An extracellular serine protease of an isolate of ...

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An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus Fabio R. Braga

a b

a

c

, Jackson V. Araújo , Filippe E.F. Soares , c

Hugo L.A. Geniêr & José H. Queiroz

c

a

Departamento de Veterinária, Universidade Federal deViçosa, Viçosa, Brazil b

Universidade Vila-Velha (UVV), Espirito Santo

c

Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Brazil Accepted author version posted online: 24 Jul 2012. Version of record first published: 21 Aug 2012

To cite this article: Fabio R. Braga, Jackson V. Araújo, Filippe E.F. Soares, Hugo L.A. Geniêr & José H. Queiroz (2012): An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus, Biocontrol Science and Technology, 22:10, 1131-1142 To link to this article: http://dx.doi.org/10.1080/09583157.2012.713912

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Biocontrol Science and Technology, Vol. 22, No. 10, October 2012, 11311142

RESEARCH ARTICLE An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus Fabio R. Bragaa,b*, Jackson V. Arau´joa, Filippe E.F. Soaresc, Hugo L.A. Genieˆrc and Jose´ H. Queirozc a

Departamento de Veterina´ria, Universidade Federal deViçosa, Viçosa, Brazil; bUniversidade Vila-Velha (UVV), Espirito Santo; cDepartamento de Bioquı´mica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, Brazil

Downloaded by [Filippe Soares] at 14:42 22 August 2012

(Received 18 May 2012; final version received 17 July 2012) This study aimed to present a protease produced by Duddingtonia flagrans fungus (AC001), and to evaluate its activity in the biological control of cyathostomin infective larvae (L3). The crude extract from D. flagrans grown in liquid medium was applied first to a DEAE-SepharoseTM and later to a CM-SepharoseTM ion exchange column. Protease activity was determined under different pHs and temperatures. Subsequently, the effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) inhibitor on activity were evaluated. Next, the protease activity in the biological control of nematodes was tested. A new 38 kDa serine protease (Df1) was purified. Optimum activity was obtained at pH 8.0 and 608C; CuSO4, ZnSO4 and PMSF strongly inhibited the activity. Df1 (AC001) showed an L3 reduction rate of 58%. In conclusion, a serine protease produced by D. flagrans (AC001) has been isolated, which is effective in the in vitro destruction of cyathostomin L3. Keywords: nematophagous fungi; cyathostomin; Duddingtonia flagrans; serine protease; biological control

1. Introduction Nematophagous fungi are an important group of antagonist micro-organisms that can suppress populations of parasitic nematodes through the destruction of their infective forms (larvae and/or eggs) (Braga et al. 2009, 2010). However, Tunlid and Jansson (1991) mention that the pathogenesis caused by nematode-trapping fungi is a complex process that includes the adhesion, penetration and digestion of nematodes. This penetration process takes place after one hour and is associated with the presence of dense bodies rich in enzymes, which act as peroxisomes and are found only in traps (Jansson and Nordbring-Hertz 1988). By this reasoning, one realises that the cuticle of parasitic nematodes is a complex structure that represents a significant barrier against penetration and infection by natural antagonists of these organisms in the environment. However, the second cuticle of these animals does not seem to protect them against predation by fungi, since they are less skillful at destroying L3 without an outer cuticle (Wharton and Murray 1990). Thus, to penetrate the cuticle of their host, both the predacious and entomopathogenic fungi *Corresponding author. Email: [email protected] ISSN 0958-3157 print/ISSN 1360-0478 online # 2012 Taylor & Francis http://dx.doi.org/10.1080/09583157.2012.713912 http://www.tandfonline.com


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depend on hydrolytic enzymes such as chitinases, collagenases and other proteases (Morton, Hirsch, and Kerry 2004; Yang, Tian, Liang, and Zhang 2007). Among these enzymes, proteases have received more attention, since serine proteases secreted by nematophagous fungi, especially by predacious and ovicidal fungi, are one of the most important virulence factors in the infection process of larvae and eggs (Lopez-Llorca and Robertson 1992; Bonants et al. 1995; Meyer and Wiebe 2003; Huang et al. 2005). The serine protease PII was seen as an important factor in the pathogenicity of nematodes by the nematode-trapping fungus Arthrobotrys oligospora, being found at relatively high levels, especially in the final stages of adhesion and penetration (Ahman et al. 2002). Duddingtonia flagrans is the most studied predacious species in the control of gastrointestinal helminthosis in domestic animals (Larsen 1999). It produces serine proteases (Meyer and Wiebe 2003). Wang et al. (2006) mentioned that molecular knowledge of the infection process by nematophagous fungi is still unclear and thus further study in this area is important. In a recent study, Braga et al. (2011) reported that isolate AC001 of D. flagrans produced proteases and its activity was evaluated in an experimental model consisting of nematodes of horses. On the other hand, Soares et al. (2012) reported that the fungi Monacrosporium thaumasium (NF34a) produced a serine protease Mt1 when this isolate was grown using first-stage larvae of Angiostrongylus vasorum (nematode of domestic dogs and wild canids) as the only source of carbon and nitrogen. These results show that the enzyme may have a possible role in the infection process of the larvae. However, the authors recommended further studies regarding the characterisation of the metabolites produced by nematode-trapping fungi. The present study aimed to purify a novel extracellular serine protease produced by an isolate of D. flagrans (AC001) nematophagous fungus and test its activity against parasitic nematodes of horses.

2. Material and methods 2.1. Fungus An isolate of D. flagrans (AC001) nematophagous fungus from Brazilian soil was used for protease production. This fungus had been maintained on solid media in the Laboratory of Parasitology, Veterinary Department, Federal University of Viç osa, in test tubes at 48C containing 2% corn meal agar (2% CMA) in the dark and for 10 days.

2.2. Serine protease production by Duddingtonia flagrans For serine protease production, fungal mycelia were obtained by transferring culture plates (about 5 mm in diameter) of the fungus maintained in 2% CMA in Erlenmeyer flasks (250 ml) containing 50 ml liquid medium according to the methodology described by Braga et al. (2011). The liquid medium was composed (g/l) of: glucose, 10; casein, 18.409; bibasic potassium phosphate (K2HPO4), 5; magnesium sulphate (MgSO4), 0.10; zinc sulphate (ZnSO4), 0.005; ferrous sulphate (FeSO4), 0.001; copper sulphate (CuSO4), 0.0005. The fungus grew in Erlenmeyer flasks under conditions of shaking at 120 g and pH 9.0. After 6 days, the supernatant fraction was collected and filtered using Whatman filter paper no. 1 at 48C, forming the crude extract.

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2.3. Serine protease purification


The crude extract was applied to a DEAE-SepharoseTM Fast Flow (Amersham Biosciences† ) ion exchange column previously equilibrated in 50 mM-TrisHCl (pH 8.0) buffer connected to a peristaltic pump and an automatic fraction collector. Flow was adjusted to 0.5 ml per minute. Proteins bound to the column were eluted with a linear gradient of the same buffer with sodium chloride (in 1M NaCl). Fractions with high protease activity were pooled and applied to a CM-SepharoseTM Fast Flow (Amersham Biosciences† ) ion exchange column previously equilibrated in 50 mMTrisHCl (pH 8.0) buffer connected to a vacuum pump and an automatic collector. Flow was adjusted to 0.5 ml per minute. Proteins bound to the column were eluted using the same buffer with sodium chloride (NaCl) in increased concentration linearly until 1 M concentration. Fractions with high protease activity were collected in a pool, which was the purified enzyme. The protease elution was monitored by enzyme assay and protein content.

2.4. Enzyme assay and protein content Protease activity was measured using the modified methodology of Braga et al. (2011). The volumes of solutions used were: 20 ml of enzyme (protease), 480 ml of 50 mM-TrisHCl (pH 8.0) and 500 ml of 1% casein (pH 8.0). The reaction medium was incubated for 15 minutes and the reaction was stopped by adding 1 ml of 10% trichloroacetic acid (TCA). After 10 minutes, the reaction medium was centrifuged at 10,000 g for 5 minutes. The collected supernatant fraction and its absorbance were determined by spectrophotometry at 280 nm. A tyrosine standard curve was built, varying the tyrosine concentration. One protease unit was defined as the amount of enzyme required to liberate 1.0 mg tyrosine per minute under the test conditions used. Protein content was determined according to the technique described by Bradford (1976). A standard curve was built for the quantification of protein content, using bovine serum albumin.

2.5. Electrophoresis Purification steps were monitored by electrophoresis (sodium dodecyl sulphatepolyacrylamide gel electrophoresis; SDS-PAGE) as described by Laemmli (1970), using 10% polyacrylamide gel. The gel was silver stained to allow the visualisation of proteins. The standard molecular weights used were: myosin, 194.859 kDa; bgalactosidase, 104.290 kDa; bovine serum albumin, 59.281 kDa; egg albumin, 41.93 kDa; carbonic anhydrase, 27.885 kDa; soy trypsin inhibitor, 20.834 kDa; lysozyme, 15.266 kDa; aprotinin, 6.528 kDa.

2.6. Serine protease characterisation 2.6.1. Effect of pH Serine protease activity was determined at different pH values, with 50 mMphosphate (pH 5.0 and 6.5) and 50 mM-TrisHCl (pH 8.0 and 9.0) buffers. The assay temperature was 408C. All characterisation tests were performed in triplicate.

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2.6.2. Effect of temperature Serine protease activity was determined at 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 and 808C. Furthermore, the thermostability of serine protease after incubation for 6 months at 48C was evaluated. All characterisation tests were performed in triplicate.

2.6.3. Effect of metal ions and inhibitors


To study the effect of different metal ions and inhibitors on proteolytic activity, protease was incubated at a concentration of 10 mM with the salts calcium chloride (CaCl2), copper sulphate (CuSO4), zinc sulphate (ZnSO4) and magnesium sulphate (MgSO4), and the inhibitors phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetra-acetic acid (EDTA). A control incubation (in the absence of substances) was performed; the activity in this incubation was considered as 100%. All characterisation tests were performed in triplicate.

2.7. Investigation of Duddingtonia flagrans serine protease production during the infection process of cyathostomins For serine protease production, fungal mycelia of AC001 were transferred to Erlenmeyer flasks (250 ml) containing 50 ml liquid medium according to a modified methodology described by Braga et al. (2011) and Tunlid and Jansson (1991). The liquid medium was composed of (g/l): bibasic potassium phosphate (K2HPO4), 5; magnesium sulphate (MgSO4), 0.10; zinc sulphate (ZnSO4), 0.005; ferrous sulphate (FeSO4), 0.001; copper sulphate (CuSO4), 0.0005; and 2 105 cyathostomin L3. The larvae were used as a carbon and nitrogen source. The fungus was cultured in Erlenmeyer flasks under conditions of shaking at 120 g and pH 9.0. After 6 days, the supernatant fraction was collected and filtered using Whatman filter paper no. 1 at 48C, centrifuged at 10,000 g for 5 minutes, and subjected to lyophilisation. The precipitate was re-suspended in 1 ml distilled water and used for electrophoresis analysis and subsequent enzyme assay.

2.8. Experimental larvicidal assay Fresh faeces were collected directly from the rectum of naturally infected crossbred horses (Equus caballus). Eggs were counted in the faeces (eggs per g; EPG) according to Gordon and Whitlock (1939) in order to find animals positive for infection. Subsequently, the coproculture was made; after 7 days, third-stage larvae (L3) were obtained, which were identified and quantified according to the criteria described by Bevilaqua, Rodrigues, and Concordet (1993), in an optical microscope (10 objective lens). Baermann’s reading showed that 100% of the L3 visualised were cyathostomins. Subsequently, two groups were formed in sterile tubes, a treated group and a control group over 24 hours, performing six replicates for each group. In the treated group, 100 cyathostomin L3 were poured into sterile tubes containing 150 ml of D. flagrans purified serine protease. The control group contained 100 cyathostomin L3 in sterile tubes and boiled purified serine protease. The tubes for the treated and control group were incubated at 258C in a dark environment for 24 hours. After


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incubation, the total number of L3 present in each tube was counted, according to the modified methodology described by Qiuhong et al. (2006). The data obtained were statistically analysed by analysis of variance at the 1 and 5% probability levels. The efficiency of L3 predation compared with the control group was assessed by Tukey’s test at the 1% probability level. The percentage reduction in the mean number of larvae was calculated using the following equation:


% Reduction ¼

ðXC XTÞ XC

100

3. Results 3.1. Protease purification After filtering the supernatant fraction, the crude extract was purified by anion exchange chromatography at pH 8.0. Most of the proteases were observed not to become adsorbed on the DEAE-SepharoseTM column (Figure 1). The fractions with activity (tubes 1322) were pooled and applied to the cationic exchange column. Moreover, it was also observed that most of the proteases were not adsorbed on the CM-Sepharose column (Figure 2). Table 1 shows the purification factors and yields of each purification step.

3.2. Electrophoresis The purified serine protease was observed to be in apparent homogeneity in SDSPAGE. The molecular mass of the purified enzyme named Df1 was approximately 38 kDa (Figure 3a). These results were based on electrophoretic mobility according to the methodology described by Laemmli (1970).

Figure 1. Elution profile of ion exchange chromatography using DEAE-SepharoseTM resin.


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Figure 2. Elution profile of ion exchange chromatography using CM-SepharoseTM resin.

3.3. Serine protease characterisation 3.3.1. Effects of pH and temperature The effect of pH on the serine protease activity of D. flagrans is shown in Figure 4a. The optimum activity value of Df1 (120.8 U/ml) was obtained at pH 8.0. On the other hand, at pH 9.0 there was a decline in proteolytic activity. Regarding the activity of this enzyme at different temperatures, it was shown that the highest activity value was observed in the range of 60658C (120.8 and 117.5 U/ ml). Moreover, it was noted that its activity increased up to 608C. Above this temperature, there was a sharp decline (67.4 U/ml) (Figure 4b). Thermostability analysis showed that after the incubation period (6 months) at 48C, the enzyme retained about 25% of residual activity.

3.3.2. Effect of metal ions and inhibitors Results for the effects of different metal ions and inhibitors on Df1 activity of D. flagrans are shown in Table 2. It was observed that CuSO4, ZnSO4 and PMSF inhibited the activity. Moreover, Ca2 and EDTA caused an increase in its activity. Table 1. Procedure for purification of serine protease (total activity (U), protein (mg), specific activity (U/mg), yield (%) and purification factor) produced by Duddingtonia flagrans nematophagous fungus (AC001). Purification Steps

Activity

Protein

Activity Specific

Yield

Purification (fold)

Crude extract DEAE-sepharose CM-sepharose

417.1 192.3 28.3

4599.1 461.1 37.4

0.1 0.4 0.8

100 46.11 6.78

1 4.60 8.34

Note: One protease unit (U) was defined as the amount of enzyme required to liberate 1.0 mg tyrosine per minute under the test conditions used.



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Figure 3. (a) Purification analysis of serine protease produced by Duddingtonia flagrans nematophagous fungus (AC001) through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 10% gel. Lines: M, molecular weight marker; lines 1 and 2, serine protease (Df1) fraction obtained from CM-SepharoseTM; line 3, crude extract. (b) Df1 production investigation during the infection process of cyathostomin L3 larvae.

3.4. Animal experimentation model (nematode larvae of horses) Df1 production was probably important in the infection process of cyathostomin larvae since they served as the sole source of carbon and nitrogen (Figure 3b). In addition, an enzyme assay was performed demonstrating its proteolytic activity (40 U/ml). Another protein that also had probable importance in the infection process was observed in the gel.

4. Discussion The serine protease (Df1) produced by isolate AC001 was effective at destroying the nematode larvae from the horses. After the study period (24 hours), a percentage 58% reduction of cyathostomin L3 of the treated group with Df1 (P B0.01) was observed compared with the control group (denatured enzyme). Several serine proteases from predacious nematophagous and ovicidal fungi have been purified and characterised, exhibiting low molecular weight and high activity at alkaline pH (Lopez-Llorca and Robertson 1992; Segers, Butt, Kerry, and Peberdy 1994; Tunlid, Rosen, Ek, and Rask 1994; Bonants et al. 1995; Zhao, Mo, and Zhang 2004; Soares et al. 2012). According to Lopez-Llorca, Macia´ -Vicente, and Jansson

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Figure 4. (a) Effect of different studied pH (5, 6.5, 8 and 9) on serine protease activity produced by Duddingtonia flagrans (AC001) fungus. (b) Effect of different studied temperatures (20, 30, 40, 50, 60, 70 and 808C) on serine protease activity produced by D. flagrans (AC001) fungus.

(2008), an important virulence factor of nematophagous fungi is the production of extracellular proteases. However, so far there are no literature reports about purification of these enzymes derived from D. flagrans isolate AC001. However, in the present study it has been demonstrated that this fungus produced a serine protease (named here as Df1) and this is the first report of its purification, characterisation, and therefore presentation. We observed that the optimum Df1 activity was found at pH 8.0 (120.8 U/ml). These results are in agreement with those of Meyer and Wiebe (2003), who reported


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Table 2. Effect of metal ions (Ca2, Cu2, Zn2 and Mg2) and inhibitors (EDTA and PMSF) on protease activity. Chemical compounds


Control Ca2 Cu2 Zn2 Mg2 EDTA PMSF

Relative activity (%) 100 110.4 31.1 45.7 82.9 112.8 0

that the highest amount of proteolytic activity of D. flagrans was at alkaline pH, while activity was low at acid pH values. Furthermore, it was found that Df1 shares the characteristic of having high activity at an alkaline pH, like other serine proteases such as PII (Tunlid et al. 1994), Aoz1 (Zhao et al. 2004), Mlx (Wang, Yang, and Zhang 2006) and Ds1 (Wang et al. 2006) obtained from predacious fungi. We observed that the Df1 activity was optimum at 608C. However, it has been reported that some nematophagous fungi possess an optimal enzymatic activity at high temperatures (Bonants et al. 1995; Wang et al. 2006). Df1 of D. flagrans (AC001) was sensitive to the inhibitor PMSF, indicating that it belongs to the serine proteases family (Siezen and Leunissen 1997) and class I of nematophagous fungi proteases, since it is a serine protease obtained from a predacious fungus (Yang et al. 2007). Nematophagous fungi in general use gastrointestinal parasitic nematodes as their source of nutrition (carbon and nitrogen). According to Eren and Pramer (1965), the periodic provision of nematodes to nematophagous fungi in a poor culture medium of nutrients could reduce its saprophytic growth, increasing its activity as a natural antagonist during the infection process. These reports are consistent with the results of the present study, since we used a poor medium of nutrients supplemented with cyathostomin L3 that served as a nutrition source. Moreover, we observed Df1 production (Figure 3b), suggesting that this enzyme is important for the fungus infection process on the nematode. Concerning the expression of enzymes of nematophagous fungi during the infection process of eggs or larvae, Lopez-Llorca and Robertson (1992) demonstrated using immunocytochemistry localisation that a protease of Verticillium chlamydosporia fungus named P32 was responsible for destroying phytonematode eggs in an in vitro assay. Thus, those reports demonstrate the importance of the study and characterisation of the enzymes produced by nematophagous fungi during fungi interaction with nematode eggs and larvae. Several studies in the laboratory and field have referred to the predatory activity of D. flagrans under a high variety of larval genera from gastrointestinal nematodes (Knox and Faedo 2001; Chandrawathani et al. 2003; Braga et al. 2009). However, there is a lack of studies that show the molecular interaction of fungi with nematodes. In the present study, it was observed that Df1 showed activity on cyathostomin L3. As previously mentioned, Braga et al. (2011) showed that the crude enzymatic extract of isolate AC001 was effective in reducing (95%) in vitro when applied on the same type of cyathostomin L3. However, when comparing the action of this crude extract with Df1 enzyme, some questions can be raised. In the crude extract used by


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Braga et al. (2011), they drew attention to the presence of a number of hydrolytic enzymes and other factors that contribute to the hydrolysis of the nematode cuticle. However, in the present study, Df1 was produced and purified, showing a lower activity on the L3 used. However, Df1 caused destruction of the larvae (58%) at 24 hours after the interaction, demonstrating its importance in the infection process. The authors agree that further studies about the interaction of enzymes v. nematodes should be carried out. The literature reports a series of papers citing the use of conidia (Braga et al. 2010), chlamydospores (Waller and Faedo 1996; Larsen 1999; Campos, Arau´ jo, and Guimara˜ es 2008), mycelial mass (Carvalho et al. 2009) and pellets on sodium alginate matrix containing D. flagrans fungus (Braga et al. 2009). These studies agree that any structure or product derived from this fungus can be used for the biological control of nematodes, as was also suggested by the authors of this paper. The present study aimed to demonstrate the possible applicability of D. flagrans serine protease (Df1) in an animal experimental model using parasitic nematode L3 of horses. The results presented here can be viewed as a support tool that can help to control pests in general through the discovery of new methodologies that can be used synergistically with chemical control, starting from the current worrying situation of parasitic resistance. However, analysis of N-terminal amino acid residues and of the gene of the enzyme (Df1) will provide more information about the degree of similarity of the purified enzyme with other proteases previously described in the literature. Acknowledgements The authors would like to thank CNPq scholarship, Capes and Fapemig for financial support and grant concession.

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