Gastrointestinal (GI) parasites are one of the main causes of diseases, death and economic loss in small ruminant production.
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
The somatic proteins of
Proteomics studies have contributed to elucidate the role of somatic proteins in the modulation of cellular immune response against
In this study, we report a list of somatic proteins of
The infective larvae of
The infection was carried out when the lamb reached 2.5 months of age (11.1 kg live weight, LW). The infection was performed using an oral dose of 400 L3/kg LW. L3 were obtained from coprocultures made of faeces of the infected lamb and weekly collected starting at 21 days after infection, according to the Corticelli and Lai technique (1963).
On day 35 post-infection, faeces were collected to calculate FEC. Subsequently, the lamb was sacrificed following the official national standards (Norma Oficial Mexicana NOM-033-ZOO-1995) to collect adult parasites directly from the abomasum during the necropsy. Parasites were washed with phosphate buffer solution (PBS, 0.01 M, pH 7.4) to eliminate cellular detritus. Afterwards, parasites were frozen at -20 °C until use.
Faecal samples were taken directly from the rectum of the lamb and collected in polyethylene bags. The faecal samples were processed by modifying the McMaster technique to determine FEC. The number of eggs per gram of faeces (EPG) was adjusted with a correction factor of 50 (Cringoli et al., 2004). Moreover, coprocultures were performed according to Corticelli and Lai (1963) to obtain
The L3 and adult parasites were pulverised to a fine powder with the mortar and pestle technique at liquid nitrogen temperature. Pulverised samples were resuspended in 500 μl of protein extraction buffer (500 mM Tri-HCl, pH 7.5, 100 mM KCl, 100 mM sucrose, 50 mM EDTA, 50 mM DTT, 1 mM PMSF and 1X complete Roche protease inhibitor cocktail). Proteins were recovered from the clarified homogenate by precipitation using a solution of TCA/acetone at 20 % cold at a volume proportion of 1:1 followed by two washes with 80 % acetone solution (Niu et al., 2018). Protein pellets were air-dried for 3 min and kept at -20 °C. Then, they were resuspended in 300 μl of 50 mM ammonium bicarbonate solution. Subsequently, the protein concentration was determined by the Peterson method using 10 μl of the extract and bovine serum albumin protein as reference (Peterson, 1977).
The protein content of 100 μg was reduced with 15 mM DTT for 1 h at 25 °C followed by alkylation with 30 mM iodoacetamide dithiothreitol for 1 h at 25 °C in darkness. Subsequently, protein samples were quenched with 30 mM DTT for 10 min at 25 °C in darkness. Trypsin Gold (PromegaTM, Madison, Wisconsin, USA) was added at a ratio of 1:30 trypsin/protein (w/w). Afterwards, protein digestion was carried out for 16 h at 37 °C, and more trypsin was added at a ratio of 1:30 trypsin/protein (w/w). The samples were left to incubate for 4 h at 37 °C. Digestion was quenched by making samples with 1 % formic acid that were dried using a SpeedVac Vacuum Concentrations (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) and stored at -70 °C until fractionation of peptides using Strong Cation Exchange (SCX) chromatography.
Peptides were resuspended in equilibration buffer consisting of 5 mM KH2PO4 (pH 3)/25% acetonitrile (ACN) for SCX fractionation using a 50 mg sorbent HyperSep TM SCX cartridge (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA). The SCX cartridge was conditioned with 2 ml milli-Q water, washed with 1 ml of 10 mM KH2PO4 (pH 3)/25% ACN and subsequently washed twice with 2 ml of milli-Q water. The cartridge was placed in equilibration buffer before sample loading. After three washes with 1 ml of equilibration buffer, peptides were eluted in a stepwise gradient of increasing salt concentrations (75, 250 and 500 mM KCl) in equilibration buffer. Eluted peptide fractions were then dried in SpeedVac Vacuum Concentrations (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA). The resulting peptides were re-suspended in 1 ml of 0.1 % formic acid for desalting using 25 mg HyperSep C18 cartridges (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA). Eluted peptides were then desalted using Zip-Tip pipette tips (Millipore, Billerica, Massachusetts, USA). Desalted peptides were vacuum-dried and resuspended in 0.1 % formic acid for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.
The desalted peptide samples were analysed in an LC-MS/MS system consisting of EASY-nLC 1000 nano-HPLC (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) coupled to an LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA). Chromatographic separation was performed using a reversed-phase Acclaim PepMap-100 trap column (20 × 0.075 mm i.d., 3 μm particle size, 100 Å pore size; Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) coupled to an EASY-Spray C18 analytical column (150 × 0.075 mm i.d., 2 μm particle size, 100 Å pore size; Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) at 40 °C with a flow of 300 nl min-1, from 5 % to 40 % ACN in 0.1 % formic acid, in a 60 min gradient. The mass spectrometer in data-dependent acquisition mode and the OrbitrapTM analyser (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) were used. Full scan MS spectra were acquired with a resolution of R = 12,000 followed by MS/MS acquisition with a resolution of R = 15,000. The 15 most intense precursor ions from the full scan were fragmented using higher-energy C-trap dissociation. Fragmented masses were excluded for 90 s. Three independent biological replicates were performed. Carbamidomethylation was used as fixed modification.
The mass spectrometry data files were processed using the Proteome Discoverer 2.2 software (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA), and protein identification was performed with the MASCOT search engine (Matrix ScienceTM) and the SEQUESTTM software (Eng et al., 1994). A total of 24,642 amino acid sequences were retrieved from the WormBase ParaSite database (Laing et al., 2013) and used for spectra matching. The database search allowed static carbamidomethylated cysteine residues (+57.02 Da), dynamic oxidised methionine residues (+15.99 Da), up to two missed trypsin cleavages, a tolerance of 10 ppm for precursor ions and 0.6 Da for fragmented ions. The masses of both precursor and fragmented ions treated as monoisotopic and multiple charges were considered. The data were filtered using an estimation of 1 % false discovery rate (FDR) according to a previous study (Rohrbough et al., 2006). The determination of protein abundance was evaluated by spectral counts (Arike & Peil, 2014).
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
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
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.
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.
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
On the other hand, it is known that gut antigens of
Somatic proteins of
In our study, a long list of proteins and their differences between the L3 and adult stages are presented. We focused our attention on L3 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
The regulation of the initial immune response in the host seems to be directed by L3 and L4 stages of
Catalase is an enzyme with antioxidant effects, used by
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
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
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