Cysteine protease is a major component in the ... - Springer Link

Parasitol Res (2014) 113:65–71 DOI 10.1007/s00436-013-3627-5

ORIGINAL PAPER

Cysteine protease is a major component in the excretory/ secretory products of Euclinostomum heterostomum (Digenea: Clinostomidae) P. A. Ahammed Shareef & S. M. A. Abidi

Received: 15 July 2013 / Accepted: 26 September 2013 / Published online: 18 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Cysteine proteases of parasite organisms play numerous indispensable roles in tissue penetration, feeding, immunoevasion, virulence, egg hatching and metacercarial excystment. They are critical key enzymes in the biology of parasites and have been exploited as serodiagnostic markers, therapeutic and vaccine targets. In the present study, the cysteine proteases in the in vitro released excretory/secretory (E/S) products of the digenetic trematode parasite, Euclinostomum heterostomum have been analysed. The encysted progenetic metacercariae of E. heterostomum collected from the infected liver and kidney of Channa punctatus were excysted in vitro and incubated in phosphate buffer at 37±1 °C, and the E/S products released were analysed. The spectrophotometric analysis of the proteases revealed active hydrolysis of chromogenic substrate, azocoll, in a time-, temperature- and pH-dependent manner. Optimum activity was observed at pH 7.0 at 37±1 °C, and with 1 mM each of various protease inhibitors (Mini Protease Inhibitor Cocktail, ethylene diaminetetraacetic acid, phenyl methyl sulphonyl fluoride, iodoacetamide and 1,10phenanthroline) used, significant inhibition was observed by iodoacetamide and 85 % of inhibition at a concentration of 2 mM, suggesting that cysteine protease is a major component in the E/S of this parasite. Four discrete protease bands of Mr 36, 39, 43 and 47 kDa were identified by gelatin-substrate zymography. Maximum gelatinolytic activity was observed at pH 7.0, and among various inhibitors used, almost complete disappearance of protease bands was observed by 2 mM iodoacetamide. The proteolytic cleavage of bovine serum P. A. Ahammed Shareef : S. M. A. Abidi (*) Molecular and Immunoparasitology Research Laboratory, Section of Parasitology, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202 002, Uttar Pradesh, India e-mail: [email protected] P. A. Ahammed Shareef e-mail: [email protected]

albumin, bovine haemoglobin and human haemoglobin in vitro were also studied.

Introduction Parasitic infections in fish impose immense economic burden to the fisheries industry. They can cause considerable impact on growth, reproduction, behaviour and even mortality (Scholz 1999). Euclinostomum heterostomum (Rudolphi 1809) is a haemophagic clinostomid trematode parasite found in many regions of Asia, Africa and Europe (Yamaguti 1971). Clinostomid trematodes are known for zoonotic potential (Park et al. 2009). In Indian subcontinent, the freshwater snail Indoplanorbis exustus and the snakeheaded fish Channa punctatus (Bloch) serve as the first and second intermediate hosts, respectively, while the adults are found in the oesophagus of piscivorous birds like pond herons Ardeola grayii and cattle egrets Bubulcus ibis (Jhansilakshmibai and Madhavi 1997). Helminth parasites have evolved enormous adaptive survival strategies to locate and evade each specific microenvironment with in the intermediate and definitive hosts, modulating the defence mechanisms, while deriving the nourishment from them. In order to complete the life cycle, E. heterostomum has to successfully cross the life stages through free-living molluscan, piscine and avian hosts, and proteases are thought to make substantial contribution towards these processes (Rizvi et al. 2010). Parasitic cysteine proteases (peptide hydrolases or peptidases) take part in numerous indispensable roles in virulence, tissue penetration, extracorporeal digestion, feeding, immunoevasion, egg hatching and metacercarial excystment (Chung et al. 1995; Mckerrow et al. 2006; Knox 2007; Kaewpitoon et al. 2008; Rizvi et al. 2010; Sajid and Mckerrow 2002). Due to the highly immunogenetic nature, they have been exploited as immunodiagnostic markers, chemotherapeutic and vaccine targets (Mckerrow et al. 1999; Knox 2007; Sajid and Mckerrow

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2002; Selzer et al. 1999; Jørgensen and Buchmann 2011). Cathepsin L-like cysteine protease has been used as a potent candidate molecule for the serodiagnosis of Fasciola gigantica, and vaccination with Cathepsin B and L have shown high protection up to 80 % against Fasciola hepatica (Jayaraj et al. 2009). Rizvi et al. (2010) reported on the cysteine proteases of Clinostomum complanatum, another closely related species, which degraded many host molecules including haemoglobins, reflecting their key role in the parasitic mode of life and survival within the host; however, the cysteine proteases of E. heterostomum are not known. Characterisation of key proteases of E. heterostomum would enable to broaden our knowledge on the basic biology of this parasite. The nature, biochemical properties and possible biological functions of these enzymes are poorly understood. In the present undertaking, the cysteine proteases in the excretory/secretory (E/S) products of the newly excysted progenetic metacercariae of E. heterostomum were studied.

Materials and methods Chemicals and buffers Azocoll, ethylene diaminetetraacetic acid (EDTA), phenyl methyl sulphonyl fluoride (PMSF), iodoacetamide (IA), 1,10phenanthroline (1,10-P), bovine serum albumin (BSA), bovine haemoglobin (BHb) and human haemoglobin (HHb) were purchased from Sigma (USA). Gelatin and standard molecular weight marker (BioRad) and complete mini protease inhibitor cocktail tablets (COK) (Roche) were procured from the local distributors. To investigate the effect of pH, 100 mM each of glycine–HCl (pH 3), sodium acetate–acetic acid (pH 4, 5), sodium phosphate (pH 6, 7, and 8), Tris–HCl (pH 9) or Na2HPO4–NaOH (pH 10 and 11) buffers were used. Collection of parasites and excretory/secretory products The encysted progenetic metacercariae of E. heterostomum were collected from the infected liver and kidney of the snakeheaded fish C. punctatus and mechanically excysted in normal saline (0.74 %) by using fine forceps and needle by carefully tearing up the cyst wall to liberate the flukes. After a short wash in 0.5 % of penicillin–streptomycin solution and three subsequent washes in 100 mM of phosphate buffer solution (PBS) at pH 7.0, the worms were incubated (ten worms per millilitre) in the same buffer at 37±1 °C for 2 h. After the incubation, all the worms were active, and the medium containing E/S products was centrifuged at 8,000×g for 20 min at 4 °C. The supernatant was aliquoted and stored at −80 °C until use. The protein concentration was spectrophotometrically

Parasitol Res (2014) 113:65–71

estimated following the method of Bradford (1976) by using BSA as standard. Proteolytic assay using azocoll substrate: effect of time, inhibitors, pH and temperature Nonspecific proteolytic activity of E/S products of E. heterostomum was estimated using the chromogenic substrate azocoll (Knox and Kennedy 1988). Briefly, the reaction was carried out at 37±1 °C in a total volume of 500 μl. The E/S containing 35 μg of protein was pre-incubated at 37±1 °C for 3 min, then 2 mg of azocoll was added, and the volume was adjusted using 100 mM PBS at pH 7.0. For time course assay, the reaction was carried out for 0, 15, 30, 45, 60, 75 and 90 min. The reaction was stopped by adding an equal volume of 20 % trichloroacetic acid (TCA). The undigested substrate was removed by centrifugation at 5,000×g for 15 min at 4 °C. The changes in the absorbance at 520 nm as a measure of proteolytic activity were measured using a spectrophotometer. To determine the effect of various protease inhibitors, the E/S was preincubated at 37±1 °C for 15 min with 1 mM each of COK, EDTA, PMSF, 1,10-P and IA at 1 or 2 mM. Subsequently, 2 mg of azocoll substrate was added and incubated for 45 min. To measure the effect of pH, the assay was carried out in different buffers, i.e. 3, 4, 5, 6, 7, 8, 9, 10 or 11, for 45 min. To determine the effect of temperature, the assay was carried out at 20, 30, 35, 37, 40 or 50 °C for 45 min. Subsequent procedures were done as above. All the assays were done in triplicate and expressed as mean ± standard deviation (SD). Gelatin-substrate zymography: effect of inhibitors, temperature and pH Zymography was carried out following the method of Heussen and Dowdle (1980). In brief, 1.5 μg of E/S proteins per lane was separated using 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) co-polymerized with 0.1 % gelatin. Standard molecular weight markers were also simultaneously loaded. After electrophoresis, the gel was sliced across each lane, and after four washes of 15 min each at 4 °C with 2.5 % Triton X-100, they were incubated in 100 mM PBS at pH 7.0 at 37±1 °C for 1.5 h. Then the gels were stained overnight with 0.2 % Coomassie Brilliant Blue R-250 (CBBR250) and then destained to visualise white gelatinolytic bands of E/S proteases. Molecular weight determination of proteases was performed following standard procedures. To determine the effect of protease inhibitors, the gel slices were incubated for 1.5 h in PBS containing 1 mM each of COK, EDTA, PMSF, 1,10-P and IA at 1 or 2 mM. To determine the effect of pH and temperature, the gel slices were incubated in different buffers of pH 3, 4, 5, 6, 7, 8, 9, 10 and 11 or at 20, 30, 35, 37, 40 and 50 °C in PBS at pH 7.0 for 1.5 h. After staining and destaining as described above, the gels were photographed using automated


Gel-Doc System (BioRad), and representative gel images of each set are presented. All the experiments were conducted at least three times, and consistent profile was repeated.

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10 % SDS-PAGE and stained with CBBR-250. This experiment was repeated at least three times, and consistent results were obtained.

Degradation of protein molecules Proteolytic cleavage of BSA, BHb and HHb by the E/S proteases of E. heterostomum was conducted and subsequently analysed by SDS-PAGE. In short, the assay was performed in a total volume of 500 μl PBS at pH 7.0 containing 250 μg BSA and incubated with the E/S (12.5 μg protein) at 37±1 °C for 4, 12 or 24 h. Similarly, for BHb or HHb, the reaction was set up with 500 μl (PBS pH 7.0) containing 2 mg BHb or HHb and incubated with E/S (12.5 μg protein) for 4 h at 37±1 °C. For each sample, control was also run simultaneously incubated without E/S. For inhibitor sensitivity, the E/S products were pre-incubated with IA (1 or 2 mM) at 37±1 °C for 30 min, followed by the addition of substrate and incubated further for 4 h. The reactions were stopped by the addition of an equal volume of Laemmli sample buffer. These samples were run on

Results

Fig. 1 Nonspecific proteolytic activity of E/S products of E. heterostomum on azocoll degradation. The E/S was incubated with 2 mg of azocoll at 37± 1 °C at pH 7.0 which degraded azocoll substrate in a time-dependent manner (a). The reaction was carried out for 45 min in the presence of 1 mM each of the various protease inhibitors, and significant inhibition was observed by iodoacetamide (b). C, control; COK, complete mini protease inhibitor

cocktail tablets; EDTA, ethylene diaminetetraacetic acid; PMSF, phenyl methyl sulphonyl fluoride; 1,10-P, 1,10-phenanthroline; IA, iodoacetamide (IA-1, 1 mM; IA-2, 2 mM). The same reaction was carried out in different pH buffers (3–11) demonstrating a pH optimum of 7.0 (c) and at different temperatures showing maximum activity over 37–40 °C (d)

The proteolytic enzymes in the E/S of E. heterostomum degraded azocoll substrate in a time-dependent manner (Fig. 1a). Among the various protease inhibitors tested, IA significantly inhibited (>80 and >85 % at 1 and 2 mM of concentrations, respectively) azocoll degradation by the E/S proteases (Fig. 1b), suggesting that cysteine proteases are the major component in the E/S of this parasite. The enzyme was found to degrade azocoll over a broad range of pH (Fig. 1c). More than 75 % of activity has been shown over the pH of 4–8; however, the highest activity was recorded at neutral pH. Similarly, more than 70 % of enzyme activity has been observed over 30–50 °C; however, 37–40 °C is the range of

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temperature that showed peak enzyme activity (Fig. 1d), reflecting wide thermostability of the enzyme. Gelatin-substrate zymography of E/S revealed four discrete protease bands of molecular weight (Mr) approximately 36, 39, 43 and 47 kDa (Fig. 2a). Gelatinolytic activity was found to be affected by the protease inhibitors, temperature and pH of the incubation medium. Among the various protease inhibitors checked, pronounced inhibition of gelatinolysis was noticed by IA (Fig. 2b), further confirming the major involvement of cysteine proteases in proteolysis. COK also significantly reduced the enzyme activity. The zymogram revealed comparable effects of pH (Fig. 2c) and temperature (Fig. 2d) on the proteolytic activity as that of azocoll. They are stable and active over a broad range of pH and temperature, but optimal activity was demonstrated at pH 7.0 and 37 °C. In order to investigate the ability of proteases in the E/S of E. heterostomum to degrade macromolecules, protein digestion analyses were performed (Fig. 3). These proteases actively degraded BSA, and longer incubation displayed its timedependent digestion (Fig. 3a). Similarly, BHb and HHb also accomplished well-defined proteolytic degradation (Fig. 3b, c). The metacercarial E/S of E. heterostomum showed typical

Fig. 2 Representative images of gelatin-substrate zymography of E/S products of E. heterostomum. E/S proteins of 1.5 μg per lane was separated using 10 % SDSPAGE co-polymerized with 0.1 % gelatin, and then Triton X-100treated gel strips were incubated in 100 mM PBS for 90 min. a Zymogram showing standard molecular weight markers (M), proteolytic bands (P) with approximate molecular weight labelled. b Effect of various protease inhibitors on gelatinolysis. Control (C), phenyl methyl sulphonyl fluoride (PMSF), 1,10-phenanthroline (1, 10-P), complete mini protease inhibitor cocktail tablets (COK), ethylene diaminetetraacetic acid (EDTA), iodoacetamide (IA-1, 1 mM; IA-2, 2 mM of concentrations). c Effect of pH (3–11) on enzyme activity. d Effect of temperature on the gelatinolytic activity


properties of cysteine proteases, and IA (1or 2 mM) completely inhibited the proteolytic cleavage of all the proteins, and the band appeared intact in the SDS-PAGE.

Discussion The whole life cycle, growth and development of free-living, intramolluscan, piscine and avian stages of E. heterostomum have been well described (Jhansilakshmibai and Madhavi 1997); however, apart from which, this parasite has not received much attention, and no more studies have been found to be reported. In the present report, we describe the preliminary analysis and partial characterisation of cysteine proteases in the E/S of the encysted progenetic metacercariae of E. heterostomum . Cysteine proteases have been considered to be the most abundantly expressed protease family in many digenetic trematode parasites such as C . complanatum, F. hepatica, Schistosoma japonicum and Clonorchis sinensis (Dvořák et al. 2008; Kang et al. 2010; Norbury et al. 2011; Rizvi et al. 2010; Xiaoli et al. 2012).


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Fig. 3 Host substrate cleavage assay using E/S proteases of E . heterostomum. Each of BSA, BHb and HHb were incubated with E/S proteases at 37±1 °C for 4 h and subsequently analysed using 10 % SDSPAGE. a Proteolytic analysis of BSA. Standard molecular weight markers (M), BSA alone (lane 1), BSA incubated with E/S proteases (showing the proteolytic cleavage; lane 2), reaction in presence of iodoacetamide (showing the inhibition of proteolytic cleavage; lane 3, 1 mM; lane 4, 2 mM), BSA incubated with E/S for 12 h (lane 5) and 24 h

(lane 6). b Proteolytic analysis of BHb. Standard molecular weight markers (M), BHb alone (lane 1), BHb with E/S (showing the proteolytic digestion; lane 2), reaction with iodoacetamide (showing the inhibition of proteolytic cleavage; lane 3, 1 mM; lane 4, 2 mM). c Proteolytic analysis of HHb. Standard molecular weight markers (M), HHb alone (lane 1), HHb with E/S (showing the proteolysis; lane 2 ), reaction with iodoacetamide (showing the inhibition of proteolytic cleavage; lane 3, 1 mM; lane 4, 2 mM)

Recently, cysteine proteases of parasitic organisms have been extensively studied in large number of species, as reflected in the literature. Due to their characteristic biochemical properties and the vital roles they play in the biology of these organisms, these proteases are key targets for diagnostic, vaccine or drug development (Sajid and Mckerrow 2002). The results of azocoll degradation obtained in the present study are in agreement with the previous studies on C. complanatum by Rizvi et al. (2010). Among various protease inhibitors tested, 80 and 85 % of inhibitions were observed by IA at 1- and 2-mM concentrations, respectively, suggesting that the cysteine proteases constitute the major role in the azocoll degradation. Rizvi et al. (2010) reported proteolytic bands of 33, 39, 45 and 47 kDa by the E/S of closely related species of C. complanatum; however, in the present study, the Mr of 36, 39, 43 and 47 kDa was displayed by E. heterostomum in gelatin-substrate zymography. Though the individual Mr varied, the molecular weights of both the species lie within the same range, reflecting their similar mode of life cycle, and common functional and evolutionary

significance. The inhibition of gelatinolysis and disappearance of protease bands by IA further confirms the involvement of cysteine proteases. Cysteine proteases of parasites have been extensively studied and are divided into clan CA (papain-like and calpain-like) and clan CD (legumain-like). Cathepsin L- and B-like cysteine proteases of the papain family are the most abundantly expressed/characterised cysteine proteases in trematode parasites (Sajid and Mckerrow 2002). However, further studies are required on E. heterostomum to characterise the specific cysteine protease. Analysis of influence of pH on the activity and stability of proteases can provide an insight into their possible roles in the free-living, intermediate or definitive hosts. Cysteine proteases of many trematode parasites have stability over a broad range of pH values, but the optimum is acidic (Lowther et al. 2009; Dvořák et al. 2008). In contrast, the results of the present study revealed that both azocoll degradation and gelatinolysis were maximum at neutral pH, as reported in C. complanatum (Rizvi et al. 2010). Nevertheless, >75 % of the activity was retained

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over a broad pH range of 4–8. In the natural course of life cycle, the infected fish bearing the metacercarial cysts of E . heterostomum are being eaten by piscivorous birds. In the crop/stomach where the pH is about 3–4, the cysts are believed to excyst and migrate back to the buccal cavity or oesophagus where they tenaciously attach and transform into adult ovigerous state. The broad pH stability of the cysteine proteases released by the metacercariae of E. heterostomum might be helping them for excystment, invasion and immune modulation in the definitive host. Similarly, these proteases may be involved in the snail tissue penetration by miracidia, migration through fish tissues and encystment in the liver or kidney. These assumptions of invasion, encystment/excystment, nutrient acquisition, host immuno-evasion/modulation and miracidial penetration of snails, have been well established in many other trematodes as well (Li et al. 2004; Collins et al. 2004; Cotton et al. 2012; Sajid and Mckerrow 2002; Dalton et al. 2003; Dixit et al. 2008; Robinson et al. 2008; Kašný et al. 2007). Cysteine proteases also take part in the digestion of host blood extracorporeally as well as in the gut (Rizvi et al. 2010; Dvořák et al. 2008; Dalton et al. 2004). Histological sections of cysts of E. heterostomum revealed the presence of ingested blood meal within the gut as well as in the cyst cavity, but outside the worm (Shareef and Abidi, unpublished). Here, it can be inferred that the secreted cysteine proteases of this parasite possibly catabolise the host blood into absorbable components to meet their nutritional requirement. In addition, these proteases readily hydrolysed BSA, BHb and HHb in vitro, as evident in Fig. 3, further confirming their possible involvement in the degradation of host blood meal in vivo in fish and bird hosts. The inhibition of these proteases in vivo might compromise the survival as it can impair nutrient acquisition and possibly other provital roles in the parasitic mode of life. The optimal activity of cysteine proteases of metacercariae of E. heterostomum observed at temperatures of 37–40 °C may be an early preparatory strategy for survival, excystment, extracorporeal digestion, invasion and immunomodulation in the definitive host, piscivorous birds, whose body temperature is about 40 °C. In conclusion, the proteases in the E/S of newly excysted progenetic metacercariae of E . heterostomum actively degraded azocoll and gelatin in a pH- and temperaturedependent manner. They were capable of digesting proteins analogous to their hosts giving hints to their possible roles in the extracorporeal digestion and nutrient acquisition in their natural hosts. All the proteolytic activities analysed were significantly inhibited by the class protease inhibitor IA, clearly indicating that cysteine proteases are the major constituent in the E/S of this parasite. The suppression of these enzymes by (inhibitor-based or other) chemotherapy or immunoprophylaxis may compromise the normal biology of this parasite. Some immunostimulants or natural products may also be tested. Comprehensive understanding and further


characterisation of these proteases may help in the development of novel strategies for the control of this infection, but extended studies are to be carried out to achieve this goal. Acknowledgments The authors are thankful to the chairman of the Department of Zoology, A.M.U., for providing laboratory facilities, and Prof. Iqbal Parwez and Dr. Arif Ahmad for kindly extending the instrumentation facility. The technical assistance of Mr. Azam and the financial assistance received from BBSRC-UK-CIDLID project are also gratefully acknowledged.

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