Ana Cristina Nogueira de Melo

Detection of matrix metallopeptidase-9-like proteins in Trypanosoma cruzi

In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitmans complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97kDa protein band in cellular extract and an 85kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid.

In vitro evidence for metallopeptidase participation in hepatocyte damage induced by Leishmania chagasi-infected macrophages

Leishmania (Leishmania) chagasi infection activates macrophages, which release several microbicidal agents, including peptidases, to eliminate the parasite. Leishmanicidal mediators released in large amounts may cause morphological and/or functional injuries to the liver. In order to investigate the involvement of peptidases in this phenomenon, an in vitro co-culture model of peritoneal macrophages infected with L. chagasi and hepatocytes was used. High levels of released hepatic transaminases were found in supernatants from infected co-cultures at the same time point in which alterations in hepatocyte morphology and maximum proteolytic activity were observed. The largest proteolytic activity being at pH 10 as well as the greatest efficiency of treatment with 1,10-phenantroline observed in supernatants from the infected co-cultures suggests the presence of metallopeptidases during the leishmanicidal activity by infected macrophages. Furthermore, TNF-alpha levels and high levels of TGF-beta were increased at this time point, and this can be related to the synthesis of metallopeptidases and the conversion of the latent form to the active form. Metallopeptidase activities were detected by gelatin SDS-PAGE in higher amounts in infected macrophages and co-culture supernatant; moreover, one metallopeptidase migrating at 85 kDa produced in excess (41% more) by infected macrophages was identified as MMP-9. This metallopeptidase may be participating in this phenomenon together with other leishmanicidal factors released by these host cells.

Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases.

The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.

Biological Roles of Peptidases in Trypanosomatids

In this review, we report the recent developments in the characterization of peptidases and their possible biological functions in the Trypanosomatidae family. The focus will be on peptidases from Trypanosoma cruzi, Leishmania spp., African trypanosomes and plant and insect trypanosomatids. There are numerous events in parasite development where the involvement of peptidases has been established, and they will be approached in the present review. Also in this review we will discuss the central roles have been proposed for peptidases in diverse processes such as virulence, host cell interaction and invasion, catabolism of host proteins, differentiation, cell cycle progression and both stimulation and evasion of host immune responses.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Peptidase Inhibitors as a Possible Therapeutic Strategy for Chagas Disease

Chagas disease, caused by Trypanosoma cruzi, is one of the neglected parasitic tropical diseases affecting thir- teen million people annually. At present, there are only two compounds used in the clinical treatment to this disease and they were introduced in the 1960s and 1970s. Nifurtimox and Benznidazole have limited effectiveness and serious side ef- fects. New strategies and targets for an effective chemotherapy for American trypanosomiasis need to be developed. Within this framework, the peptidases have aroused great interest as a chemotherapeutic target against Chagas disease. This is because some T. cruzi peptidases have been implicated, among other processes, in host-parasite interactions and parasitic survival in their hosts like cruzipain (cysteine peptidase) and prolyl oligopeptidase (serine peptidase). Besides, some of these peptidases are expressed in all life cycle stages of the parasite and it is essential for replication of the intra- cellular forms. The inhibition of these enzymes has shown high anti-T. cruzi activity in vitro and in vivo. In this review, we describe some peptidase inhibitors that have potential in the fight against T. cruzi. Emphasis is given to cruzipain in- hibitors and the inhibition mechanism proposed for some them. Among these inhibitors are peptidyl irreversible (halo- methyl ketone, diazomethane ketones and vinyl sulfones derivatives) and reversible (aryl ureas and oxadiazoles deriva- tives) inhibitors, besides non-peptidyl inhibitors (thiosemicarbazones, triazole, triazine nitriles and vinyl sulfones deriva- tives). Some serine peptidase inhibitors are also described (tic-based peptides, prolylprolylisoxazoles and prolylprolyli- soxazolines). Other peptidases, including the metallo-, aspartic and threonine (proteosome) peptidases are discussed and along with blocking cruzipain are targets for future therapeutic strategies.

Trypanosoma cruzi Peptidases: An Overview

Peptidases are a group of enzymes which have a catalytic function that is to hydrolyze peptide bonds of pro- teins. The enzymes that hydrolyze peptide bonds at the amino- or carboxy- terminus are classified as exopeptidases, and those that cleave peptide bonds inside the polypeptide are endopeptidases. Endopeptidases, such as cysteine-, metalo-, ser- ine- and threonine peptidases as well as some exopeptidases, have been characterized in Trypanosoma cruzi. Understand- ing the pathogenesis of T. cruzi requires the identification of functional properties of those peptidases, as they are implied in virulence, are important for host-parasite interactions and are critical for successful survival in their hosts. Here we examine the main T. cruzi peptidases, focusing on their biological roles, especially concerning the parasite-mammalian host relations.