A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania ( Viannia) braziliensis strains P. CUERVO 1,2,5 , L. SABO ´ IA-VAHIA 3,4 , F. COSTA SILVA-FILHO 4 , O. FERNANDES 1 , E. CUPOLILLO 5 and J. B. DE JESUS 3,4 * 1 Departamento de Medicina Tropical, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil 2 Centro Internacional de Entrenamiento e Investigaciones Me ´dicas CIDEIM, Cali, Colombia 3 Departamento de Bioquı ´mica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil 4 Instituto de Biofisica, Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil 5 Departamento de Imunologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brasil (Received 13 April 2005; revised 24 June and 3 August 2005; accepted 4 August 2005; first published online 3 October 2005) SUMMARY Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and ex- tracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5 . 5 to 9 . 0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L.(V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation. Key words: parasitic protozoa, trypanosomatids, Leishmania (Viannia) braziliensis, leishmaniasis, proteases, metalloproteases. INTRODUCTION The genus Leishmania includes parasitic protozoa responsible for a species-dependent spectrum of diseases extending from localized, self-healing cu- taneous lesions to more severe visceral infections. In endemic areas of the Americas, Leishmania (Viannia) braziliensis is the major causative agent of tegumen- tary leishmaniasis (Grimaldi, Tesh and McMahon- Pratt, 1989 ; Canto-Lara et al. 1998). This parasite is genetically heterogeneous (Cupolillo et al. 2003), exhibiting substantial enzyme polymorphism. This protozoa has been associated to a broad range of clinical manifestations – from simple cutaneous ulcers to disseminated lesions and mucosal involve- ment, a very destructive form of leishmaniasis (Saravia et al. 1985 ; Marsden, 1986 ; Weigle et al. 1986; Grimaldi and Tesh 1993; Cupolillo et al. 2003). The plasticity of L.(V.) braziliensis and its interaction with the human host, producing distinct clinical forms, represents a challenge to researchers looking for a better understanding of the infection’s natural history. The life-cycle of Leishmania parasites involves 2 principal developmental stages : the amastigote form, usually confined in mammalian macrophages, and the promastigote form, found within the sand fly insect vector. The digenetic parasite’s mechanism of differentiation presented in diverse vertebrate and invertebrate hosts requires the parasite to possess the enzymatic tools necessary for quick and efficient re- sponse to dramatic environmental change. Proteases have been implicated to play crucial roles in parasitic cytoadherence, tissue invasion activities, survival and proliferation in host cells and virulence (Coombs, 1982; McGrath et al. 1995; Klemba and Goldberg, 2002; Sajid and McKerrow, 2002). Both metalloproteases and cysteine proteases have been implicated as putative determinants for infection and virulence factors of several species of Leishmania (Coombs, 1982 ; Chang and Chang, 1986 ; Russell and Wilhelm, 1986 ; Liu and Chang, 1992 ; Brittingham et al. 1995 ; Mottram et al. 1996, Mottram, Brooks and Coombs, 1998 ; Mahmoudzzadeh-Niknam and McKerrow, 2004). In addition, differential expression of protease * Corresponding author : Departamento de Bioquı ´mica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, CEP: 21045-900, Rio de Janeiro, RJ, Brazil. Tel: +55 21 3865 8153. Fax: +55 21 2590 3495. E-mail : jbj@ioc.fiocruz.br 177 Parasitology (2006), 132, 177–185. f 2005 Cambridge University Press doi:10.1017/S0031182005008942 Printed in the United Kingdom