Comparative zymographic analysis of metallopeptidase of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis isolates from Peru

Abstract

American tegumentary leishmaniasis (ATL) in Peru is mainly associated with Leishmania (Viannia) peruviana and L. (V.) braziliensis. These parasites are genetically related, and their characterization as distinct species is controversial. Despite their genetic similarity, each species is associated with different clinical manifestations of ATL; L. (V.) peruviana causes only cutaneous leishmaniasis, whereas L. (V.) braziliensis can cause both cutaneous and mucocutaneous leishmaniasis. Because the primary cutaneous lesions caused by infection with these species are indistinguishable, it is necessary to develop a suitable method to differentiate them in order to prevent possible metastasis to oropharyngeal mucosa. In the present study, we investigated the proteolytic profile of L. (V.) peruviana and L. (V.) braziliensis isolates from Peru by zymographic analysis in SDS-PAGE copolymerized with gelatin. Enzymes were characterized according to their pH range of activity and sensitivity to distinct peptidase inhibitors. We observed that L. (V.) peruviana isolates displayed three proteolytic bands with molecular masses ranging from 55 to 80 kDa, whereas L. (V.) braziliensis isolates showed six proteolytic activities between 55 and 130 kDa. Using specific inhibitors, we determined that these proteolytic activities are due to metallopeptidases and present optimal activity between the pH range 5.5 and 10.0. Our results suggest that the expression of metallopeptidases in L. (V.) peruviana and L. (V.) braziliensis isolates from Peru is species-specific.

Introduction

American tegumentary leishmaniasis (ATL) is a disease caused by protozoa of the Leishmania genus. This disease comprises a broad range of clinical manifestations ranging from mild skin ulcers that may spontaneously heal to disfiguring mucosal lesions that imply parasite dissemination from the primary cutaneous lesion [1]. It has been suggested that clinical outcome depends on the infecting Leishmania species, host and parasite genetic factors, and the immunological response of the host [1], [2]. More recently, the presence of a high load of RNA virus has been reported as another factor for severe mucocutaneous lesions [3]. In tropical and subtropical regions of South America, where the disease is endemic, Leishmania species of the Leishmania (Viannia) subgenus are predominant [4]. In Peru, the two most frequent species are L. (V.) peruviana and L. (V.) braziliensis [5]. The close genetic relationship between these species has encouraged several studies of their taxonomic status, and the results of these studies have been controversial. Despite their genetic similarity, these species are associated with different clinical outcomes of ATL; L. (V.) peruviana only causes cutaneous leishmaniasis, whereas L. (V.) braziliensis can cause both cutaneous and mucocutaneous leishmaniasis [6]. Because the primary cutaneous lesions caused by the infection with these species are indistinguishable, the development of a suitable method to determine the infecting Leishmania species is imperative in order to assign the appropriate treatment and prevent possible metastasis to oropharyngeal mucosa in cases of L. (V.) braziliensis infection [7], [8].

The first approach used for distinguishing between L. (V.) peruviana and L. (V.) braziliensis was based on different electrophoretic patterns of the mannose phosphate isomerase (MPI) isoenzyme [9]. Recently, a real-time PCR assay that distinguishes between the two species was developed based on a single nucleotide polymorphism in the MPI coding gene that accounts for different migration rates in isoenzymatic electrophoresis [10]. In addition, multi-locus PCR [11], molecular karyotyping [12], [13] and PCR-RFLP techniques targeting Gp63 and HSP70 genes have been proposed for distinguishing between these two species [14], [15], [16].

Though some methodologies are able to distinguish between these two species, the taxonomic validity of L. (V.) peruviana is still a point of debate [17], [18]. In addition to genetic characteristics, phenotypic traits can help in understanding differences observed between organisms presenting distinct biological behaviors. Peptidases play crucial roles during host–parasite interaction, affecting processes that include cytoadherence, tissue invasion, survival, proliferation, and differentiation [19], [20], [21], [22]. In the present study, we conduct a comparative analysis of peptidase activities from L. (V.) peruviana and L. (V.) braziliensis isolates from Peru. The results indicate that these species exhibit proteolytic profiles of metallopeptidases that are species-specific and that allow the distinction between these species.