- Email: [email protected]
Proteolytic and nematicidal potential of the compost colonized by Hypsizygus marmoreus
Experimental Parasitology 197 (2019) 16–19
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Proteolytic and nematicidal potential of the compost colonized by Hypsizygus marmoreus
T
Filippe Elias de Freitas Soaresa,b,∗, Vânia Mayumi Nakajimaa, Bruna Leite Sufiatea, Luana Aparecida Simões Satirob, Elias Honorato Gomesb, Frederico Vieira Fróesc, Fabio Porto Senac, Fabio Ribeiro Bragac, José Humberto de Queiroza a
Federal University of Viçosa, Biochemistry and Molecular Biology Department, Viçosa, Minas Gerais, Cep: 3657000, Brazil State University of Minas Gerais, Campus Ubá, Av. Olegário Maciel, 1427, Ubá, Minas Gerais, Cep: 36500-000, Brazil c Vila Velha University, Rua Viana, Soteco, Vila Velha, ES, CEP 29106-091, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Nematicidal Spent mushroom compost Hypsizygus marmoreus Protease In silico
Spent mushroom compost (SMC) is a residue generated in edible mushrooms production, such as Hypsizygus marmoreus. Its genome was recently sequenced, demonstrating cuticle-degrading protease genes. The present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae. The extraction of the crude extract directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ± 18.38 U g−1 of compound. Moreover, the zymogram and SDS-PAGE indicated the presence of two proteases with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, there was a significant reduction in the number of Panagrellus redivivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively. A0A151VWY3 mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.
1. Introduction Diseases caused by nematodes constitute the major parasitic diseases of plants, animals and humans, having great economic impact on agriculture and animal husbandry (Lustigman et al., 2012; Singh et al., 2013; Charlier et al., 2014). The chemical control of these parasites using anthelmintics in large scale can induce parasitic resistance and has potential for bioaccumulation, which can lead to soil and groundwater contamination (Horvat et al., 2012). Given this context, it is necessary to use forms of control less aggressive to the environment, demanding investigations of these alternatives feasibility. In addition, the reuse of agro-industrial waste also plays a key role in avoiding environmental contamination. Spent mushroom compost (SMC), a residue generated in edible mushrooms production, can be reused as animal feed, organic fertilizer, soil conditioner, plant substrate, and in bioremediation and cleaning processes of contaminated soil and water (Phan and Sabaratnam, 2012; Grujić et al., 2015). Our ∗
research group aims to evaluate SMC's potential in control nematode infested soils. Many microorganisms can attack and destroy nematodes through several processes such as capture, parasitism and production of toxins and enzymes (Yang et al., 2013; Sivanandhan et al., 2017). Hypsizygus marmoreus is an edible mushroom consumed worldwide whose genome was recently sequenced, demonstrating cuticle-degrading protease genes (Zhang et al., 2010). However, there are no studies regarding the action of these proteases on nematodes. Thus, the present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae.
Corresponding author. Federal University of Viçosa, Biochemistry and Molecular Biology Department, Viçosa, Minas Gerais, Cep: 3657000, Brazil. E-mail address: fi[email protected] (F.E.d.F. Soares).
https://doi.org/10.1016/j.exppara.2018.12.006 Received 11 February 2018; Received in revised form 1 November 2018; Accepted 28 December 2018 Available online 05 January 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.
Experimental Parasitology 197 (2019) 16–19
F.E.d.F. Soares et al.
2. Materials and methods
larvae present in each tube was counted using an optical microscopy according to the modified methodology described by Soares et al. (2013). Statistical difference between the groups was evaluated by unpaired T test. Differences were considered significant when p ≤ 0.01. All analyses were performed using the software GraphPad Prism 5 for Windows version 5.00 (GraphPad Software, Inc.). Subsequently, the larvae reduction percentage average was calculated according to the following equation:
2.1. Spent mushroom compost crude extract Hypsizygus marmoreus SMC was obtained from the Urakami Group, Mogi das Cruzes, São Paulo – Brazil. Five grams (wet weight) were mixed with 50 ml distilled water. These mixtures were incubated at 30 °C for 1 h with shaking at 180 rpm, filtered and centrifuged at 10,000×g at 4 °C for 10 min. The supernatant was used as crude extract.
%Reduction ( Number of larvae in Control − Number of larvae in Treatment ) = x Number of larvae in Control
2.2. Obtaining bovine infective larvae (L3) Infective larvae (L3) of nematodes belonging to superfamilies Trichostrongyloidea (Haemonchus spp., Cooperia spp.) and Strongyloidea (Oesophagostomum spp.) were obtained from naturally infected bovine faeces, which were collected and mixed with vermiculite. The coproculture was moistened and kept in an incubator at 28 °C, where it remained for 14 days, for the development of infective larvae (L3). The larvae were recovered by the Baermann technique.
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2.6. In silico analysis FASTA sequences of cuticle-degrading proteases from H. marmoreus were retrieved from UNIPROTKB (ID A0A151VWY3, A0A151WD28 and A0A151VEK8). Signal sequences were predicted using SignalP 4.0 server. Theoretical protein isoelectric point (pI) and molecular weight were calculated using the pI/MW tool (http://web.expasy.org/ compute_pi/). H. marmoreus cuticle-degrading proteases 3D structures were predicted by protein modeling using SWISS-MODEL (http://swissmodel. expasy.org/). Verticillium psalliotae Cuticle-Degrading Protease (ver112) (UniProt Acession N° Q68GV9) was used as template.
2.3. Enzymatic activity Protease activity was determined by the caseinolytic method. Five hundred μL of casein substrate (1% w/v, pH 8), 450 μL of 50 mM citrate-phosphate buffer (pH 5), and 50 μL of the crude extract were incubated for 20 min, at 50 °C. The reaction was stopped by adding 1 mL 10% w/v trichloroacetic acid (TCA). Then the reaction medium was centrifuged at 10,000×g for 5 min and the supernatant collected to determine the absorbance at 280 nm in a spectrophotometer. The enzyme unit (U) was expressed as the amount of enzyme capable of releasing 1 μg of tyrosine per minute. To estimate the amount of tyrosine released after the reaction, a standard curve was constructed using increasing amounts of tyrosine diluted in aqueous solution. The reaction blank consisted of 500 μl of buffer, 500 μl of 1% w/v casein and 1 ml of 10% w/v TCA solution.
3. Results The crude extract extraction directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ± 18.38 U g−1 of compound. Moreover, the formation of unstained regions in the zymogram (Fig. 1) indicated the presence of two proteases. The corresponding bands were excised and analyzed again by SDS-PAGE. Thus, two proteins (Fig. 2) were observed, with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, its nematicidal effect on P. redvivus and bovine infective larvae were evaluated. After 24 h of treatment, there was a significant reduction in
2.4. Zymography and SDS-PAGE Protease zymograms of the crude extracts were performed in 10% SDS-polyacrylamide gel co-polymerized with 0.1% (w/v) gelatin and 0.1% (w/v) casein. After electrophoresis, the gel was washed with 2.5% Triton X-100 solution for 1 h at room temperature. Then, the gel was incubated in citrate-phosphate buffer pH 8 for 1 h at 50 °C, subsequently stained with Coomassie brilliant blue R-250 0,1% (w/v) dissolved in ethanol: acetic acid: water (30:7:63), and then destained. The digested bands were visualized as unstained regions in the gels. The digested protein bands were then excised from the gel, grounded in a mortar using 50 mM Tris–HCl (pH 8.0) and centrifuged at 8000×g for 15 min at 4 °C to remove gel particles. The supernatant was used in a new 10% SDS-PAGE analysis (Laemmli, 1970). Electrophoresis was performed at 100 V, and the gel was stained with silver nitrate to allow protein visualization. 2.5. Nematicidal activity Nematicidal activities of SMC crude extract were evaluated on Panagrellus redivivus and bovine infective larvae (L3). The assay was composed of treatment and control groups. Six replicates were performed for each group using sterile tubes. In the treated group, about 40 P. redvivus or 50 bovine infective larvae (L3) were poured into sterile tubes containing H. marmoreus SMC crude extract. The control group contained approximately 40 P. redvivus or 50 bovine infective larvae (L3) in the presence of the boiled extract, containing the denatured proteases (no activity). The tubes were incubated at 28 °C in the dark for 24 h. After this period, the total number of
Fig. 1. Protease zymogram of the extract from Hypsizygus marmoreus spent mushroom compost. A: SDS-PAGE co-polymerized with 0.1% (w/v) gelatin. B: SDS-PAGE co-polymerized with 0.1% (w/v) casein. 17
Experimental Parasitology 197 (2019) 16–19
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Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of excised band from zymogram of the extract from Hypsizygus marmoreus spent mushroom compost.
the number of P. redvivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively (Fig. 3). Considering the nematicidal activity, the proteases that could be acting were investigated. H. marmoreus has three cuticle-degrading proteases sequences deposited in UNIPROTKB (ID A0A151VWY3, A0A151WD28 and A0A151VEK8). A0A151VWY3 has 394 amino acid residues, with a peptide signal of 19 residues. Mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 has 387 amino acid residues, with a peptide signal of 19 residues. Mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. A0A151VEK8 has 559 amino acid residues, the mature protein is composed of 248 amino acid residues, with molecular weight of 24.1 kDa and pI 7.15. However, since A0A151VEK8 is a transmembrane protein, the possible H. marmoreus cuticle-degrading proteases in the extract are A0A151VWY3 and A0A151WD28. These two proteases 3D structures can be seen in Fig. 4.
Fig. 4. Protease structure models predicted in silico for the both proteases present in the extract from Hypsizygus marmoreus spent mushroom compost, using Verticillium psalliotae Cuticle-Degrading Protease (ver112) as template.
4. Discussion SMCs contain many residual nutrients and enzymes, such as proteases, cellulases, hemicellulases, lignin peroxidase, manganese
*
*
Fig. 3. Nematicidal activity of crude extract from Hypsizygus marmoreus spent mushroom compost on Panagrellus redivivus and bovine infective larvae (L3). The asterisk denotes a difference (p < 0.01). 18
Experimental Parasitology 197 (2019) 16–19
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References
peroxidase and laccase (Lau et al., 2003). The present work showed that SMC from H. marmoreus has protease content, which can be quickly and easily extracted. Edible mushrooms and their proteases presented activity against nematodes (Barron and Thorn, 1987; Genier et al., 2015; Sufiate et al., 2017). However, this is the first work demonstrating protease activity of SMC extract on nematodes. P. redivivus is a free living nematode usually used as a model in bioassays evaluating plant extracts or compounds for nematicidal action (Herbert-Doctor et al., 2016). Since the nematicidal effect in this nematode is indicative of action in another nematodes species, our results in P. redivivus show the potential of SMC extract to control plant and animal parasitic nematodes. In order to evaluate the potential nematicidal activity against animal parasitic nematodes, we used bovine infective larvae. These nematodes are difficult to control due to anthelmintics resistance development, demanding alternative treatments. Here we present the possibility of using an extract obtained from an agroindustrial residue, presenting low cost. Still, the use of enzymes from SMC extract for the biocontrol has the advantage of using an environmentally friendly, effective and affordable alternatives for nematode control (Geng et al., 2016). Chemical nematicides that are commonly used to control nematodes are toxic and costly to the environment and human health (Riga, 2011), highlighting the advantage of the extract. H. marmoreus has three known cuticle-degradating proteases sequences; however A0A151VEK8 is a transmembrane protein. Once the extraction method recovers only the extracellular enzymes, we suggest that A0A151VWY3 and A0A151WD28 (Fig. 4) were the proteases observed in this article. Furthermore, the molecular weights estimated by SDS-PAGE are similar to A0A151VWY3 and A0A151WD28 molecular weights obtained by in silico analysis. The SignalP 4.0 server predicts that the most likely N-terminal cleavage sites of the sequences A0A151VWY3 and A0A151WD28 are between 19 and 20 residues for both, and for sequence A0A151VEK8 there was no predicted signal peptide. A propeptide was identified for each sequence. This propeptide acts as a temporary inhibitor to assist in the mature peptidase folding, and it is removed by proteolytic cleavage, resulting in the mature enzyme (Zhang et al., 2010). In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.
Barron, G.L., Thorn, R.G., 1987. Destruction of nematodes by species of Pleurotus. Can. J. Bot. 774–778. https://doi.org/10.1139/b87-103. Charlier, J., van der Voort, M., Kenyon, F., Skuce, P., Vercruysse, J., 2014. Chasing helminths and their economic impact on farmed ruminants. Trends Parasitol. 30 (7), 361–367. https://doi.org/10.1016/j.pt.2014.04.009. Geng, C., Nie, X., Tang, Z., Zhang, Y., Lin, J., Sun, M., Peng, D., 2016. A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins. Sci. Rep. 6, 1–12. https://doi.org/ 10.1038/srep25012. Genier, H.L.A., Soares, F.E.F., Queiroz, J.H., Gouveia, A.S., Araujo, J.V., Braga, F.R., Kasuya, M.C.M., 2015. Activity of the fungus Pleurotus ostreatus and of its proteases on Panagrellus sp. larvae. Afr. J. Biotechnol. 14 (17), 1496–1503. https://doi.org/10. 5897/AJB2015.14447. Grujić, M., Dojnov, B., Potočnik, I., Duduk, B., Vujčić, Z., 2015. Spent mushroom compost as substrate for the production of industrially important hydrolytic enzymes by fungi Trichoderma spp. and Aspergillus niger in solid state fermentation. Int Biodeterior Biodegradation 104, 290–298. https://doi.org/10.1016/j.ibiod.2015.04.029. Herbert-Doctor, L.A., Saavedra-Aguilar, M., Villarreal, M.L., Cardoso-Taketa, A., ViteVallejo, O., 2016. Insecticidal and nematicidal effects of Agave tequilana juice against Bemisia tabaci1 and Panagrellus redivivus. Southwest. Entomol. 41 (1), 27–40. https:// doi.org/10.3958/059.041.0105. Horvat, A.J., Babić, S., Pavlović, D.M., Ašperger, D., Pelko, S., Kaštelan-Macan, M., Petrovic Mance, A.D., 2012. Analysis, occurrence and fate of anthelmintics and their transformation products in the environment. TrAC Trends Anal. Chem. (Reference Ed.) 31, 61–84. https://doi.org/10.1016/j.trac.2011.06.023. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259), 680–685. https://doi.org/10.1038/227680a0. Lau, K.L., Tsang, Y.Y., Chiu, S.W., 2003. Use of spent mushroom compost to bioremediate PAH-contaminated samples. Chemosphere 52 (9), 1539–1546. https://doi.org/10. 1016/S0045-6535(03)00493-4. Lustigman, S., Prichard, R.K., Gazzinelli, A., Grant, W.N., Boatin, B.A., McCarthy, J.S., Basáñez, M.G., 2012. A research agenda for helminth diseases of humans: the problem of helminthiases. PLoS Negl Trop Dis 6 (4) e1582. https://doi.org/10.1371/ journal.pntd.0001582. Phan, C.W., Sabaratnam, V., 2012. Potential uses of spent mushroom substrate and its associated lignocellulosic enzymes. Appl. Microbiol. Biotechnol. 96 (4), 863–873. https://doi.org/10.1007/s00253-012-4446-9. Riga, E., 2011. The effects of Brassica green manures on plant parasitic and free living nematodes used in combination with reduced rates of synthetic nematicides. J. Nematol. 43 (2), 119–121. Singh, S.K., Hodda, M., Ash, G.J., 2013. Plant‐parasitic nematodes of potential phytosanitary importance, their main hosts and reported yield losses. EPPO Bull. 43 (2), 334–374. https://doi.org/10.1111/epp.12050. Sivanandhan, S., Khusro, A., Paulraj, M.G., Ignacimuthu, S., AL-Dhabi, N.A., 2017. Biocontrol Properties of Basidiomycetes: An Overview. J Fungi 3 (1), 2. https://doi. org/10.3390/jof3010002. Soares, F.E.F., Braga, F.R., Araújo, J.V., Geniêr, H.L., Gouveia, A.S., Queiroz, J.H., 2013. Nematicidal activity of three novel extracellular proteases of the nematophagous fungus Monacrosporium sinense. Parasitol. Res. 112 (4), 1557–1565. https://doi.org/ 10.1007/s00436-013-3304-8. Sufiate, B.L., Soares, F.E.F., Moreira, S.S., Gouveia, A.S., Monteiro, T.S.A., Freitas, L.G., Queiroz, J.H., 2017. Nematicidal action of Pleurotus eryngii metabolites. Biocatal Agric Biotechnol 12, 216–219. https://doi.org/10.1016/j.bcab.2017.10.009. Yang, J., Liang, L., Li, J., Zhang, K.Q., 2013. Nematicidal enzymes from microorganisms and their applications. Appl. Microbiol. Biotechnol. 97 (16), 7081–7095. https://doi. org/10.1007/s00253-013-5045-0. Zhang, X., Liu, Q., Zhang, G., Wang, H., Ng, T., 2010. Purification and molecular cloning of a serine protease from the mushroom Hypsizigus marmoreus. Process Biochem. 45 (5), 724–730. https://doi.org/10.1016/j.procbio.2010.01.009.
Acknowledgements The authors thank FAPEMIG, CNPq, CAPES and Agiel for financial support. The authors also are thankful to Dr. Juliana Milani Araujo.
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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Proteolytic and nematicidal potential of the compost colonized by Hypsizygus marmoreus
T
Filippe Elias de Freitas Soaresa,b,∗, Vânia Mayumi Nakajimaa, Bruna Leite Sufiatea, Luana Aparecida Simões Satirob, Elias Honorato Gomesb, Frederico Vieira Fróesc, Fabio Porto Senac, Fabio Ribeiro Bragac, José Humberto de Queiroza a
Federal University of Viçosa, Biochemistry and Molecular Biology Department, Viçosa, Minas Gerais, Cep: 3657000, Brazil State University of Minas Gerais, Campus Ubá, Av. Olegário Maciel, 1427, Ubá, Minas Gerais, Cep: 36500-000, Brazil c Vila Velha University, Rua Viana, Soteco, Vila Velha, ES, CEP 29106-091, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Nematicidal Spent mushroom compost Hypsizygus marmoreus Protease In silico
Spent mushroom compost (SMC) is a residue generated in edible mushrooms production, such as Hypsizygus marmoreus. Its genome was recently sequenced, demonstrating cuticle-degrading protease genes. The present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae. The extraction of the crude extract directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ± 18.38 U g−1 of compound. Moreover, the zymogram and SDS-PAGE indicated the presence of two proteases with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, there was a significant reduction in the number of Panagrellus redivivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively. A0A151VWY3 mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.
1. Introduction Diseases caused by nematodes constitute the major parasitic diseases of plants, animals and humans, having great economic impact on agriculture and animal husbandry (Lustigman et al., 2012; Singh et al., 2013; Charlier et al., 2014). The chemical control of these parasites using anthelmintics in large scale can induce parasitic resistance and has potential for bioaccumulation, which can lead to soil and groundwater contamination (Horvat et al., 2012). Given this context, it is necessary to use forms of control less aggressive to the environment, demanding investigations of these alternatives feasibility. In addition, the reuse of agro-industrial waste also plays a key role in avoiding environmental contamination. Spent mushroom compost (SMC), a residue generated in edible mushrooms production, can be reused as animal feed, organic fertilizer, soil conditioner, plant substrate, and in bioremediation and cleaning processes of contaminated soil and water (Phan and Sabaratnam, 2012; Grujić et al., 2015). Our ∗
research group aims to evaluate SMC's potential in control nematode infested soils. Many microorganisms can attack and destroy nematodes through several processes such as capture, parasitism and production of toxins and enzymes (Yang et al., 2013; Sivanandhan et al., 2017). Hypsizygus marmoreus is an edible mushroom consumed worldwide whose genome was recently sequenced, demonstrating cuticle-degrading protease genes (Zhang et al., 2010). However, there are no studies regarding the action of these proteases on nematodes. Thus, the present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae.
Corresponding author. Federal University of Viçosa, Biochemistry and Molecular Biology Department, Viçosa, Minas Gerais, Cep: 3657000, Brazil. E-mail address: fi[email protected] (F.E.d.F. Soares).
https://doi.org/10.1016/j.exppara.2018.12.006 Received 11 February 2018; Received in revised form 1 November 2018; Accepted 28 December 2018 Available online 05 January 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.
Experimental Parasitology 197 (2019) 16–19
F.E.d.F. Soares et al.
2. Materials and methods
larvae present in each tube was counted using an optical microscopy according to the modified methodology described by Soares et al. (2013). Statistical difference between the groups was evaluated by unpaired T test. Differences were considered significant when p ≤ 0.01. All analyses were performed using the software GraphPad Prism 5 for Windows version 5.00 (GraphPad Software, Inc.). Subsequently, the larvae reduction percentage average was calculated according to the following equation:
2.1. Spent mushroom compost crude extract Hypsizygus marmoreus SMC was obtained from the Urakami Group, Mogi das Cruzes, São Paulo – Brazil. Five grams (wet weight) were mixed with 50 ml distilled water. These mixtures were incubated at 30 °C for 1 h with shaking at 180 rpm, filtered and centrifuged at 10,000×g at 4 °C for 10 min. The supernatant was used as crude extract.
%Reduction ( Number of larvae in Control − Number of larvae in Treatment ) = x Number of larvae in Control
2.2. Obtaining bovine infective larvae (L3) Infective larvae (L3) of nematodes belonging to superfamilies Trichostrongyloidea (Haemonchus spp., Cooperia spp.) and Strongyloidea (Oesophagostomum spp.) were obtained from naturally infected bovine faeces, which were collected and mixed with vermiculite. The coproculture was moistened and kept in an incubator at 28 °C, where it remained for 14 days, for the development of infective larvae (L3). The larvae were recovered by the Baermann technique.
100
2.6. In silico analysis FASTA sequences of cuticle-degrading proteases from H. marmoreus were retrieved from UNIPROTKB (ID A0A151VWY3, A0A151WD28 and A0A151VEK8). Signal sequences were predicted using SignalP 4.0 server. Theoretical protein isoelectric point (pI) and molecular weight were calculated using the pI/MW tool (http://web.expasy.org/ compute_pi/). H. marmoreus cuticle-degrading proteases 3D structures were predicted by protein modeling using SWISS-MODEL (http://swissmodel. expasy.org/). Verticillium psalliotae Cuticle-Degrading Protease (ver112) (UniProt Acession N° Q68GV9) was used as template.
2.3. Enzymatic activity Protease activity was determined by the caseinolytic method. Five hundred μL of casein substrate (1% w/v, pH 8), 450 μL of 50 mM citrate-phosphate buffer (pH 5), and 50 μL of the crude extract were incubated for 20 min, at 50 °C. The reaction was stopped by adding 1 mL 10% w/v trichloroacetic acid (TCA). Then the reaction medium was centrifuged at 10,000×g for 5 min and the supernatant collected to determine the absorbance at 280 nm in a spectrophotometer. The enzyme unit (U) was expressed as the amount of enzyme capable of releasing 1 μg of tyrosine per minute. To estimate the amount of tyrosine released after the reaction, a standard curve was constructed using increasing amounts of tyrosine diluted in aqueous solution. The reaction blank consisted of 500 μl of buffer, 500 μl of 1% w/v casein and 1 ml of 10% w/v TCA solution.
3. Results The crude extract extraction directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ± 18.38 U g−1 of compound. Moreover, the formation of unstained regions in the zymogram (Fig. 1) indicated the presence of two proteases. The corresponding bands were excised and analyzed again by SDS-PAGE. Thus, two proteins (Fig. 2) were observed, with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, its nematicidal effect on P. redvivus and bovine infective larvae were evaluated. After 24 h of treatment, there was a significant reduction in
2.4. Zymography and SDS-PAGE Protease zymograms of the crude extracts were performed in 10% SDS-polyacrylamide gel co-polymerized with 0.1% (w/v) gelatin and 0.1% (w/v) casein. After electrophoresis, the gel was washed with 2.5% Triton X-100 solution for 1 h at room temperature. Then, the gel was incubated in citrate-phosphate buffer pH 8 for 1 h at 50 °C, subsequently stained with Coomassie brilliant blue R-250 0,1% (w/v) dissolved in ethanol: acetic acid: water (30:7:63), and then destained. The digested bands were visualized as unstained regions in the gels. The digested protein bands were then excised from the gel, grounded in a mortar using 50 mM Tris–HCl (pH 8.0) and centrifuged at 8000×g for 15 min at 4 °C to remove gel particles. The supernatant was used in a new 10% SDS-PAGE analysis (Laemmli, 1970). Electrophoresis was performed at 100 V, and the gel was stained with silver nitrate to allow protein visualization. 2.5. Nematicidal activity Nematicidal activities of SMC crude extract were evaluated on Panagrellus redivivus and bovine infective larvae (L3). The assay was composed of treatment and control groups. Six replicates were performed for each group using sterile tubes. In the treated group, about 40 P. redvivus or 50 bovine infective larvae (L3) were poured into sterile tubes containing H. marmoreus SMC crude extract. The control group contained approximately 40 P. redvivus or 50 bovine infective larvae (L3) in the presence of the boiled extract, containing the denatured proteases (no activity). The tubes were incubated at 28 °C in the dark for 24 h. After this period, the total number of
Fig. 1. Protease zymogram of the extract from Hypsizygus marmoreus spent mushroom compost. A: SDS-PAGE co-polymerized with 0.1% (w/v) gelatin. B: SDS-PAGE co-polymerized with 0.1% (w/v) casein. 17
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Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of excised band from zymogram of the extract from Hypsizygus marmoreus spent mushroom compost.
the number of P. redvivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively (Fig. 3). Considering the nematicidal activity, the proteases that could be acting were investigated. H. marmoreus has three cuticle-degrading proteases sequences deposited in UNIPROTKB (ID A0A151VWY3, A0A151WD28 and A0A151VEK8). A0A151VWY3 has 394 amino acid residues, with a peptide signal of 19 residues. Mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 has 387 amino acid residues, with a peptide signal of 19 residues. Mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. A0A151VEK8 has 559 amino acid residues, the mature protein is composed of 248 amino acid residues, with molecular weight of 24.1 kDa and pI 7.15. However, since A0A151VEK8 is a transmembrane protein, the possible H. marmoreus cuticle-degrading proteases in the extract are A0A151VWY3 and A0A151WD28. These two proteases 3D structures can be seen in Fig. 4.
Fig. 4. Protease structure models predicted in silico for the both proteases present in the extract from Hypsizygus marmoreus spent mushroom compost, using Verticillium psalliotae Cuticle-Degrading Protease (ver112) as template.
4. Discussion SMCs contain many residual nutrients and enzymes, such as proteases, cellulases, hemicellulases, lignin peroxidase, manganese
*
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Fig. 3. Nematicidal activity of crude extract from Hypsizygus marmoreus spent mushroom compost on Panagrellus redivivus and bovine infective larvae (L3). The asterisk denotes a difference (p < 0.01). 18
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References
peroxidase and laccase (Lau et al., 2003). The present work showed that SMC from H. marmoreus has protease content, which can be quickly and easily extracted. Edible mushrooms and their proteases presented activity against nematodes (Barron and Thorn, 1987; Genier et al., 2015; Sufiate et al., 2017). However, this is the first work demonstrating protease activity of SMC extract on nematodes. P. redivivus is a free living nematode usually used as a model in bioassays evaluating plant extracts or compounds for nematicidal action (Herbert-Doctor et al., 2016). Since the nematicidal effect in this nematode is indicative of action in another nematodes species, our results in P. redivivus show the potential of SMC extract to control plant and animal parasitic nematodes. In order to evaluate the potential nematicidal activity against animal parasitic nematodes, we used bovine infective larvae. These nematodes are difficult to control due to anthelmintics resistance development, demanding alternative treatments. Here we present the possibility of using an extract obtained from an agroindustrial residue, presenting low cost. Still, the use of enzymes from SMC extract for the biocontrol has the advantage of using an environmentally friendly, effective and affordable alternatives for nematode control (Geng et al., 2016). Chemical nematicides that are commonly used to control nematodes are toxic and costly to the environment and human health (Riga, 2011), highlighting the advantage of the extract. H. marmoreus has three known cuticle-degradating proteases sequences; however A0A151VEK8 is a transmembrane protein. Once the extraction method recovers only the extracellular enzymes, we suggest that A0A151VWY3 and A0A151WD28 (Fig. 4) were the proteases observed in this article. Furthermore, the molecular weights estimated by SDS-PAGE are similar to A0A151VWY3 and A0A151WD28 molecular weights obtained by in silico analysis. The SignalP 4.0 server predicts that the most likely N-terminal cleavage sites of the sequences A0A151VWY3 and A0A151WD28 are between 19 and 20 residues for both, and for sequence A0A151VEK8 there was no predicted signal peptide. A propeptide was identified for each sequence. This propeptide acts as a temporary inhibitor to assist in the mature peptidase folding, and it is removed by proteolytic cleavage, resulting in the mature enzyme (Zhang et al., 2010). In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.
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Acknowledgements The authors thank FAPEMIG, CNPq, CAPES and Agiel for financial support. The authors also are thankful to Dr. Juliana Milani Araujo.
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