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Shotgun proteomics of Strongyloides venezuelensis infective third stage larvae: Insights into host–parasite interaction and novel targets for diagnostics
Molecular & Biochemical Parasitology 235 (2020) 111249
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Shotgun proteomics of Strongyloides venezuelensis infective third stage larvae: Insights into host–parasite interaction and novel targets for diagnostics
T
Priscilla D.M. Fonsecaa,b, Marcelo A. Corrala, Miguel Cosenza-Contrerasd, Dirce M.C.L. Meisela, Gessica B. Melob, Milena M.S. Antunesa, Maria C.E. Santoa, Ronaldo C.B. Gryscheka,b, Julia M. Costa-Cruzc, William Castro-Borgesd, Fabiana M. Paulaa,b,* a
Laboratório De Investigação Médica Da Faculdade De Medicina Da Universidade De São Paulo, São Paulo, Brazil Instituto De Medicina Tropical Da Faculdade De Medicina Da Universidade De São Paulo, São Paulo, Brazil c Laboratório De Diagnostico De Parasitoses, Instituto De Ciências Biomédicas Da Universidade De Federal De Uberlândia, Uberlândia, Minas Gerais, Brazil d Laboratório De Enzimologia e Proteômica, Instituto De Ciências Exatas e Biológicas, Universidade Federal De Ouro Preto, Ouro Preto, Minas Gerais, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Strongyloides venezuelensis Shotgun proteomics Diagnostic markers
Strongyloides venezuelensis is an important alternative source of antigen for the serologic diagnosis of human strongyloidiasis. Proteomics techniques applied to the analysis of the protein content of infective third stage larvae (iL3) of S. venezuelensis provide a powerful tool for the discovery of new candidates for immunodiagnosis. This study presents an overview of the protein iL3 S. venezuelensis focusing on the diagnosis of strongyloidiasis. A total of 877 proteins were identified by shotgun proteomics. Many of these proteins are involved in different cellular processes, metabolic as well as structural maintenance. Our results point to a catalog of possible diagnostic targets for human strongyloidiasis and highlight the need for evaluation of uncharacterized proteins, especially the proteins within the CAP domain, transthyretin, and BTPI inhibitor domains, as a repertoire as yet unexplored in the context of strongyloidiasis diagnostic markers. We believe that the protein profile presented in this shotgun analysis extends our understanding of the protein composition within the Strongyloides genus, opening up new perspectives for research on biomarkers that may help with the diagnosis of human strongyloidiasis. Data are available via ProteomeXchange with identifier PXD013703.
Strongyloides stercoralis is a causative agent of human strongyloidiasis, affecting approximately 350 million people worldwide, especially in tropical and subtropical areas [1]. This helminthiasis results in an asymptomatic chronic disease, which can be maintained for decades by a process of autoinfection and may be related to hyperinfection or disseminated disease, with high mortality rates, especially in immunocompromised patients [2]. The definitive diagnosis of S. stercoralis infection is made by detecting larvae in fecal samples, but it has low sensitivity [2,3]. Thus, serologic methods have been proposed as a complement to the parasitologic diagnosis. In this context, the research using heterologous antigen, Strongyloides venezuelensis, has been highlighted in the literature [3], mainly because it can be easily obtained in large quantities, allowing the development of new diagnostic approaches of strongyloidiasis [3–5]. Recent studies show that identification and characterization of Strongyloides proteins are essential for a better understanding of the
host–parasite relationship, as well as for identification of new diagnosis targets [6,7]. Proteomic studies in Strongyloides sp. have been based on the analysis of proteins by immunoblotting [5], somatic extracts [6], or secretion/excretion products [8,9], however these efforts have been restricted. There is an urgent need for new biomarkers that can be used for diagnosis. In addition, a marker for diagnosis must be parasite specific and be recognized by the host immune system. Considering S. venezuelensis as an important alternative source of antigen for the serologic diagnosis of human strongyloidiasis, shotgun analysis of the protein content of infective third stage larvae (iL3) of S. venezuelensis provides a powerful tool for the discovery of new candidates for immunodiagnosis. The present study describes the shotgun analysis of S. venezuelensis iL3. The research was carried out on infective third stage larvae (iL3) of S. venezuelensis obtained from stool cultures of experimentally infected Rattus norvegicus. The cultures were performed with stool samples collected on the day 8 after infection, homogenized with charcoal (ratio of
⁎ Corresponding author at: Laboratório de Investigação Médica (LIM/06), Instituto de Medicina Tropical, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000, São Paulo, SP, Brazil. E-mail addresses: [email protected], [email protected] (F.M. Paula).
https://doi.org/10.1016/j.molbiopara.2019.111249 Received 30 August 2019; Received in revised form 20 December 2019; Accepted 23 December 2019 Available online 24 December 2019 0166-6851/ © 2019 Published by Elsevier B.V.
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each spectrum was later filtered with the Search Engine Processor (SEPro) with the following parameters: delta mass of 30 ppm, total delta CN of 0.001, minimum of 6 amino acids per peptide, minimum of 1 peptide per protein, 1 spectral count, PSM (peptide spectrum matches) delta mass of 10 ppm, and a normalized primary score (XCorr) ≥2.0 for proteins with 1 mass spectrum and ≥1.8 for proteins with ≥2 mass spectra. A Bayesian score (XCorr, delta CN, peak matched values, delta mass error, spectral count score, and secondary rank) was used to sort the identification in a non-decreasing order of confidence. In addition, a cutoff was applied to accept a false discovery rate of 3 % for spectra, 2 % for peptide, and 1 % for protein (based on labeled decoy identifications accepted into the discovery list). For identification, all proteins were considered in order to increase the identification of lower molecular weight proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [14] partner repository with the dataset identifier PXD013703. The proteins identified were categorized based on their molecular function as presented in the Gene Ontology (GO) database [15]. GO term to Uniprot ID mapping was performed from the S. venezuelensis reference proteome (UniProt proteome ID: UP000035680) [13]. Identified protein sequences were submitted to SignalP-5.0 [16] and TMHMM Server v.2.0 [17] for the prediction of signal peptides and transmembrane domains, respectively. Two logical variables were created to indicate whether a protein passed the criteria to be considered a signal peptide and/or presented transmembrane helice(s). A few proteomic datasets were recently published for members of genus Strongyloides [6–9]. On the other hand, the S. venezuelensis proteome has not yet been reported, especially in the context of new targets for diagnosis. Considering the good results for the sensitivity and specificity of the heterologous antigen for the diagnosis of human strongyloidiasis, mainly of iL3 S. venezuelensis, the present study evaluated the shotgun proteomic analysis of this developmental stage. Shotgun proteomics analyses identified a total of 877 proteins in S. venezuelensis iL3 within the Uniprot database (Supplementary Table 1). In addition, 87.34 % of the proteins obtained had > 2 unique peptides. The dynamic range of protein abundance revealed a variation of more than 3 orders of magnitude in terms of the spectral counts of proteins in the extract (Fig. 1A). Similarly, the cumulative abundance plot defined 82 proteins representing 50 % of the total mass, and 385 proteins made up 90 % of the extract. A set of 491 lowly abundant proteins were also identified, accounting for 10 % of the total spectral abundance (Fig. 1B). The number total of proteins identified in the soluble extract of iL3 S. venezuelensis reinforces the intense adaptation activity of this developmental stage within the host. GO enrichment analysis of the larval protein set within the whole S. venezuelensis predicted proteome identified 85 molecular function-related GO (GO-MF) terms. Among these, 5 GO-MF terms presented the higher protein counts: oxidoreductase activity (101 identities), cofactor binding (80 identities), protein binding (61 identities), and coenzyme binding (58 identities) (Fig. 2). Infective third stage larvae of Strongyloides actively penetrate the skin or mucosa of the host and migrate through the host body to reach the small intestine [4]. That might explain the large amount of enzyme, which makes up half of the total mass of the protein extract. Among the 82 most abundant proteins, actin is a dominant constituent. Not surprisingly, structural proteins, such as actin and myosin, were the most abundant in iL3 S. venezuelensis extract. These proteins can play important roles in maintaining the body shape and muscle integrity of the nematodes [18], and are probably involved in migration in the host at this developmental stage. In addition, our group demonstrated recognition of structural proteins by antibodies present in patients with strongyloidiasis [5]. However, we believe that the structural proteins, due to their high similarity among organisms, cannot be considered as a good diagnostic marker, and further studies are necessary.
1 g of stool to 3 g of charcoal) and water, followed by incubation at 28 °C for 48 h. After this procedure, iL3 was recovered according to the modified Baermann method [10] and stored at −20 °C for later extraction of protein. All procedures were conducted in accordance with the ethical guidelines adopted by the Comissão de Ética no Uso de Animais (CEUA) of Instituto de Medicina Tropical, Universidade de São Paulo (protocol CEP-IMT 317A and 0356A). Soluble proteins of S. venezuelensis were obtained with approximately 200,000 iL3 in 1 mL of 25 mM Tris−HCl (pH 7.5) containing 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The samples were homogenized on ice in a tissue homogenizer (TissueTearor, BioSpec Products, Bartlesville, OK) for 5 cycles of 5 min each. The resulting suspension was centrifuged at 12,400 × g for 30 min at 4 °C followed by recovery of the supernatant containing the soluble proteins. Protein content was obtained using a Bio-Rad RC DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Three replicates of soluble proteins of S. venezuelensis iL3 were prepared. Fifty micrograms of soluble proteins were diluted in 25 mM ammonium bicarbonate (0.06 % w/v) and then used for the digestion procedure. Proteins samples were incubated with 1 % RapiGest (Waters, Milford, MA) at 80 °C for 5 min. Then, the proteins were reduced using dithiothreitol (Sigma-Aldrich) to a final concentration of 3.3 mM at 60 °C for 10 min and alkylated using iodoacetamide (SigmaAldrich) to a final concentration of 9.4 mM in the dark for 45 min. After this process, the samples were digested by addition of trypsin (Promega, Madison, WI) at a ratio of 50:1 (protein/trypsin) followed by incubation for 16 h at 37 °C. Tryptic digestion was halted by acidifying the sample by adding 0.5 % (v/v) acetic acid (J.T. Baker, Center Valley, PA). The samples were incubated at 37 °C for 45 min for RapiGest acid-induced cleavage, and the salt was removed by centrifugation at 20,000 × g for 15 min at 7 °C. The samples were then ready for mass spectrometry analysis. Liquid chromatography was performed on a nanoUHPLC Dionex Ultimate 3000 (Thermo Scientific, Bremen, Germany) equipped with an Acer PepMap100 C18 Nano-Trap column (100 μm ×2 cm, 5 μm beads, 100 Å pores; Thermo Scientific) followed by an analytical Acclaim PepMap100 C18 column (75 μm ×15 cm, 2 μm beads, 100 Å pores; Thermo Scientific). A nonlinear gradient of eluent A (0.1 % formic acid) to B (0.1 % formic acid in 80 % acetonitrile) was applied. Peptides were ionized using a nano electrospray ion source and detected on a QExactive mass spectrometer (Thermo Scientific). The ion source operated at 3.8 kV in positive mode with a capillary temperature of 250 °C. The precursor ions were measured with the resolution set at 70,000 (range, 300–2000 m/z) and were selected for fragmentation by a datadependent method in which the top 12 most intense ions with charges between +2 and +4 were selected, given an exclusion time of 40 s. Then, a high-energy collisional dissociation method was applied with a normalized collision energy of 28–30 V to obtain the fragments. Tandem mass spectra were obtained with the resolution set at 17,500 (minimum of 120 m/z and isolation widow of 1.2 m/z; maximum injection time of 60 ms). The mass spectrometric data obtained from three independent nanoLC-MS/MS runs were combined using the Patternlab software to increase the chance of protein identification and therefore provide an improved compositional analysis of the iL3 soluble fraction. Database searching was performed using the Patternlab for proteomics software [11] and the UniProt compilation database [12], considering S. venezuelensis and totaling 16,639 sequences. In addition, spectra were searched in the S. venezuelensis database [9,13]. The search was performed using the Comet algorithm, applying in silico digestion parameters as follows: search performed in a semi-specific tryptic digestion space, up to 2 missed cleavage sites, cysteine carbamidomethylation (+57.02146 Da) as fixed and methionine oxidation (+15.9949 Da) as variable posttranslational modifications (up to 3 variable modifications per peptide), MS1 mass range of 550–5500 Da (10 ppm mass tolerance), and B, Y, and neutral loss MS2 fragmentation. The list of candidates for 2
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Fig. 1. Protein distribution in extracts from S. venezuelensis iL3. (A) Dynamic abundance plot based on the number of peptide spectral counts, normalized using the spectral abundance factor (NSAF). There is variation of more than 3 orders of magnitude in terms of protein abundance in the extract. (B) Protein abundance in extracts represented as a cumulative frequency plot revealed that 82 constituents contributed to 50 % of the total mass.
Even among the abundance of constituents, we can highlight different enzymes, particularly kinases (arginine, nucleoside diphosphate, phosphoenolpyruvate carboxykinase, pyruvate and phosphoglycerate), aldolase (fructose biphosphate aldolase), isomerases (triosephosphate, protein disulfide, peptidylprolyl and glucose 6-phosphate), dehydrogenases (malate, glyceraldehyde-3-phosphate and retinal dehydrogenase 2), aminotransferases (aspartate, alanine aminotransferase 1,4-aminobutyrate), and synthases (citrate, ATP synthase subunit beta, hematopoietic prostaglandin D). The kinases are known to act in a variety of cellular signaling processes [19], and isomerase are keys enzymes in glycolysis and catalyze the reversible isomerization of different molecules [20]. Aldolase has been associated with antigenic properties and mediating interactions with the host [21]. The identification of these proteins may suggest that they play a fundamental role in the parasite–host interaction. In addition, the most abundant components include the following proteins: CAP domain protein, profilin, 14-3-3 zeta protein, nucleoredoxin-like protein 2, calmodulin, superoxide dismutase, enolase, ferritin, and heat shock proteins (HSP60 and HSP70). Antioxidant enzymes such as superoxide dismutase can act to protect the innate immune response of the host [19], favoring parasite survival inside the host body. Some other components were identified such as galectin and 14-3-3 protein. The biological function of galectins in nematodes is not well understood but has been related to host survival and interaction, as well as to negative regulation of host innate immunity [22]. The 14-3-3 protein may be related to proliferation, migration, and morphologic changes during the life cycle of the parasite [23]. Galectin and 14-3-3
Fig. 2. Molecular function by Gene Ontology analysis of identified proteins in extracts from S. venezuelensis iL3.
protein have been indicated in recent research as possible diagnostic markers for human strongyloidiasis [5,8,24]. The SignalP prediction analysis showed that 137 proteins (15.34 % of the total) identified can be considered as secreted proteins, and 50 of these (36.5 % of SignalP+ proteins) were also predicted to have transmembrane domain(s). Overall, these putatively secreted proteins were observed to participate in a range of biological processes within this parasitic life stage. Eighty-seven proteins (9.7 % of the total) were positive exclusively for SignalP analyses. Among them, we can highlight cathepsins B, D, Z, and L.1., one metalloendopeptidase, and 2 CAP domain-containing proteins. When considering only the TMHMM analysis, 42 (4.7 %) identities were categorized as transmembrane proteins, such as peptidylprolyl isomerase, putative metalloproteinase inhibitor tag-225, CD09 antigen, cytochrome b5, laminin subunit alpha-2, and some phosphatases and oxidases. Some proteins were positive for both SignalP and TMHMM analyses, including a series of metalloproteases 3
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Fig. 3. Domains and TMHMM/SignalP predictions for uncharacterized proteins within the UniProt S. venezuelensis iL3 proteome.
fatty acid binding protein (8 identities; 7 SignalP+/1 Both+), zinc binding protein (7 identities; 3 SignalP+/3 TMHMM+/1 Both+), transthyretin-like (7 identities; 4 Both+/3 SignalP+), col-cuticle-like N (6 identities; all TMHMM+), MOLO1 (4 identities; 2 Both+/2 SignalP+), EGF-like (4 identities; 3 Both+/1 SignalP+), BPTI Kunitz inhibitor (4 identities; 3 SignalP+/1 Both+), and lysozyme-like (3 identities; all SignalP+) (Fig. 3). Our data complement the results of recently published reports on secretome analysis of S. venezuelensis [9] and S. ratti [8]. Comparing the current results with a previous proteomic investigation of iL3 S. venezuelensis [9], the list of proteins identified has increased. The published data [9] represent the excretory/secretory products, whereas the present results demonstrate the somatic content. Considering only proteins positive for the SignalP criteria, a total of 144 proteins were identified in the present study compared with 68 obtained previously [9]; both studies share 49 proteins. Of the 94 different proteins that have been identified, 50 are uncharacterized proteins. Based on specificity as an important criterion for a good diagnostic marker, we can emphasize the importance of the analysis of uncharacterized proteins specific to a parasite. Thus, the research on the domains revealed proteins with great parasitic importance, such as CAP domain, transthyretin, BTPI inhibitors, astacins, among others, which may be further studied in future in the context of the Strongyloides genus. Proteins in the CAP domain have demonstrated great diversity; some in the TMHMM domain, SignalP, or both, consequently representing several functions inside or outside the cell. CAP domains are among the most prevalent domains across the helminth secretomes and may be associate with larval migration and evasion of the host immune
(4 zin. metalloproteinases and 1 matrix metalloendopeptidase protein), laminin subunit alpha-5, 3 CAP domain-containing proteins, and peptidylprolyl cis trans isomerase. Of the 5 galectins identified, only one has the transmembrane domain. In the present study, several proteases were identified that, in nematodes, can be involved in the digestion of proteins associated with the old cuticle [19] and in the degradation of host tissues [25]. The metalloproteases were identified within the genus Strongyloides [7,8,13] and described as immunogenic in strongyloidiasis [5] and angiostrongyliasis [18]. Cathepsins are cysteine-proteases and involved in different proteolytic activities of helminths [26], such as in the penetration process and migration of iL3 into the host organism. The identification of different proteases in the soluble extract of iL3 S. venezuelensis reinforces that this developmental stage involves several processes, acting on parasite and host-derived proteins. However, evidence of their value in the diagnosis of strongyloidiasis has not yet been elucidated. A good diagnostic marker can be defined as a protein capable of activating the immune system of hosts and be specific to the parasite. Considering the contact with the host, secreted proteins represent a large study repertoire to be explored. The identification of secreted proteins of helminths may provide a knowledge base for the development of new parasite control measures based on vaccines and new diagnostic tools [25]. A set of 93 proteins that were considered positive by SignalP, TMHMM, or both analyses are still described as uncharacterized within the UniProt database, although 80 of these are annotated within 31 different protein domains. Forty-three of these uncharacterized proteins can be categorized within 8 protein domains: 4
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response [27]. In addition, CAP domains have also been associated with immunomodulatory and immunogenic activity [28], which may be explored in the future as a diagnostic marker for strongyloidiasis. There is strong evidence that proteinase inhibitors, such as BTPI Knutiz, may be secreted by parasitic nematodes to counteract attack by host proteinases, providing the parasite with protection [13]. These inhibitors are among the upregulated protein family in the parasite females of S. ratti and S. stercoralis [7,8] and are highly abundant in S. venezuelensis iL3 E/S products [9]. Recently, the importance of Knutiz inhibitors in Schistosoma mansoni infection has been described by Morais et al. [29] as a key protein in parasite survival and maintenance inside their mammalian hosts. Transthyretin is a nematode-specific protein and is highly expressed in the parasitic stages of S. ratti and S. stercoralis [7]. Among the uncharacterized proteins, many have the transthyretin domain. This observation might lead to novel markers for strongyloidiasis. We believe that this group of proteins can be explored in the future as possible diagnostic markers. Our results point to a catalog of possible diagnostic targets for human strongyloidiasis, reinforce new evaluations of galectins and 143-3 zeta protein, as well as the need for studies on the effective applicability of metalloproteinases and cathepsins in the diagnosis of strongyloidiasis. In addition, they highlight the need for evaluations of uncharacterized proteins, especially the proteins within the CAP domain, transthyretin, and BTPI inhibitor domains, as a repertoire as yet unexplored in the context of strongyloidiasis diagnostic markers. This study provides an overview of the iL3 protein S. venezuelensis focusing on the diagnosis of strongyloidiasis. We believe that the protein profile presented in this shotgun analysis extends our understanding of the protein composition within the genus Strongyloides, opening up new perspectives for biomarker research that may help in the diagnosis of human strongyloidiasis.
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Financial support This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (grant no.2016/06185-0),Brazil (to F.M.P). Declaration of competing interest The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.molbiopara.2019. 111249. References [1] Z. Bisoffi, D. Buonfrate, A. Montresor, A. Requena-Méndez, J. Muñoz, A.J. Krolewiecki, E. Gotuzzo, M.A. Mena, P.L. Chiodini, M. Anselmi, J. Moreira, M. Albonico, Strongyloides stercoralis: a plea for action, PLoS Negl. Trop. Dis. 7 (2013) e2214. [2] R. Toledo, C. Munoz-Antoli, J.G. Esteban, Strongyloidiasis with emphasis on human infections and its different clinical forms, Adv. Parasitol. 88 (2015) 165–241. [3] M.A. Levenhagen, J.M. Costa-Cruz, Update on immunologic and molecular diagnosis of human strongyloidiasis, Acta Trop. 135 (2014) 33–43. [4] M. Viney, T. Kikuchi, Strongyloides ratti and S. Venezuelensis - rodent models of Strongyloides infection, Parasitology 144 (2017) 285–294. [5] M.A. Corral, F.M. Paula, Meisel DMCL, V.L.P. Castilho, E.M.N. Gonçalves, D. Levy, S.P. Bydlowski, P.P. Chieffi, W. Castro-Borges, R.C.B. Gryschek, Potential immunological markers for diagnosis of human strongyloidiasis using heterologous antigens, Parasitology 144 (2017) 124–130. [6] A. Marcilla, J. Sotillo, A. Perez-Garcia, R. Igual-Adell, M.L. Valero, M.M. SánchezPino, D. Bernal, C. Muñoz-Antolí, M. Trelis, R. Toledo, J.G. Esteban, Proteomic analysis of Strongyloides stercoralis L3 larvae, Parasitology 137 (2010) 1577–1583.
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Shotgun proteomics of Strongyloides venezuelensis infective third stage larvae: Insights into host–parasite interaction and novel targets for diagnostics
T
Priscilla D.M. Fonsecaa,b, Marcelo A. Corrala, Miguel Cosenza-Contrerasd, Dirce M.C.L. Meisela, Gessica B. Melob, Milena M.S. Antunesa, Maria C.E. Santoa, Ronaldo C.B. Gryscheka,b, Julia M. Costa-Cruzc, William Castro-Borgesd, Fabiana M. Paulaa,b,* a
Laboratório De Investigação Médica Da Faculdade De Medicina Da Universidade De São Paulo, São Paulo, Brazil Instituto De Medicina Tropical Da Faculdade De Medicina Da Universidade De São Paulo, São Paulo, Brazil c Laboratório De Diagnostico De Parasitoses, Instituto De Ciências Biomédicas Da Universidade De Federal De Uberlândia, Uberlândia, Minas Gerais, Brazil d Laboratório De Enzimologia e Proteômica, Instituto De Ciências Exatas e Biológicas, Universidade Federal De Ouro Preto, Ouro Preto, Minas Gerais, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Strongyloides venezuelensis Shotgun proteomics Diagnostic markers
Strongyloides venezuelensis is an important alternative source of antigen for the serologic diagnosis of human strongyloidiasis. Proteomics techniques applied to the analysis of the protein content of infective third stage larvae (iL3) of S. venezuelensis provide a powerful tool for the discovery of new candidates for immunodiagnosis. This study presents an overview of the protein iL3 S. venezuelensis focusing on the diagnosis of strongyloidiasis. A total of 877 proteins were identified by shotgun proteomics. Many of these proteins are involved in different cellular processes, metabolic as well as structural maintenance. Our results point to a catalog of possible diagnostic targets for human strongyloidiasis and highlight the need for evaluation of uncharacterized proteins, especially the proteins within the CAP domain, transthyretin, and BTPI inhibitor domains, as a repertoire as yet unexplored in the context of strongyloidiasis diagnostic markers. We believe that the protein profile presented in this shotgun analysis extends our understanding of the protein composition within the Strongyloides genus, opening up new perspectives for research on biomarkers that may help with the diagnosis of human strongyloidiasis. Data are available via ProteomeXchange with identifier PXD013703.
Strongyloides stercoralis is a causative agent of human strongyloidiasis, affecting approximately 350 million people worldwide, especially in tropical and subtropical areas [1]. This helminthiasis results in an asymptomatic chronic disease, which can be maintained for decades by a process of autoinfection and may be related to hyperinfection or disseminated disease, with high mortality rates, especially in immunocompromised patients [2]. The definitive diagnosis of S. stercoralis infection is made by detecting larvae in fecal samples, but it has low sensitivity [2,3]. Thus, serologic methods have been proposed as a complement to the parasitologic diagnosis. In this context, the research using heterologous antigen, Strongyloides venezuelensis, has been highlighted in the literature [3], mainly because it can be easily obtained in large quantities, allowing the development of new diagnostic approaches of strongyloidiasis [3–5]. Recent studies show that identification and characterization of Strongyloides proteins are essential for a better understanding of the
host–parasite relationship, as well as for identification of new diagnosis targets [6,7]. Proteomic studies in Strongyloides sp. have been based on the analysis of proteins by immunoblotting [5], somatic extracts [6], or secretion/excretion products [8,9], however these efforts have been restricted. There is an urgent need for new biomarkers that can be used for diagnosis. In addition, a marker for diagnosis must be parasite specific and be recognized by the host immune system. Considering S. venezuelensis as an important alternative source of antigen for the serologic diagnosis of human strongyloidiasis, shotgun analysis of the protein content of infective third stage larvae (iL3) of S. venezuelensis provides a powerful tool for the discovery of new candidates for immunodiagnosis. The present study describes the shotgun analysis of S. venezuelensis iL3. The research was carried out on infective third stage larvae (iL3) of S. venezuelensis obtained from stool cultures of experimentally infected Rattus norvegicus. The cultures were performed with stool samples collected on the day 8 after infection, homogenized with charcoal (ratio of
⁎ Corresponding author at: Laboratório de Investigação Médica (LIM/06), Instituto de Medicina Tropical, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000, São Paulo, SP, Brazil. E-mail addresses: [email protected], [email protected] (F.M. Paula).
https://doi.org/10.1016/j.molbiopara.2019.111249 Received 30 August 2019; Received in revised form 20 December 2019; Accepted 23 December 2019 Available online 24 December 2019 0166-6851/ © 2019 Published by Elsevier B.V.
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each spectrum was later filtered with the Search Engine Processor (SEPro) with the following parameters: delta mass of 30 ppm, total delta CN of 0.001, minimum of 6 amino acids per peptide, minimum of 1 peptide per protein, 1 spectral count, PSM (peptide spectrum matches) delta mass of 10 ppm, and a normalized primary score (XCorr) ≥2.0 for proteins with 1 mass spectrum and ≥1.8 for proteins with ≥2 mass spectra. A Bayesian score (XCorr, delta CN, peak matched values, delta mass error, spectral count score, and secondary rank) was used to sort the identification in a non-decreasing order of confidence. In addition, a cutoff was applied to accept a false discovery rate of 3 % for spectra, 2 % for peptide, and 1 % for protein (based on labeled decoy identifications accepted into the discovery list). For identification, all proteins were considered in order to increase the identification of lower molecular weight proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [14] partner repository with the dataset identifier PXD013703. The proteins identified were categorized based on their molecular function as presented in the Gene Ontology (GO) database [15]. GO term to Uniprot ID mapping was performed from the S. venezuelensis reference proteome (UniProt proteome ID: UP000035680) [13]. Identified protein sequences were submitted to SignalP-5.0 [16] and TMHMM Server v.2.0 [17] for the prediction of signal peptides and transmembrane domains, respectively. Two logical variables were created to indicate whether a protein passed the criteria to be considered a signal peptide and/or presented transmembrane helice(s). A few proteomic datasets were recently published for members of genus Strongyloides [6–9]. On the other hand, the S. venezuelensis proteome has not yet been reported, especially in the context of new targets for diagnosis. Considering the good results for the sensitivity and specificity of the heterologous antigen for the diagnosis of human strongyloidiasis, mainly of iL3 S. venezuelensis, the present study evaluated the shotgun proteomic analysis of this developmental stage. Shotgun proteomics analyses identified a total of 877 proteins in S. venezuelensis iL3 within the Uniprot database (Supplementary Table 1). In addition, 87.34 % of the proteins obtained had > 2 unique peptides. The dynamic range of protein abundance revealed a variation of more than 3 orders of magnitude in terms of the spectral counts of proteins in the extract (Fig. 1A). Similarly, the cumulative abundance plot defined 82 proteins representing 50 % of the total mass, and 385 proteins made up 90 % of the extract. A set of 491 lowly abundant proteins were also identified, accounting for 10 % of the total spectral abundance (Fig. 1B). The number total of proteins identified in the soluble extract of iL3 S. venezuelensis reinforces the intense adaptation activity of this developmental stage within the host. GO enrichment analysis of the larval protein set within the whole S. venezuelensis predicted proteome identified 85 molecular function-related GO (GO-MF) terms. Among these, 5 GO-MF terms presented the higher protein counts: oxidoreductase activity (101 identities), cofactor binding (80 identities), protein binding (61 identities), and coenzyme binding (58 identities) (Fig. 2). Infective third stage larvae of Strongyloides actively penetrate the skin or mucosa of the host and migrate through the host body to reach the small intestine [4]. That might explain the large amount of enzyme, which makes up half of the total mass of the protein extract. Among the 82 most abundant proteins, actin is a dominant constituent. Not surprisingly, structural proteins, such as actin and myosin, were the most abundant in iL3 S. venezuelensis extract. These proteins can play important roles in maintaining the body shape and muscle integrity of the nematodes [18], and are probably involved in migration in the host at this developmental stage. In addition, our group demonstrated recognition of structural proteins by antibodies present in patients with strongyloidiasis [5]. However, we believe that the structural proteins, due to their high similarity among organisms, cannot be considered as a good diagnostic marker, and further studies are necessary.
1 g of stool to 3 g of charcoal) and water, followed by incubation at 28 °C for 48 h. After this procedure, iL3 was recovered according to the modified Baermann method [10] and stored at −20 °C for later extraction of protein. All procedures were conducted in accordance with the ethical guidelines adopted by the Comissão de Ética no Uso de Animais (CEUA) of Instituto de Medicina Tropical, Universidade de São Paulo (protocol CEP-IMT 317A and 0356A). Soluble proteins of S. venezuelensis were obtained with approximately 200,000 iL3 in 1 mL of 25 mM Tris−HCl (pH 7.5) containing 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The samples were homogenized on ice in a tissue homogenizer (TissueTearor, BioSpec Products, Bartlesville, OK) for 5 cycles of 5 min each. The resulting suspension was centrifuged at 12,400 × g for 30 min at 4 °C followed by recovery of the supernatant containing the soluble proteins. Protein content was obtained using a Bio-Rad RC DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Three replicates of soluble proteins of S. venezuelensis iL3 were prepared. Fifty micrograms of soluble proteins were diluted in 25 mM ammonium bicarbonate (0.06 % w/v) and then used for the digestion procedure. Proteins samples were incubated with 1 % RapiGest (Waters, Milford, MA) at 80 °C for 5 min. Then, the proteins were reduced using dithiothreitol (Sigma-Aldrich) to a final concentration of 3.3 mM at 60 °C for 10 min and alkylated using iodoacetamide (SigmaAldrich) to a final concentration of 9.4 mM in the dark for 45 min. After this process, the samples were digested by addition of trypsin (Promega, Madison, WI) at a ratio of 50:1 (protein/trypsin) followed by incubation for 16 h at 37 °C. Tryptic digestion was halted by acidifying the sample by adding 0.5 % (v/v) acetic acid (J.T. Baker, Center Valley, PA). The samples were incubated at 37 °C for 45 min for RapiGest acid-induced cleavage, and the salt was removed by centrifugation at 20,000 × g for 15 min at 7 °C. The samples were then ready for mass spectrometry analysis. Liquid chromatography was performed on a nanoUHPLC Dionex Ultimate 3000 (Thermo Scientific, Bremen, Germany) equipped with an Acer PepMap100 C18 Nano-Trap column (100 μm ×2 cm, 5 μm beads, 100 Å pores; Thermo Scientific) followed by an analytical Acclaim PepMap100 C18 column (75 μm ×15 cm, 2 μm beads, 100 Å pores; Thermo Scientific). A nonlinear gradient of eluent A (0.1 % formic acid) to B (0.1 % formic acid in 80 % acetonitrile) was applied. Peptides were ionized using a nano electrospray ion source and detected on a QExactive mass spectrometer (Thermo Scientific). The ion source operated at 3.8 kV in positive mode with a capillary temperature of 250 °C. The precursor ions were measured with the resolution set at 70,000 (range, 300–2000 m/z) and were selected for fragmentation by a datadependent method in which the top 12 most intense ions with charges between +2 and +4 were selected, given an exclusion time of 40 s. Then, a high-energy collisional dissociation method was applied with a normalized collision energy of 28–30 V to obtain the fragments. Tandem mass spectra were obtained with the resolution set at 17,500 (minimum of 120 m/z and isolation widow of 1.2 m/z; maximum injection time of 60 ms). The mass spectrometric data obtained from three independent nanoLC-MS/MS runs were combined using the Patternlab software to increase the chance of protein identification and therefore provide an improved compositional analysis of the iL3 soluble fraction. Database searching was performed using the Patternlab for proteomics software [11] and the UniProt compilation database [12], considering S. venezuelensis and totaling 16,639 sequences. In addition, spectra were searched in the S. venezuelensis database [9,13]. The search was performed using the Comet algorithm, applying in silico digestion parameters as follows: search performed in a semi-specific tryptic digestion space, up to 2 missed cleavage sites, cysteine carbamidomethylation (+57.02146 Da) as fixed and methionine oxidation (+15.9949 Da) as variable posttranslational modifications (up to 3 variable modifications per peptide), MS1 mass range of 550–5500 Da (10 ppm mass tolerance), and B, Y, and neutral loss MS2 fragmentation. The list of candidates for 2
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Fig. 1. Protein distribution in extracts from S. venezuelensis iL3. (A) Dynamic abundance plot based on the number of peptide spectral counts, normalized using the spectral abundance factor (NSAF). There is variation of more than 3 orders of magnitude in terms of protein abundance in the extract. (B) Protein abundance in extracts represented as a cumulative frequency plot revealed that 82 constituents contributed to 50 % of the total mass.
Even among the abundance of constituents, we can highlight different enzymes, particularly kinases (arginine, nucleoside diphosphate, phosphoenolpyruvate carboxykinase, pyruvate and phosphoglycerate), aldolase (fructose biphosphate aldolase), isomerases (triosephosphate, protein disulfide, peptidylprolyl and glucose 6-phosphate), dehydrogenases (malate, glyceraldehyde-3-phosphate and retinal dehydrogenase 2), aminotransferases (aspartate, alanine aminotransferase 1,4-aminobutyrate), and synthases (citrate, ATP synthase subunit beta, hematopoietic prostaglandin D). The kinases are known to act in a variety of cellular signaling processes [19], and isomerase are keys enzymes in glycolysis and catalyze the reversible isomerization of different molecules [20]. Aldolase has been associated with antigenic properties and mediating interactions with the host [21]. The identification of these proteins may suggest that they play a fundamental role in the parasite–host interaction. In addition, the most abundant components include the following proteins: CAP domain protein, profilin, 14-3-3 zeta protein, nucleoredoxin-like protein 2, calmodulin, superoxide dismutase, enolase, ferritin, and heat shock proteins (HSP60 and HSP70). Antioxidant enzymes such as superoxide dismutase can act to protect the innate immune response of the host [19], favoring parasite survival inside the host body. Some other components were identified such as galectin and 14-3-3 protein. The biological function of galectins in nematodes is not well understood but has been related to host survival and interaction, as well as to negative regulation of host innate immunity [22]. The 14-3-3 protein may be related to proliferation, migration, and morphologic changes during the life cycle of the parasite [23]. Galectin and 14-3-3
Fig. 2. Molecular function by Gene Ontology analysis of identified proteins in extracts from S. venezuelensis iL3.
protein have been indicated in recent research as possible diagnostic markers for human strongyloidiasis [5,8,24]. The SignalP prediction analysis showed that 137 proteins (15.34 % of the total) identified can be considered as secreted proteins, and 50 of these (36.5 % of SignalP+ proteins) were also predicted to have transmembrane domain(s). Overall, these putatively secreted proteins were observed to participate in a range of biological processes within this parasitic life stage. Eighty-seven proteins (9.7 % of the total) were positive exclusively for SignalP analyses. Among them, we can highlight cathepsins B, D, Z, and L.1., one metalloendopeptidase, and 2 CAP domain-containing proteins. When considering only the TMHMM analysis, 42 (4.7 %) identities were categorized as transmembrane proteins, such as peptidylprolyl isomerase, putative metalloproteinase inhibitor tag-225, CD09 antigen, cytochrome b5, laminin subunit alpha-2, and some phosphatases and oxidases. Some proteins were positive for both SignalP and TMHMM analyses, including a series of metalloproteases 3
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Fig. 3. Domains and TMHMM/SignalP predictions for uncharacterized proteins within the UniProt S. venezuelensis iL3 proteome.
fatty acid binding protein (8 identities; 7 SignalP+/1 Both+), zinc binding protein (7 identities; 3 SignalP+/3 TMHMM+/1 Both+), transthyretin-like (7 identities; 4 Both+/3 SignalP+), col-cuticle-like N (6 identities; all TMHMM+), MOLO1 (4 identities; 2 Both+/2 SignalP+), EGF-like (4 identities; 3 Both+/1 SignalP+), BPTI Kunitz inhibitor (4 identities; 3 SignalP+/1 Both+), and lysozyme-like (3 identities; all SignalP+) (Fig. 3). Our data complement the results of recently published reports on secretome analysis of S. venezuelensis [9] and S. ratti [8]. Comparing the current results with a previous proteomic investigation of iL3 S. venezuelensis [9], the list of proteins identified has increased. The published data [9] represent the excretory/secretory products, whereas the present results demonstrate the somatic content. Considering only proteins positive for the SignalP criteria, a total of 144 proteins were identified in the present study compared with 68 obtained previously [9]; both studies share 49 proteins. Of the 94 different proteins that have been identified, 50 are uncharacterized proteins. Based on specificity as an important criterion for a good diagnostic marker, we can emphasize the importance of the analysis of uncharacterized proteins specific to a parasite. Thus, the research on the domains revealed proteins with great parasitic importance, such as CAP domain, transthyretin, BTPI inhibitors, astacins, among others, which may be further studied in future in the context of the Strongyloides genus. Proteins in the CAP domain have demonstrated great diversity; some in the TMHMM domain, SignalP, or both, consequently representing several functions inside or outside the cell. CAP domains are among the most prevalent domains across the helminth secretomes and may be associate with larval migration and evasion of the host immune
(4 zin. metalloproteinases and 1 matrix metalloendopeptidase protein), laminin subunit alpha-5, 3 CAP domain-containing proteins, and peptidylprolyl cis trans isomerase. Of the 5 galectins identified, only one has the transmembrane domain. In the present study, several proteases were identified that, in nematodes, can be involved in the digestion of proteins associated with the old cuticle [19] and in the degradation of host tissues [25]. The metalloproteases were identified within the genus Strongyloides [7,8,13] and described as immunogenic in strongyloidiasis [5] and angiostrongyliasis [18]. Cathepsins are cysteine-proteases and involved in different proteolytic activities of helminths [26], such as in the penetration process and migration of iL3 into the host organism. The identification of different proteases in the soluble extract of iL3 S. venezuelensis reinforces that this developmental stage involves several processes, acting on parasite and host-derived proteins. However, evidence of their value in the diagnosis of strongyloidiasis has not yet been elucidated. A good diagnostic marker can be defined as a protein capable of activating the immune system of hosts and be specific to the parasite. Considering the contact with the host, secreted proteins represent a large study repertoire to be explored. The identification of secreted proteins of helminths may provide a knowledge base for the development of new parasite control measures based on vaccines and new diagnostic tools [25]. A set of 93 proteins that were considered positive by SignalP, TMHMM, or both analyses are still described as uncharacterized within the UniProt database, although 80 of these are annotated within 31 different protein domains. Forty-three of these uncharacterized proteins can be categorized within 8 protein domains: 4
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response [27]. In addition, CAP domains have also been associated with immunomodulatory and immunogenic activity [28], which may be explored in the future as a diagnostic marker for strongyloidiasis. There is strong evidence that proteinase inhibitors, such as BTPI Knutiz, may be secreted by parasitic nematodes to counteract attack by host proteinases, providing the parasite with protection [13]. These inhibitors are among the upregulated protein family in the parasite females of S. ratti and S. stercoralis [7,8] and are highly abundant in S. venezuelensis iL3 E/S products [9]. Recently, the importance of Knutiz inhibitors in Schistosoma mansoni infection has been described by Morais et al. [29] as a key protein in parasite survival and maintenance inside their mammalian hosts. Transthyretin is a nematode-specific protein and is highly expressed in the parasitic stages of S. ratti and S. stercoralis [7]. Among the uncharacterized proteins, many have the transthyretin domain. This observation might lead to novel markers for strongyloidiasis. We believe that this group of proteins can be explored in the future as possible diagnostic markers. Our results point to a catalog of possible diagnostic targets for human strongyloidiasis, reinforce new evaluations of galectins and 143-3 zeta protein, as well as the need for studies on the effective applicability of metalloproteinases and cathepsins in the diagnosis of strongyloidiasis. In addition, they highlight the need for evaluations of uncharacterized proteins, especially the proteins within the CAP domain, transthyretin, and BTPI inhibitor domains, as a repertoire as yet unexplored in the context of strongyloidiasis diagnostic markers. This study provides an overview of the iL3 protein S. venezuelensis focusing on the diagnosis of strongyloidiasis. We believe that the protein profile presented in this shotgun analysis extends our understanding of the protein composition within the genus Strongyloides, opening up new perspectives for biomarker research that may help in the diagnosis of human strongyloidiasis.
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