Fasciola hepatica proteolytic activity in liver revealed ... - Springer Link

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May 26, 2005 - Abstract Fasciola hepatica secretes cysteine proteases that play a role in facilitating parasite migration. The aim of this study was to detect the ...
Parasitol Res (2005) 96: 308–311 DOI 10.1007/s00436-005-1367-x

O R I GI N A L P A P E R

Yazmı´ n Alcala´-Canto Æ Froyla´n Ibarra-Velarde Jesu´s Gracia-Mora Æ He´ctor Sumano-Lo´pez

Fasciola hepatica proteolytic activity in liver revealed by in situ zymography

Received: 23 February 2005 / Accepted: 23 March 2005 / Published online: 26 May 2005 Ó Springer-Verlag 2005

Abstract Fasciola hepatica secretes cysteine proteases that play a role in facilitating parasite migration. The aim of this study was to detect the inhibition of the proteolytic activity of F. hepatica cysteine proteases in the liver of C57BL/6 cathepsin B knockout mice (cat B / ) and wild-type controls (cat B+/+) by intraperitoneal administration of N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine, (E-64) using the film in situ zymography (FIZ) technique and image analysis. The FIZ technique revealed that intraperitoneal administration of E-64 dramatically reduced (85%) F. hepatica proteolytic activity in the liver of experimentally infected mice with no discernable side effects. These results suggest the usefulness of the FIZ for determining in vivo activity of F. hepatica proteases, as well as their inhibition by intraperitoneal administration of E-64 in hepatic tissue of infected mice.

Introduction Fasciolosis caused by F. hepatica causes worldwide economic losses of approximately two billion dollars per year (Torgerson and Claxton 1999). Proteases of both excretory–secretory and somatic origin from F. hepatica possess a wide range of actions, from tissue digestion and facilitation of migration to immune suppression or evasion from host defence mechanisms (Smith et al. 1993). Dalton and Heffernan (1989) demonstrated that

Y. Alcala´-Canto (&) Æ F. Ibarra-Velarde Æ H. Sumano-Lo´pez Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, Me´xico, DF, 04510 E-mail: [email protected] J. Gracia-Mora Facultad de Quı´ mica, Universidad Nacional Auto´noma de Me´xico, Me´xico, DF, 04510

F. hepatica releases thiol proteases by analyzing immature fluke in vitro-released products. They suggested that further purification of the identified individual proteases was needed. Smith et al. (1993) demonstrated that liver flukes secrete cathepsin L-like proteases. Further research showed that cathepsin L proteases are synthesized and secreted into the parasite lumen from vesicles within the gastrodermal epithelial cells (Collins et al. 2004), and these proteases are being tested as potential targets to produce vaccines against fasciolosis in cattle and sheep (Dalton et al. 2003). Cathepsin B protease activity has also been shown in F. hepatica . These proteases are the major enzymes secreted by the newly excysted juvenile fluke (Wilson et al. 1998) and it is suggested that cathepsin B is involved in the migration process of this through liver tissue (Law et al. 2003). Hence, it has been postulated that parasite proteases are attractive targets for the development of new chemotherapeutic agents, using protease inhibitors, as it has been shown for Leishmania spp and Trypanosoma cruzi (McKerrow et al. 1999). Among the protease inhibitors are the epoxysuccinyl peptides that interact with cysteine proteases, such as N-[ N-(L-3-transcarboxyoxirane-2-carbonyl)-L-leucyl]-agmatine (E-64), a natural inhibitor initially isolated from Aspergillus japonicus (Hanada et al. 1978). E-64 is potent, specific and irreversibly inhibits cysteine proteases. It is used as a diagnostic reagent for their identification (Rawlings et al. 2004). The interaction of F. hepatica proteases released in vitro with specific low molecular-weight inhibitors has been widely reported over the last decade (Dalton and Heffernan 1989; McGinty et al. 1993; Carmona et al. 1994; Wilson et al. 1998; Acosta et al. 1998; Hawthorne et al. 2000). Results postulate cathepsin L (Dalton et al. 2003) and cathepsin B (Law et al. 2003) as targets to develop immunoprophylaxis. Nevertheless, control of fasciolosis requires chemotherapy and targeting parasite proteases may probe to be a useful approach. Considering that the in vivo interaction of F. hepatica cysteine proteases with a specific cathepsin B and L-protease inhibitor has not yet been demonstrated, the present study was carried out in order to evaluate the inhibitory activity of

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E-64 on F. hepatica cysteine proteases during the migration period of the parasite through the host liver. To discriminate mammalian cathepsin B activity in liver injury due to fasciolosis, liver sections from mice with a genetic inactivation of cathepsin B (Cat B / ) were included in this experiment, and compared with corresponding activity of wild-type mice genetically able to secrete cathepsin B (Cat B+/+).

Material and methods Animal model of infection C57BL/6 cathepsin B knockout (Cat B / ) mice (18–21 g body weight), were used for this study. Generation of these animals was done as described by Deussing et al. (1998) and were kindly provided by Dr. Wong, of the Social Insurance Mexican Institute (IMSS). Wild type (WT) mice, with no genetic inhibition of cathepsin B (Cat B +/+) were used as controls. Throughout the experiment, animals were fed a standard rodent diet (Rodent Lab Chow, Purina) and water supplied ad libitum. Metacercariae of F. hepatica were obtained by infecting Lymnaea cubensis snails under laboratory conditions using the method described by Vera (1994). An amount of 0.2 mM of the cysteine protease inhibitor N-[N-(L-3-trans-carboxyoxirane-2carbonyl)-L-leucyl]-agmatine (E-64) (Sigma) were dissolved in dimethyl sulfoxide (DMSO) and the final concentration adjusted with distilled sterile water 70:30 (v/v DMSO/H2O). Treatments with E-64 were done intraperitoneally at a dose of 50 mg/kg. Two hours later, 20 metacercariae were administered in suspension per os to each mouse. Four hours after the experimental infection, mice were again treated with the same dose of E-64. Thereafter, administration was repeated once a day, for 5 weeks. Twelve groups of ten mice each were established: a group of Cat B / mice infected and E-64-treated; a group of WT mice infected and E-64-treated; a group of Cat B / mice infected and DMSO-treated; a group of WT mice infected and DMSO-treated; a group of Cat B / mice infected and untreated; a group of WT mice infected and untreated; a group of Cat B / mice uninfected and E-64-treated; a group of WT mice uninfected and E-64-treated; a group of Cat B / mice uninfected and DMSO-treated; a group of WT mice uninfected and DMSO-treated; a group of Cat B / mice uninfected and untreated; and a group of WT mice uninfected and untreated. All animals were observed daily for evidence of side effects of the inhibitor. Mice were humanely sacrificed 35 days after the beginning of the study. Film in situ zymography (FIZ) To assess the proteolytic activity of F. hepatica cathepsins, three liver sections of each mouse included in this

trial were analyzed by film in situ zymography as decribed by Cheung et al. (1991). Briefly, 4 lm sections of fresh mice liver tissue were embedded in cryomold O.C.T. (Tissue-Tek, Miles Inc., Elkhart, IN, USA) and frozen on dry ice. The frozen sections were dipped into NTB 2 photographic emulsion (Eastman Kodak Co., NY, USA) which form a gelatin surface that serves as substrate. Sections were incubated into humidified chambers at 37 °C for 12 h. After incubation, emulsioncovered slides were air-dried and the development of the photographic process carried out. Protease activity was assessed using light microscopy (Olympus BX40 with camera PM 10 AK, Japan) as white spots on a dark background. Image analysis The intensity of in situ gelatinolysis was measured using Image J software 1.33 u (National Institutes of Health, USA). Statistics Results were statistically analyzed by means of a Chisquare test.

Results Cathepsin activity was dramatically reduced (85%), in Cat B / and WT mice treated with E-64 when compared to infected untreated mice. Figure 1 shows the comparative proteolytic activity observed in infecteduntreated Cat B / and WT mice, and its reduction in the corresponding infected and E-64-treated groups. Gelatinolysis was not reduced in infected mice treated with the vehicle only. Liver sections from uninfected controls, treated or not, did not exhibit any proteolytic effect. A Chi-square comparison of images obtained by film in situ zymography, reveal that intraperitoneal administration of E-64 significantly inhibited gelatin degradation in fasciolosis in both Cat B / and WT mice (P>0.05). No significant differences were observed between proteolytic areas from both Cat B / and WT mice liver sections. Daily inoculation of mice with E-64 during 5 weeks failed to reveal discernable side effects.

Discussion In contrast with various techniques that determine indiscriminately the presence of proteases in tissues, whether in its active or its inactive proform phase, in situ zymography utilized in this trial detects only the active form by revealing specific proteolytic activity in tissue sections. This selectivity allowed the detection of cysteine protease activity and its interaction with an

310 Fig. 1 Inhibition of in situ gelatinolytic activity of Fasciola hepatica cysteine proteases in Cat B / and WT mice exerted by the injection of E-64. Proteolytic activity is observed as white spots on a dark background. The background is dark due to exposure of emulsion to ambient light during processing, while gelatinolysis in the emulsion produces spots. a Strong gelatin degradation in liver sections of Cat B / mice infected with Fasciola hepatica metacercariae. b Complete lysis of gelatin was observed in liver sections of Fasciola hepatica-infected WT mice. c Reduction of gelatinolytic activity in infected Cat B / mice treated with E64. d Reduction of gelatinolytic activity by the cysteine protease inhibitor E-64 in liver of infected and treated WT mice

expoxysuccinyl cysteine protease inhibitor in liver of F. hepatica-infected mice, for the first time. Furthermore, when E-64 was injected to the F. hepatica-infected mice, proteolytic activity was statistically significantly inhibited (85%). The persistence of some proteolysis in the presence of the inhibitor E-64 could indicate the participation of other enzymes, such as serine or aspartyl proteases. Indeed, other enzymes have been isolated from the metabolic products of F. hepatica, and these include a dipeptidylpeptidase (Carmona et al. 1994), a leucine aminopeptidase (Acosta et al. 1998) and glycosidases (Irwin et al. 2004). In contrast, the F. hepaticainfected mice, not treated with E-64 had considerable gelatinolysis in liver. The main areas where activity for cysteine proteases were observed coincide with the surroundings of F. hepatica bodies within the liver slide and support the view that F. hepatica cathepsins are important for the process of tissue invasion (Berasain et al. 1997). To determine whether the increase in gelatinolytic activity was due to release of F. hepatica cathepsins and not mammalian liver cathepsin B, as this protease is released from lysosomes into the cytosol in hepatic injury (Canbay et al. 2003), a comparison between genedeleted mice unable to activate this protease and wildtype mice, were included. Lack of differences between these two animal populations suggests that the mammalian hepatic cells retain or fail to activate most of their lysosomal proteases during fasciolosis in this murine model.

To the best of our knowledge, this is the first time that the FIZ technique has been used to detect proteolytic activity in vivo in a model of fasciolosis; yet, it appears safe to recommend the FIZ technique to directly assess the biological activity of cysteine proteases inhibitors in tissues. Furthermore, such information could help to determine effective dose of cysteine protease inhibitors. However, to further enhance selectivity to target cysteine proteases, specific inhibitors of aspartyl, serine and metalloproteases could be included in the gelatin-coated film. Moreover, FIZ can be complementary to gelatin (in vitro) zymography to evaluate local effects of the inhibitors. The degree of clarity in the results observed with the FIZ method leads us to speculate that this technique may be of use to predict reduction in the extent of liver damage by direct correlation with the proteolytic activity of F. hepatica enzymes. Acknowledgements This study complies with the current laws of Mexico.

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