Wang et al., 2019b). Thus, a study reported that proteins such as rHcp26/23 have a protective effect only with challenges against low and moderate parasite burden (García-Coiradas et al., 2010). The immunization of lambs using the recombinant protein rHc23 produced low of egg excretion and low parasites burden (Gonzalez-Sanchez et al., 2019). However, highest protection of lambs was achieved using aluminum hydroxide as adjuvant and 200 μg/dose rHc23 reaching a reduction of over 70 % of the abomasal parasitic burden (Gonzalez-Sanchez et al., 2019). The recombinant protein rHc23 (23 kDa) was patented for induce more than 85 % of immunity in lambs (Alunda et al., 2014). However, the efficacy of the immunization in lambs seems to depend of the parasite burden used as challenge or by the use of adjuvants (Fawzi et al., 2014; González-Sánchez et al., 2018, 2019). In other study, recombinant HcGAPDH protein expressed on probiotic Bacillus subtilis spores protected sheep from Haemonchus contortus infection by inducing both humoral and cellular responses which reduced the eggs excretion in feces by 84.1 % and adult worms by 71.5 % (Yang et al., 2020). Other somatic protein of H. contortus used as vaccine antigen is actin although the parasite burdens were reduced only 34 % in vaccinated animals (Yan et al., 2014). The HcGAPDH and actin were found in protein extracts from L₃ and adult parasites in present study,

On the other hand, it is known that gut antigens of H. contortus (H-gal-GP) inoculated in lambs induce an immune response mediated by CD4 and T lymphocytes, reducing the FEC in the infected animals (Karanu et al., 1997). The main components of H-gal-GP are metalloproteases and pepsinogen-like aspartyl proteinases that H. contortus use to digest the ingested blood (Scarff et al., 2020). H-gal-GP and gut-derived antigens H11 (aminopeptidases) were antigens with good vaccination response against H. contortus and therefore, they were used in the commercial Barbervax^TM vaccine (Nisbet et al., 2016). Despite its effectiveness, the vaccine has disadvantages such as the lack of the global availability of the product; repeated vaccination is required to stimulate high amount of antigen-specific circulating antibody levels and special management of peri-parturient ewes and young lambs are limiting of the vaccine (Nisbet et al., 2016; Bassetto et al., 2018; Kebeta et al., 2021). A vaccine targeting multiple stages of H. contortus (L_{3 4} surface antigens and gut antigens of Land adult stages) has been proposed (Nisbet et al., 2016) and likely, the integration with recombinant proteins such as 15- and 24-kDa ES proteins, galectins, Hc 23, enolase and cysteine could improve the protective immunity inclusive in young lambs, peri-parturient ewes or in animals with infections with high parasite burdens.

Somatic proteins of H. contortus have also been used as somatic antigens for immunodiagnosis of the infection (Javare, 2017). The recombinant H. contortus adhesion-regulating molecule protein (rHCADRM) was evaluated as potential antigen for diagnosis of early infection in goats. The results showed that rHcADRM can be recognized in the serum as early as 14 post-infection days and antibodies were maintained for over 89 days. The ELISA test developed using this recombinant protein obtained a sensitive (90.6 %) and specific (93.75 %), no showing cross-reaction with other common parasites, which places it as an important antigen for diagnosis of H. contortus infection in goats. Similarly, recombinant cold shock domain containing protein (rHc-CS) was evaluated by immunoblotting and ELISA assays during early and late infections of H. contortus in goats. Results showing specific antibodies from 14 days to 103 post-infection days and specificity and sensibility values of 100 % in both techniques (Naqvi et al., 2020b). However, the use of these recombinants proteins should be validated in sheep.

In our study, a long list of proteins and their differences between the L₃ and adult stages are presented. We focused our attention on L_{3 4} and adult parasites that live in mammalian hosts. Both the Land adult stages utilise catabolic proteins especially in degradation of haemoglobin, meaning that several of these proteins should be present in these parasitic stages (Wang et al., 2019b). In the present study, peptidase S28, putative zinc metallopeptidase precursor, peptidase C1A, peptidase M13 and proteinase inhibitor I25 were present only in the adult stage, whereas proteinase inhibitor I33, aminopeptidase, ubiquitin conjugation factor E4, gelsolin, calpain clp-1, peptidase M16 and peptidase M12A were present only in the L₃ stage. These findings show that the catabolism of proteins is carried out using different enzymes according to the developmental stage.

Differences in proteins involved in the metabolism of carbohydrates and lipids were found in L₃ and adult parasites, in line with the report by Wang et al. (2019b). In the present study, enzymes involved in the catalysis of ATP, such as adenylyl-sulfate kinase, ATP-sulfurylase domain-containing protein, Fructose-1 domain-containing protein and ATP synthase subunit delta, were not found in the L₃ stage. However, it is known that L₃ stage synthetizes glycogen from lipids and them are able to degrade carbohydrates via Krebs cycle (Harder, 2016). Therefore, many of the enzymes involved in glycolysis are not present in this stage, as shown in this study.

Controversial findings were observed in the case of enzymes involved in lipid metabolism. Acetyl-CoA hydrolase, acyl-CoA-binding protein and crotonase domain-containing protein were found only in L. ₃Similar results were previously found in the L₃ stage of H. contortus (Wang et al., 2019b). Therefore, the assumption of Wang et al. (2018), arguing that H. contortus alters its lipidome by adapting metabolism to take advantage of the fatty acids in the blood of the host, seems to be true. Additionally, the protein dhs-3 involved in several lipid functions, such as storage and transportation, metabolic regulation and serving as a source of membrane lipid precursors through cellular organelles named “lipid droplets” (organelles constituted by a neutral lipid core covered by a monolayer of phospholipids and proteins), were exclusively found in L₃₃ , suggesting that lipid metabolism has different routes in Land adult parasites (Xie et al., 2019). Similarly, thiolase, an enzyme catalysing acetyl-CoA, was present only in the larval stage, in agreement with the peaks of the transcriptional levels of its encoding gene Hc-daf-22 in H. contortus L₃ and L₄, suggesting an important role in these larval stages (Guo et al., 2016). Other proteins found exclusively in Lin this study were vinculin, ₃ alpha-catenin, calsequestrin, proteinase inhibitor I33, macrophage migration inhibitory factor, catalase, galectin and calponin. These proteins seem to have an important role in the structure and motility of the parasite. Vinculin and alpha-catenin form a complex of proteins named cadherins that contribute to cellular adhesion and contractibility, both proteins are important in the motility of the parasite (Dufour et al., 2013). The presence of calsequestrin and calponin, involved in Ca²⁺ regulation and highly related to myosin and actin in the tissue contraction (El-Mezgueldi, 1996; Wang & Michalak, 2020), reinforces this assumption.

The regulation of the initial immune response in the host seems to be directed by L₃ and L₄ stages of H. contortus and several structural proteins are involved in this process. Proteinase inhibitor I33 inhibits trypsin and anti-coagulation of blood, favouring in this way the feeding and the survival of L₄ (Yi et al., 2010). The macrophage migration inhibitory factor is a cytokine that regulates the inflammatory response (Lang et al., 2018). Similarly, galectin modulates host-pathogen interactions (Sakthivel et al., 2018). Its functions involve modulating the production of Th2 and Th9 cells, IL-4, IL-9, blood mononuclear cell proliferation, nitric oxide and cell migration in the host (Naqvi et al., 2020).

Catalase is an enzyme with antioxidant effects, used by H. contortus as a defence against hydrogen peroxide and other oxygen reactive species produced by phagocytes (e.g., macrophages, eosinophils and neutrophils) of the host (Kotze, 2003). Notably, in our study, catalase was only present in L₃ but not in the adult stage. Although catalase effects were previously studied in the larval and adult stages of H. contortus (Kotze & McClure, 2001; Kotze, 2003), this effect was indirectly measured by monitoring the disappearance of hydrogen peroxide after the addition of glucose oxidase in cultures of L₄ and adult H. contortus. Therefore, in these studies, the presence of catalase in both parasitic stages was assumed but not directly determined.

In general, our findings show that protein abundance was high in adult parasites. However, most proteins identified in both stages involved the categories of catalytic activity, binding, metabolic and cellular processes.

Some proteins found in the present study were not found in previous reports of the H. contortus proteome (C05D9.9, isoform b, T10G3.3, C29F5.1, dhs-3, DUF148 domain-containing protein, Y48B6A.5 and p15). Moreover, the functions of the unnamed protein products such as VDO47405.1, CDJ93528.1 and VDO21904.1 should be elucidated in future research to contribute to identifying them.

In the present study, specific proteins of the L₃ and adult stages were identified and compared with the purpose of understanding the biological processes involved in the establishment of infection and the role of somatic proteins in the survival of the parasite. The list of proteins provides a database that can be used to identify target proteins that may serve as biomarkers of the infection, to generate anthelmintic drugs that inhibit proteins essential for the establishment of the infection and the survival of adult parasites, or to introduce new candidates for vaccine research. Future studies should be performed to discover the functions of unknown proteins of H. contortus until today, as well as to unravel the recognition and specific role of somatic proteins in the immunological responses generated in the infected sheep.