Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas' disease/AIDS co-infection, we decided to explore the ...effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas' disease.
HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages.
The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.
The introduction of the HIV aspartic peptidase inhibitors (HIV-PIs) has revolutionized the medical arena, since they have drastically reduced the number and the severity of opportunistic infections, ...including the protozoal diseases that afflict the HIV-infected individuals worldwide. HIV-PIs rapidly and profoundly diminish the viral load, which is paralleled by increase in the CD4+ T lymphocyte counts and stimulation of the survival and activation of neutrophil, monocyte and endothelial cells, culminating in a vigorous reduction in the number of deaths due to the AIDS, in the number of new cases of AIDS and in the number of hospitalization days. Many research groups around the globe are trying to decipher both the in vitro and in vivo antiprotozoal mechanisms behind the use of HIVPIs. These studies have been supported by the urgent need to discover novel active compounds able to treat incurable parasitoses, including three major neglected diseases: malaria, leishmaniasis and Chagas' disease. The present review summarizes the recent advances on the effects of HIV-PIs against Plasmodium spp., Leishmania spp. and Trypanosoma cruzi.
Chagas disease, caused by the flagellate parasite Trypanosoma cruzi, is a wellknown neglected tropical disease. This parasitic illness affects 6-7 million people and can lead to severe myocarditis ...and/or complications of the digestive tract. The changes in its epidemiology facilitate co-infection with the Human Immunodeficiency Virus (HIV), making even more difficult the diagnosis and prognosis. The parasitic infection is reactivated in T. cruzi/HIV co-infection, with the appearance of unusual manifestations in the chronic phase and the exacerbation of classical clinical signs. The therapeutic arsenal to treat Chagas disease, in all its clinical forms, is restricted basically to two drugs, benznidazole and nifurtimox. Both drugs are extremely toxic and the therapeutic efficacy is still unclear, making the clinical treatment a huge issue to be solved. Therefore, it seems obvious the necessity of new tangible approaches to combat this illness. In this sense, the repositioning of approved drugs appears as an interesting and viable strategy. The discovery of Human Immunodeficiency Virus Aspartyl Peptidase Inhibitors (HIV-PIs) represented a milestone in the treatment of Acquired Immune Deficiency Syndrome (AIDS) and, concomitantly, a marked reduction in both the incidence and prevalence of important bacterial, fungal and parasitic co-infections was clearly observed. Taking all these findings into consideration, the present review summarizes the promising and beneficial data concerning the effects of HIV-PIs on all the evolutionary forms of T. cruzi and in important steps of the parasite's life cycle, which highlight their possible application as alternative drugs to treat Chagas disease.
We report the characterization of cell-associated and extracellular peptidases of Bodo sp., a free-living flagellate of the Bodonidae family, order Kinetoplastida, which is considered ancestral to ...the trypanosomatids. This bodonid isolate is phylogenetically related to Bodo caudatus and Bodo curvifilus. The proteolytic activity profiles of Bodo sp. were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing co-polymerized gelatin, casein, hemoglobin, or bovine serum albumin as substrates. The enzymatic complex degraded gelatin better in acidic pH, and under these conditions four proteolytic bands (120, 100, 90, and 75 kDa) were detected in the cellular or extracellular extracts. Two peptidases (250 and 200 kDa) were exclusively detected with the substrate casein. All these enzymes belong to the serine peptidase class, based on inhibition by aprotinin and phenylmethylsulfonyl fluoride. This is the first biochemical characterization of peptidases in a free-living Bodo sp., potentially providing insight into the physiology of these protozoa and the evolutionary importance of peptidases to the order Kinetoplastida as some of these enzymes are important virulence factors in pathogenic trypanosomatids.
The cell-associated and extracellular peptidases of
Herpetomonas megaseliae grown in brain–heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on ...gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115–100, 40 and 35
kDa) and metallopeptidase (70 and 60
kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50
kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition,
H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the
H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from
Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52
kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in
H. megaseliae on the interaction with explanted guts of its original host,
Megaselia scalaris, and with an experimental model employing
Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to
M. scalaris and
A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues.
Abstract
Any actual understanding of trypanosomatids in general requires a comprehensive analysis of the less‐specialized species as thorough as our knowledge of the more specialized Leishmania and ...Trypanosoma. In this context, we have shown by antibody cross‐reactivity that purified extracellular metallopeptidases from Phytomonas françai, Crithidia deanei (cured strain) and Crithidia guilhermei share common epitopes with the leishmanial gp63. Flow cytometry and fluorescence microscopy analyses indicated the presence of gp63‐like molecules on the cell surface of these lower trypanosomatids. Binding assays with explanted guts of Aedes aegypti incubated with purified gp63 and the pretreatment of trypanosomatids with anti‐gp63 antibodies indicated that the gp63‐like molecules are involved in the adhesive process of these trypanosomatids to the A. aegypti gut wall. In addition, our results indicate for the first time that the gp63‐like molecule binds to a polypeptide of 50 kDa on the A. aegypti gut epithelium extract.
The glycoprotein profiles of seven choanomastigote-shaped trypanosomatids (six
Crithidia spp. and one
Herpetomonas sp.), which have been suggested to form three distinct taxonomic groups (
Crithidia,
...Angomonas and
Strigomonas), were analyzed by Western blotting using the lectins
Limax flavus (LFA),
Sambucus nigra (SNA) and
Maackia amurensis (MAA), which specifically recognize sialic acid residues, and concanavalin A (Con A) that recognizes mannose-like residues in glycoconjugates. All lectins showed a sugar-inhibited recognition with the parasite extracts, with the exception of LFA, which did not show any reactivity with the studied species. The SNA agglutinin presented a characteristic and specific pattern for each taxonomic group. The MAA lectin showed an identical profile for all species analyzed, while Con A grouped the choanomastigote-shaped species in two different patterns, one specific for the
Angomonas group, and the other comprehending both
Strigomonas and
Crithidia groups. These results illustrate the heterogeneity of the genus
Crithidia. The possible taxonomic redistribution of the choanomastigote-shaped trypanosomatids is also discussed.
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