CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection.

Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 µm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 µm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 µm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.

Cysteine peptidases in Herpetomonas samuelpessoai are modulated by temperature and dimethylsulfoxide-triggered differentiation.

Cysteine peptidases of protozoa have been implicated in a variety of biological events, and the expression of these enzymes is modulated in response to distinct stimuli, including environmental changes and differentiation. In the present work, we have examined the expression of cysteine peptidases from Herpetomonas samuelpessoai grown at distinct temperatures and during dimethylsulfoxide (DMSO)-elicited differentiation. We demonstrated that a 45 kDa cysteine peptidase had its activity reduced during the parasite growth at 37 degrees C in comparison to 26 degrees C, and when cultured up to 72 h in the presence of DMSO. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and DMSO are able to trigger the promastigote to paramastigote transformation in H. samuelpessoai. The possible immunological similarity of H. samuelpessoai proteins with well-known cysteine peptidases produced by trypanosomatid pathogens, including cruzipain (Trypanosoma cruzi) and cysteine peptidase b (cpb) from Leishmania mexicana, was also investigated, as well as with calpain molecules. The protein cellular lysate of H. samuelpessoai reacted with antibodies raised against cpb of L. mexicana and calpain of Drosophila melanogaster; however, no reaction was observed against cruzipain. The 35 kDa cpb-like protein had its expression diminished in DMSO-treated parasites, while the 80 kDa calpain-like molecule was enhanced and an additional 30 kDa calpain-related polypeptide was exclusively observed in these cells. Fluorescence microscopy and flow cytometry analyses corroborated these data. The results described above add H. samuelpessoai to the list of parasites whose differentiation seems to be correlated with cysteine peptidase expression.

Arrested growth of Trypanosoma cruzi by the calpain inhibitor MDL28170 and detection of calpain homologues in epimastigote forms.

In this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms of Trypanosoma cruzi isolate Dm28c. MDL28170 at 70 microM promoted a powerful reduction in the growth rate after 48 h. The IC50 value was calculated to be 31.7 microM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect. T. cruzi CALPs presented a strong cross-reactivity with anti-Drosophila melanogaster calpain and anti-cytoskeleton-associated protein from Trypanosoma brucei antibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TcII), Dm28c (lineage TcI) [corrected] and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

Cruzipain: An Update on its Potential as Chemotherapy Target against the Human Pathogen Trypanosoma cruzi.

Chagas' disease is one of the most impactful and prevalent neglected tropical diseases in the Americas, specially affecting the poor and underdeveloped areas in Latin America. Aggravating this scenario, the medicines used in the current chemotherapy are old, toxic and present a low efficacy to treat the chronic stage of this disease. In addition, resistant strains of Trypanosoma cruzi, the etiological agent, are frequently reported. So, there is an imperative requirement for novel chemotherapeutic options to treat this debilitating disease. In this context, peptidases have emerged as potential targets and, consequently, proteolytic inhibitors have confirmed to be valuable drugs against several human pathologies. In this line of thinking, T. cruzi produces a major multifunctional cysteine peptidase, named cruzipain, which directly and/or indirectly orchestrates several physiological and pathological processes, which culminate in a successful parasitic infection. Taken together, these findings point out that cruzipain is one of the most important targets for driving a chemotherapy approach against the human pathogen T. cruzi. The present review summarizes some of the recent advances and failures in this area, with particular emphasis on recently published studies.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species.

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Aspartic peptidases of human pathogenic trypanosomatids: perspectives and trends for chemotherapy.

Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas' disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in trypanosomatid cells and aspartic proteolytic inhibitors can be benefic chemotherapeutic agents against these human pathogenic microorganisms.

Calpains: potential targets for alternative chemotherapeutic intervention against human pathogenic trypanosomatids.

The treatment for both leishmaniasis and trypanosomiasis, which are severe human infections caused by trypanosomatids belonging to Leishmania and Trypanosoma genera, respectively, is extremely limited because of concerns of toxicity and efficacy with the available anti-protozoan drugs, as well as the emergence of drug resistance. Consequently, the urgency for the discovery of new trypanosomatid targets and novel bioactive compounds is particularly necessary. In this context, the investigation of changes in parasite gene expression between drug resistant/sensitive strains and in the up-regulation of virulence-related genes in infective forms has brought to the fore the involvement of calpain-like proteins in several crucial pathophysiological processes performed by trypanosomatids. These studies were encouraged by the publication of the complete genome sequences of three human pathogenic trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which allowed in silico analyses that in turn directed the identification of numerous genes with interesting chemotherapeutic characteristics, including a large family of calpain-related proteins, in which to date 23 genes were assigned as calpains in T. brucei, 40 in T. cruzi and 33 in L. braziliensis. In the present review, we intend to add to these biochemical/biological reports the investigations performed upon the inhibitory capability of calpain inhibitors against human pathogenic trypanosomatids.

Antimicrobial action of chelating agents: repercussions on the microorganism development, virulence and pathogenesis.

Infections caused by resistant microorganisms often fail to respond to conventional therapy, resulting in prolonged illness, increased treatment costs and greater risk of death. Consequently, the development of novel antimicrobial drugs is becoming more demanding every day since the existing drugs either have too many side-effects or they tend to lose effectiveness due to the selection of resistant strains. In view of these facts, a number of new strategies to obstruct vital biological processes of a microbial cell have emerged; one of these is focused on the use of metal-chelating agents, which are able to selectively disturb the essential metal metabolism of the microorganism by interfering with metal acquisition and bioavailability for crucial reactions. The chelation activity is able to inhibit the biological role of metal-dependent proteins (e.g., metalloproteases and transcription factors), disturbing the microbial cell homeostasis and culminating in the blockage of microbial nutrition, growth and development, cellular differentiation, adhesion to biotic (e.g., extracellular matrix components, cell and/or tissue) and abiotic (e.g., plastic, silicone and acrylic) structures as well as controlling the in vivo infection progression. Interestingly, chelating agents also potentiate the activity of classical antimicrobial compounds. The differences between the microorganism and host in terms of the behavior displayed in the presence of chelating agents could provide exploitable targets for the development of an effective chemotherapy for these diseases. Consequently, metal chelators represent a novel group of antimicrobial agents with potential therapeutic applications. This review will focus on the anti-fungal and anti-protozoan action of the most common chelating agents, deciphering and discussing their mode of action.

Differential influence of gp63-like molecules in three distinct Leptomonas species on the adhesion to insect cells.

Parasites belonging to the Leptomonas genus have been used as model organisms for studying biochemical, cellular, and genetic processes unique to members of the Trypanosomatidae family. In the present study, the cell-associated and extracellular peptidases of three Leptomonas species, Leptomonas collosoma, Leptomonas samueli, and Leptomonas wallacei, were assayed and characterized by gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis. All parasites released metallopeptidases, whereas no cell-associated proteolytic activity could be detected in the cellular extracts from L. collosoma. Western blotting probed with a polyclonal antibody raised against gp63 from Leishmania amazonensis revealed two major reactive polypeptides of apparent molecular masses of 63 and 52 kDa, with different intensities in cellular extracts and released proteins from the studied trypanosomatids. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. This is the first report on the presence of gp63-like molecules in L. collosoma, L. samueli, and L. wallacei. The pretreatment of L. samueli and L. wallacei with anti-gp63 antibody significantly diminished their association index to Aedes albopictus cell line (C6/36), suggesting a potential involvement of the gp63-like molecules in the interaction process of these insect trypanosomatids with the vector.

Proteolytic expression in Blastocrithidia culicis: influence of the endosymbiont and similarities with virulence factors of pathogenic trypanosomatids.

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.