Functional Analysis of Cathepsin B-like Cysteine Proteases fromLeishmania donovani Complex

Cathepsin B-like genes from Leishmania donovani and Leishmania chagasi have been isolated and characterized. It is a single gene, which is constitutively expressed in all the life cycle stages of the parasite. Studies using cathepsin B-specific inhibitor treatment suggested that cathepsin B does not seem to play a role in the promastigote stages of the parasite, however it aids in the parasite survival within the host macrophages. Antisense mRNA inhibition of cathepsin B gene also revealed that it plays an important role in the parasite survival within the host macrophages. Furthermore, for the first time, we have shown that Leishmania whole cell lysates as well as the recombinant cathepsin B protein cleaved human recombinant latent transforming growth factor (TGF)-β1 into a mature peptide releasing the latency associated protein, in a cell-free incubation system. Mink lung epithelial cell growth inhibition assay revealed that the cleaved TGF-β1 was biologically active, suggesting thatLeishmania cathepsin B can cleave latent TGF-β1 into mature and active form. These results suggest that cathepsin B plays an important role in Leishmania survival within the host macrophages by activating latent TGF-β1. Cathepsin B-like genes from Leishmania donovani and Leishmania chagasi have been isolated and characterized. It is a single gene, which is constitutively expressed in all the life cycle stages of the parasite. Studies using cathepsin B-specific inhibitor treatment suggested that cathepsin B does not seem to play a role in the promastigote stages of the parasite, however it aids in the parasite survival within the host macrophages. Antisense mRNA inhibition of cathepsin B gene also revealed that it plays an important role in the parasite survival within the host macrophages. Furthermore, for the first time, we have shown that Leishmania whole cell lysates as well as the recombinant cathepsin B protein cleaved human recombinant latent transforming growth factor (TGF)-β1 into a mature peptide releasing the latency associated protein, in a cell-free incubation system. Mink lung epithelial cell growth inhibition assay revealed that the cleaved TGF-β1 was biologically active, suggesting thatLeishmania cathepsin B can cleave latent TGF-β1 into mature and active form. These results suggest that cathepsin B plays an important role in Leishmania survival within the host macrophages by activating latent TGF-β1. transforming growth factor β l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-l-isoleucyl-l-proline juvenile hormone esterase benzoylcarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin latency-associated protein interferon minimum essential medium Protozoan parasites of Leishmania sp. are diploid eukaryotes, causing a variety of human diseases (1Herwaldt B.L. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar). These organisms live alternately as extracellular promastigotes within the insect vector and as intralysosomal amastigotes in the host macrophages (2Alexander J. Russell D.G. Adv. Parasitol. 1992; 31: 175-254Crossref PubMed Scopus (269) Google Scholar). Among the variety of disease manifestations, visceral leishmaniasis caused by Leishmania donovani complex is fatal and is a serious health problem in many tropical and subtropical countries (3Tselentis Y. Gilkas A. Chaniotis B. Lancet. 1994; 343: 8913Abstract Scopus (25) Google Scholar).Leishmania parasites contain high levels of cysteine proteases, belonging to cathepsin L and cathepsin B families (4Mottram J.C. Brooks D.R. Coombs G.H. Curr. Opin. Microbiol. 1998; 1: 455-460Crossref PubMed Scopus (139) Google Scholar). Cysteine proteases have been shown to play critical roles in the pathogenesis of parasitic protozoan infections (5McKerrow J.H. Sun E. Rosenthal P.J. Bouvier J. Annu. Rev. Microbiol. 1993; 47: 821-853Crossref PubMed Scopus (346) Google Scholar). Studies using CA074, a cathepsin B-specific inhibitor-treated BALB/c mice, showed resistance against Leishmania major infection and also showed the shift of immune responses from Th2 to protective Th1 type (6Maekawa Y. Himeno K. Ishikawa H. Hisaeda H. Sakai T. Dainichi T. Asao T. Good R.A. Katunuma N. J. Immunol. 1998; 161: 2120-2127PubMed Google Scholar). However, the mechanism by which cathepsin B affects the immune response is not known. Studies involving species of Leishmania such as L. major and Leishmania amazonensis have been shown to induce the production of biologically active transforming growth factor β (TGF-β)1 by macrophages upon infection (7Barral-Netto M. Barrel A. Brownell C.E. Skeiky Y.A.W. Ellingsworth L.R. Twardzik D.R. Reed S.G. Science. 1992; 257: 545-548Crossref PubMed Scopus (392) Google Scholar). L. donovani infection is known to induce the expression of a number of cytokine genes like TNF-α, GM-CSF, TGF-β, and IL-6. Also, susceptibility to Leishmania chagasi infection in BALB/c mice is correlated with the localized production of TGF-β1 at the site of maximal parasite growth in the liver (8Wilson M.E. Young B.M. Davidson B.L. Mente K.A. McGowan S.E. J. Immunol. 1998; 161: 6148-6155PubMed Google Scholar). Application of anti-TGF-β antibodies arrested the development of lesions in mice, whereas treatment with TGF-β exacerbated the infection with L. amazonensis,Trypanosoma cruzi, and Toxoplasma gondii (9Hunter C.A. Bermudez H. Beernink H. Waegell W. Remington J.S. Eur. J. Immunol. 1995; 25: 994-1000Crossref PubMed Scopus (132) Google Scholar).L. major, Leishmania mexicana, andLeishmania braziliensis trigger the production of TGF-β and IL-10, which inhibit killing of intracellular organisms (10Bogdan C. Rollinhoff M. Parasitol. Today. 1999; 15: 22-28Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). It was also shown that TGF-β plays a role in limiting IFN-γ production during the primary infection in mice (11Bogdan C. Paik J. Vodovotz C. Nathan C. J. Biol. Chem. 1992; 267: 23301-23308Abstract Full Text PDF PubMed Google Scholar). IFN-γ, on the other hand, is known to induce the expression of inducible nitric-oxide synthase, the key effector mechanism for the killing ofLeishmania and other parasites within the mouse macrophages, both in vitro and in vivo (12Bogdan C. Behring Inst. Res. Commun. 1997; 99: 58-72PubMed Google Scholar). TGF-β isoforms are synthesized as large biologically inactive precursors, which are proteolytically processed to yield mature and active homodimer. A variety of agents and treatments are known to activate latent TGF-β, like heat, acidic pH, plasmin, subtilisin-like endopeptidases, and cathepsins (13Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3946) Google Scholar, 14Munger J.S. Harpel J.G. Gleizes P.E. Mazzieri R. Nunes I. Rifkin D.B. Kidney Int. 1997; 51: 1376-1382Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 15Chu T.M. Kawinski E. Biochem. Biophys. Res. Commun. 1998; 253: 118-134Crossref Scopus (47) Google Scholar). Earlier studies have implicated the role of proteases and sialidases in the processing of host-derived latent TGF-β (16Miyazono K. Heldin C.H. Nature. 1989; 338: 158-160Crossref PubMed Scopus (213) Google Scholar). Therefore, we hypothesize thatLeishmania cathepsin B enzymes cleave latent TGF-β into mature and active TGF-β and thus helps parasites to overcome the microbicidal activity of the macrophages. In the present study, we have isolated and characterized the cathepsin B-like gene from the L. donovani complex. The functional role of cathepsin B in parasite survival using cathepsin B-specific inhibitor CA074 and antisense mRNA inhibition revealed that it plays an important role in the amastigote survival within the macrophages. Also, we have expressed the L. chagasi cathepsin B in an insect cell expression system, in its active form, and for the first time we have demonstrated that leishmanial cathepsin B can cleave latent TGF-β into biologically active form, using parasite whole cell extracts as well as recombinant cathepsin B protein. These results suggest that cathepsin B plays an important role in Leishmania survival within the macrophages, by activating TGF-β. Modified minimum essential medium (HO-MEM), M199 medium, fetal calf serum, Lipofectin, and gentamycin were obtained from Invitrogen. Tunicamycin and phorbol myristate acetate were purchased from Sigma. Protease inhibitors, E-64, CA074, and peptide substrate Z-Phe-Arg-AMC were purchased from Peptides International, Inc., Louisville, KY. Human recombinant TGF-β1 and the neutralizing antibodies, anti-TGF-β1, anti-LAP (latency-associated protein), and anti-mouse secondary antibody, were obtained from R & D Systems. The ECL-enhanced chemiluminescent kit and Quick-Prime labeling kits were purchased from Amersham Biosciences. L. chagasi (MHOM/BR/84, kindly provided by Dr. Mary, E. Wilson, University of Iowa) was cultured at 26 °C in modified minimum essential medium (HO-MEM) supplemented with 10% heat-inactivated fetal calf serum. L. donovani (1S2D, kindly provided by Dr. Greg Matlashweshki, McGill University, Montreal, Canada) was cultured at 26 °C in modified M199 medium supplemented with 10% heat-inactivated fetal calf serum. Cultures were seeded at 1 × 106 parasites/ml and harvested at logarithmic or stationary phase of growth as defined by cell density and morphological characteristics. U937 macrophage cells were cultured in RPMI medium (Invitrogen) supplemented with 10% fetal calf serum at 37 °C and in the presence of 5% CO2. Mv1Lu mink lung epithelial cells (CCL-64, ATCC) were cultured in RPMI medium supplemented with 10% bovine calf serum, 1% MEM nonessential amino acids, and 1 mm HEPES in the presence of 5% at 37 amino was obtained by the cathepsin B-like gene from A was which site to the amino in the mature and the antisense a site to the amino in the of other cathepsin B. These were to cathepsin B-like gene from the chagasi and L. was in the The in the were at °C for 1 °C for 1 and °C for 1 A single of was and for gene was as a to an L. chagasi amastigote provided by Dr. M. E. Wilson, University of of were with and the from the gene The gene of cathepsin B from to the of was obtained from the the gene a was using a 1 in and were and amino were using provided by was isolated from stationary phase promastigotes of L. chagasi and L. using T. J. A Scholar). of was with were on and was isolated using as by the was obtained from logarithmic and stationary phase amastigote the human macrophage cell ATCC) was with stationary which into intracellular amastigotes within the macrophage was isolated from the macrophage cells using was on and were to and T. J. A Scholar). of cathepsin B were from the or the of L. chagasi and with using a Quick-Prime labeling active cathepsin B-like cysteine the was by from of L. was with and was into site of vector P.J. K. PubMed Scopus Google and by of insect cells and of recombinant protein from insect cells are as M. J. C. K. 1998; Scopus Google Scholar). insect cells were seeded into mm at a density of × and for with of and in the was and the cells were with medium and of 10% fetal bovine serum was and for The was harvested and at °C for Cathepsin B activity in the recombinant protein and the whole cell lysates of the parasites was using the peptide The were in The for the mm mm 1 and was by the of of the and for at 37 The activity was by the of from the The of the was using a at and an of inhibition cathepsin B-specific inhibitor CA074 was at a of in vitro inhibition promastigotes of L. cultured in 10% fetal calf serum, with or CA074 for at 26 The of parasites was by the parasites and whole cell lysates were to assay for the the and antisense mRNA of cathepsin B gene, the vector provided by Dr. K. was a with B. PubMed Scopus Google Scholar). The of cathepsin B was at site of the The was in both and antisense with to that of a gene which is the for The the and antisense were and was isolated for using the were by and into L. chagasi × phase promastigotes were with and in of in a of of vector with cathepsin B in and with cathepsin B in were using a at of and resistance of cells were in medium for were for resistance at and at were for all the The U937 macrophage cells were with L. chagasi promastigotes at a host to parasite of U937 cells cells in of RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, mm and of gentamycin were to at 37 °C for in the presence of 5% CO2. The cells were and seeded at the of × The cells were to with phorbol myristate acetate and to for The macrophage cells were with stationary phase parasites macrophage cell and to for The and parasites were with RPMI medium, and the macrophage cells were with RPMI medium and for cells were from at a with and on the were using and for the intracellular human latent TGF-β1 was in with of recombinant cathepsin B or the parasite whole cell lysates were at 37 °C for a active TGF-β1 was by by with for at was to all the and was to the mature TGF-β1 as a with a of TGF-β1 was using a growth inhibition assay with Mv1Lu mink lung epithelial as A. PubMed Scopus Google Scholar). Mv1Lu cells were in at to assay for TGF-β1 activity were the cells were with for was and cells were with by with were with and with was in and into was in a was to the were to using a were for with 5% the of cathepsin were at °C with of B provided by Dr. University, the of the the were with at a of and to the mature human TGF-β1 was at a of incubation in the primary antibody, were with and with of anti-mouse or secondary chemiluminescent kit was for protein the cathepsin B gene from L. were on the amino from using a of was to a of L. major, L. and L. with The results showed that a cathepsin B-like gene is present in L. chagasi and L. donovani as a single gene and that the gene is in chagasi and L. in the of L. major The was to a promastigote of L. and were revealed that were present in the the chagasi 1 into vector and a to the gene, the on the of L. chagasi cathepsin B gene, were to the L. donovani cathepsin B-like gene from L. donovani The of L. chagasi number and L. number cathepsin B genes a protein of amino showed that and are to other and to that of to and to human cathepsin B gene not in of L. major cathepsin the amino is by a in donovani and L. chagasi cathepsin which results in activity of leishmanial cathepsin B the peptide However, of leishmanial cathepsin B can cleave the cathepsin revealed a of which is constitutively expressed in all the life cycle stages of the parasite 1 with the 1 the of expression at all stages suggesting that the cathepsin B gene play a role in during the stages of the parasite. the substrate of the cathepsin B-like cysteine the of L. chagasi cathepsin B gene was into vector and into insect Cathepsin B expressed as a hormone protein is into the The was and for the presence of recombinant protein by using and The results of the are in and The antibodies a major of to the B protein. The recombinant protein was for the activity using the peptide The of the activity was by using a cathepsin B The results are in the of that the recombinant B protein is active against The and protein showed The activity is in presence of CA074, to that the recombinant cathepsin B is proteolytically active, it is with protein. the functional role of cathepsin B on the parasite growth and we have CA074, a cathepsin B-specific that the inhibitor does not the growth of the parasites during the promastigote stages of the parasite not Furthermore, inhibitor treatment not the of cathepsin both in promastigote and amastigote stages of the parasite However, the cathepsin B activity in which were in presence of CA074 showed with that of parasites in both the stages of development The activity Z-Phe-Arg-AMC was in of promastigotes as well as in The parasites in the presence of inhibitor were to the U937 macrophages, to the of cathepsin B activity on parasite The macrophage infection that the inhibitor-treated promastigotes with at all time the results from suggest that cathepsin B activity is important survival within the host macrophage does not play a role during the promastigote of the survival of L. donovani promastigotes with or number of type parasites and inhibitor-treated promastigotes were to phorbol myristate U937 macrophages. The the number of amastigotes present in a macrophage cell for macrophages on a to The from are in The with the infection with type and the with the infection with promastigotes in the presence of were and in medium with to The presence of in was by using not of with revealed that and antisense cathepsin B with the expressed the antisense which showed a in the with the type and vector parasites of is shown by the The results obtained that the presence of cathepsin B antisense mRNA the cathepsin B of showed that the expression of antisense the of cathepsin B protein The in the of cathepsin B is with in the type parasites and with the vector The is to the of the protein extracts The results obtained thus that antisense mRNA expression affects the of cathepsin B the cathepsin B protein in the of cathepsin B activity to antisense mRNA inhibition on survival of parasites in the host macrophages, cathepsin B activity substrate in the was using and also the on intracellular survival and was using U937 The results from the assay revealed that the with the antisense activity the substrate Z-Phe-Arg-AMC with that of the with and vector the macrophage infection also revealed that the with the antisense showed in the parasite survival and within the macrophage cells with with and vector results suggest that cathepsin B activity is important for the parasite survival within the macrophages. A involving human cathepsin B its role in of latent TGF-β1 in the human epithelial cell M. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Leishmania cathepsin B on TGF-β1 processing and in an assay using a cell-free incubation was TGF-β1 was by using antibodies for latent TGF-β1 and shown in latent TGF-β1 is as a of TGF-β1 in the of latent TGF-β1 into its and mature which is a for The recombinant cathepsin protein also cleaved latent TGF-β1 into the and mature TGF-β1 and the cathepsin B-specific CA074, was in the the activity was cathepsin B was with protein was as a which not activity and have also characterized cathepsin protein A. L. PubMed Scopus Google which to cleave the latent TGF-β1 In latent TGF-β1 was also cleaved whole cell lysates However, lysates from parasites with cathepsin B CA074, showed that the activity in the Leishmania whole cell was to cathepsin B. the parasite lysates from the and antisense cathepsin B to the on TGF-β1 The results that was in in of cathepsin B antisense mRNA with cathepsin B or vector and which that cathepsin B is for the TGF-β1 all the results that Leishmania cathepsin B processed latent TGF-β1 into its mature and the activity of TGF-β1 in obtained from the assay using latent TGF-β1 and cathepsin mink lung epithelial cell growth inhibition assay was The of are into of synthesized The suggest that latent cells of the cells were with the latent was a inhibition in that was of latent TGF-β1 into mature form. results were obtained from the cells with latent TGF-β1 with cathepsin protein, the of inhibition was that of TGF-β1. to the of active TGF-β1 from the by the of cathepsin B present in the cathepsin However, was with that obtained from inhibitor cathepsin as well as the and with inhibitor with latent TGF-β1 whole cell lysates also showed a in with that of the inhibitor-treated whole cell lysates These results that Leishmania cathepsin B latent TGF-β1 into mature and biologically active of a that been shown for the first time in the ofLeishmania cathepsin B. Cysteine proteases have been from species of Leishmania (4Mottram J.C. Brooks D.R. Coombs G.H. Curr. Opin. Microbiol. 1998; 1: 455-460Crossref PubMed Scopus (139) Google Scholar, J.C. Coombs G.H. Parasitol. Today. Full Text PDF PubMed Scopus Google J.H. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). have the and gene of cathepsin cysteine proteases from the L. donovani complex A. L. PubMed Scopus Google Scholar). In the present study, we have isolated and the functional of a cathepsin B-like gene from L. donovani and L. results that the cathepsin B-like gene is present as a single expressed during all the life cycle stages of the parasite. studies revealed that cathepsin B gene expression is important for the parasite survival within the host macrophages. Also, for the first time, we have demonstrated that the recombinant cathepsin B protein as well as Leishmania whole cell lysates cleaved the latent human recombinant TGF-β1 into the mature and biologically active of TGF-β1. results suggest that cathepsin B plays an important role in the survival ofLeishmania parasites within the host macrophages by activating latent TGF-β. The gene of L. chagasi and L. donovani cathepsin B that it is a single gene, which is constitutively expressed at all stages of the parasite life with a of the to the activity in leishmanial cell lysates are not to of cysteine it is not to the levels of mRNA with that of activity in life cycle showed that are to L. major to L. cathepsin and to human cathepsin high the to that of L. major L. chagasi and L. donovani cathepsin B have a at in the which is known to for the activity with the peptide Z-Phe-Arg-AMC J.H. Biochem. J. 1999; PubMed Google Scholar). The recombinant cathepsin protein was proteolytically active, and it cleaved peptide substrate showed activity not The functional role of cathepsin B on growth and survival of parasites was using a cathepsin inhibitor The inhibitor not have a on the parasite growth it the parasite survival within the U937 macrophages, suggesting that cathepsin B activity is for the parasite survival as amastigotes within the The not on macrophage infection in to the of cell of In the present study, we have also antisense to the functional role of cathepsin B in the survival of L. of antisense been as an for of the gene expression in many T. R. 1999; 67: Scholar). were to cathepsin B gene both at the mRNA and protein a we have a in parasite survival within the macrophages, which was with that of the inhibitor However, the in parasite survival was not which to the activity of other proteases like cathepsin L. results were obtained in the cells the type that cathepsin B does play in the stages of the parasite R. Coombs G.H. J.C. Biochem. Parasitol. 1997; PubMed Scopus Google Scholar). from study, as well as that of (6Maekawa Y. Himeno K. Ishikawa H. Hisaeda H. Sakai T. Dainichi T. Asao T. Good R.A. Katunuma N. J. Immunol. 1998; 161: 2120-2127PubMed Google Scholar, R. Coombs G.H. J.C. Biochem. Parasitol. 1997; PubMed Scopus Google that cathepsin B plays a role in Leishmania to live within the macrophages. the other hand, studies on cathepsin cysteine proteases have genes in a factor J.C. R. Coombs G.H. A. PubMed Scopus Google Scholar). Therefore, it that the cysteine proteases have roles in the parasite. Studies involving species of Leishmania such as L. major and L. amazonensis have been shown to induce the production of biologically active TGF-β by macrophages (7Barral-Netto M. Barrel A. Brownell C.E. Skeiky Y.A.W. Ellingsworth L.R. Twardzik D.R. Reed S.G. Science. 1992; 257: 545-548Crossref PubMed Scopus (392) Google Scholar). TGF-β macrophage and to L. chagasi infection in BALB/c mice is correlated with the localized production of TGF-β at the site of maximal parasite growth in the liver (8Wilson M.E. Young B.M. Davidson B.L. Mente K.A. McGowan S.E. J. Immunol. 1998; 161: 6148-6155PubMed Google Scholar). TGF-β is also known to a cytokine response Studies involving cathepsin B-specific CA074, treatment in leishmaniasis have a of cell from Th2 to suggesting that cathepsin B plays a role in processing and of (6Maekawa Y. Himeno K. Ishikawa H. Hisaeda H. Sakai T. Dainichi T. Asao T. Good R.A. Katunuma N. J. Immunol. 1998; 161: 2120-2127PubMed Google Scholar). However, Leishmania cathepsin B to A involving human cathepsin B its role in of latent TGF-β in human epithelial cell M. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the cathepsin B gene is known to by TGF-β treatment in cells P.J. Scopus Google Scholar). In study, for the first time, we have shown with that B can cleave latent TGF-β1 into biologically active form, using the recombinant protein as well as parasite whole cell in a cell-free system. activity was in the presence of the cathepsin B-specific inhibitor can well correlated with the survival of inhibitor as well as antisense parasites within the macrophage of of TGF-β been in of J. PubMed Google Scholar). of Biochem. Parasitol. 1997; PubMed Scopus Google Biochem. Parasitol. 1998; PubMed Scopus Google have been to play a role in activating latent TGF-β. activity been implicated to the of latent TGF-β, in the infection (16Miyazono K. Heldin C.H. Nature. 1989; 338: 158-160Crossref PubMed Scopus (213) Google Scholar). However, of the mechanism of TGF-β not been suggest that Leishmania cathepsin B play a role in survival and pathogenesis by activating latent TGF-β, the parasites to within the macrophages. The mature and active TGF-β in cytokine causing susceptibility to disease A. M. R. H. J. 1992; PubMed Scopus Google Scholar). TGF-β macrophage inhibition of inducible nitric-oxide S.E. J. 1993; PubMed Scopus Google and Y. C. Paik J. Nathan C. J. 1993; PubMed Scopus Google production and thus the of parasites within the macrophages. that cathepsin B can as a to the of a for the of intracellular parasitic are to cathepsin B gene which can with in of its role in Leishmania pathogenesis using mouse Also, studies on the mechanism of of latent TGF-β by cathepsin B. These studies to the of the of the disease in of intracellular parasites and a for of in which TGF-β is

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