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1.
Free Radic Biol Med ; 146: 392-401, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31760093

RESUMO

During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. Strigomonas culicis is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type H2O2-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of Aedes aegypti infection. Here, we assess the biological contribution of the S. culicis endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in H2O2 resistance and may be responsible for the high infection rates previously described for A. aegypti. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of S. culicis are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.

2.
Mem Inst Oswaldo Cruz ; 114: e190147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553371

RESUMO

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.


Assuntos
Calpaína/genética , Genoma de Protozoário/genética , Leishmania braziliensis/química , Macrófagos Peritoneais/metabolismo , Animais , Western Blotting , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Calpaína/ultraestrutura , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica , Imuno-Histoquímica , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Virulência
4.
PLoS One ; 14(1): e0210740, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682075

RESUMO

The enteric protist Blastocystis is one of the most frequently reported parasites infecting both humans and many other animal hosts worldwide. A remarkable genetic diversity has been observed in the species, with 17 different subtypes (STs) on a molecular phylogeny based on small subunit RNA genes (SSU rDNA). Nonetheless, information regarding its distribution, diversity and zoonotic potential remains still scarce, especially in groups other than primates. In Brazil, only a few surveys limited to human isolates have so far been conducted on Blastocystis STs. The aim of this study is to determine the occurrence of Blastocystis subtypes in non-human vertebrate and invertebrate animal groups in different areas of the state of Rio de Janeiro, Brazil. A total of 334 stool samples were collected from animals representing 28 different genera. Blastocystis cultivated samples were subtyped using nuclear small subunit ribosomal DNA (SSU rDNA) sequencing. Phylogenetic analyses and BLAST searches revealed six subtypes: ST5 (28.8%), ST2 (21.1%), ST1 and ST8 (19.2%), ST3 (7.7%) and ST4 (3.8%). Our findings indicate a considerable overlap between STs in humans and other animals. This highlights the importance of investigating a range of hosts for Blastocystis to understand the eco-epidemiological aspects of the parasite and its host specificity.


Assuntos
Blastocystis/classificação , Blastocystis/genética , Animais , Brasil , DNA de Protozoário/genética , DNA Ribossômico/genética , Epidemiologia Molecular/métodos , Filogenia
5.
Mem. Inst. Oswaldo Cruz ; 114: e190147, 2019. tab, graf
Artigo em Inglês | LILACS-Express | ID: biblio-1040618

RESUMO

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

6.
Parasitol Res ; 117(7): 2085-2094, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29728827

RESUMO

Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an interesting alternative, since Leishmania cells express calpain-related proteins. The susceptibility of six Leishmania species (L. amazonensis, L. braziliensis, L. major, L. mexicana, L. chagasi, and L. donovani) to the calpain inhibitor MDL28170 was determined. Promastigote and intracellular amastigote viability in the presence of MDL28170 was evaluated. MDL28170 was able to reduce promastigote proliferation in a dose-dependent manner for all the parasites. A significant reduction on the general parasite metabolism was detected, as judged by resazurin assay, as well as induced important morphological alterations, including rounding promastigotes and loss of the flagellum. MDL28170 was also able to reduce the number of intracellular amastigotes in RAW macrophages. The susceptibility of both parasite stages (promastigotes and amastigotes) to MDL28170 was similar for all Leishmania species tested. MDL28170 showed a much higher toxicity to Leishmania amastigotes when compared with mammalian macrophages, displaying selectivity index values varying from 13.1 to 39.8. These results suggest that the development of calpain inhibitors may represent an interesting alternative in the treatment of leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Glicoproteínas/farmacologia , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Animais , Linhagem Celular , Humanos , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Parasitária , Células RAW 264.7
7.
PLoS One ; 13(3): e0193860, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29522552

RESUMO

BACKGROUND: Intestinal parasitic infections are considered a serious public health problem and widely distributed worldwide, mainly in urban and rural environments of tropical and subtropical countries. Globally, soil-transmitted helminths and protozoa are the most common intestinal parasites. Blastocystis sp. is a highly prevalent suspected pathogenic protozoan, and considered an unusual protist due to its significant genetic diversity and host plasticity. METHODOLOGY/MAIN FINDINGS: A total of 294 stool samples were collected from inhabitants of three rural valleys in Rio de Janeiro, Brazil. The stool samples were evaluated by parasitological methods, fecal culture, nested PCR and PCR/Sequencing. Overall prevalence by parasitological analyses was 64.3% (189 out of 294 cases). Blastocystis sp. (55.8%) was the most prevalent, followed by Endolimax nana (18.7%), Entamoeba histolytica complex (7.1%), hookworm infection (7.1%), Entomoeba coli (5.8%), Giardia intestinalis (4.1%), Iodamoeba butchilii (1.0%), Trichuris trichiura (1.0%), Pentatrichomonas hominis (0.7%), Enterobius vermicularis (0.7%), Ascaris lumbricoides (0.7%) and Strongyloides stercoralis (0.7%). Prevalence of IPIs was significantly different by gender. Phylogenetic analysis of Blastocystis sp. and BLAST search revealed five different subtypes: ST3 (34.0%), ST1 (27.0%), ST2 (27.0%), ST4 (3.5%), ST8 (7.0%) and a non-identified subtype. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that intestinal parasite infection rates in rural areas of the Sumidouro municipality of Rio de Janeiro, Brazil are still high and remain a challenge to public health. Moreover, our data reveals significant genetic heterogeneity of Blastocystis sp. subtypes and a possible novel subtype, whose confirmation will require additional data. Our study contributes to the understanding of potential routes of transmission, epidemiology, and genetic diversity of Blastocystis sp. in rural areas both at a regional and global scale.


Assuntos
Infecções por Blastocystis/epidemiologia , Blastocystis/isolamento & purificação , Enteropatias Parasitárias/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/parasitologia , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos Transversais , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Feminino , Variação Genética , Helmintíase/epidemiologia , Helmintíase/parasitologia , Humanos , Enteropatias Parasitárias/parasitologia , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Infecções por Protozoários/epidemiologia , Infecções por Protozoários/parasitologia , Ribotipagem , População Rural , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Adulto Jovem
8.
Free Radic Biol Med ; 113: 255-266, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28993269

RESUMO

Reactive oxygen species (ROS) are toxic molecules involved in several biological processes such as cellular signaling, proliferation, differentiation and cell death. Adaptations to oxidative environments are crucial for the success of the colonization of insects by protozoa. Strigomonas culicis is a monoxenic trypanosomatid found in the midgut of mosquitoes and presenting a life cycle restricted to the epimastigote form. Among S. culicis peculiarities, there is an endosymbiotic bacterium in the cytoplasm, which completes essential biosynthetic routes of the host cell and may represent an intermediary evolutive step in organelle origin, thus constituting an interesting model for evolutive researches. In this work, we induced ROS resistance in wild type S. culicis epimastigotes by the incubation with increasing concentrations of hydrogen peroxide (H2O2), and compared the oxidative and energetic metabolisms among wild type, wild type-H2O2 resistant and aposymbiotic strains. Resistant protozoa were less sensitive to the oxidative challenge and more dependent on oxidative phosphorylation, which was demonstrated by higher oxygen consumption and mitochondrial membrane potential, increased activity of complexes II-III and IV, increased complex II gene expression and higher ATP production. Furthermore, the wild type-H2O2 resistant strain produced reduced ROS levels and showed lower lipid peroxidation, as well as an increase in gene expression of antioxidant enzymes and thiol-dependent peroxidase activity. On the other hand, the aposymbiotic strain showed impaired mitochondrial function, higher H2O2 production and deficient antioxidant response. The induction of H2O2 resistance also led to a remarkable increase in Aedes aegypti midgut binding in vitro and colonization in vivo, indicating that both the pro-oxidant environment in the mosquito gut and the oxidative stress susceptibility regulate S. culicis population in invertebrates.


Assuntos
Aedes/parasitologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Metabolismo Energético/genética , Interações Hospedeiro-Parasita , Peróxido de Hidrogênio/farmacologia , Proteínas de Protozoários/genética , Trypanosomatina/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Antioxidantes/metabolismo , Betaproteobacteria/metabolismo , Evolução Biológica , Resistência a Medicamentos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Trato Gastrointestinal/parasitologia , Regulação da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Simbiose/fisiologia , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/genética , Trypanosomatina/microbiologia
9.
Parasit Vectors ; 10(1): 518, 2017 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-29070053

RESUMO

BACKGROUND: Blastocystis is a cosmopolitan protist parasite found in the human gastrointestinal tract and is highly prevalent in developing countries. Recent molecular studies have revealed extensive genetic diversity, which has been classified into different subtypes (STs) based on sequence analysis of small subunit ribosomal RNA gene. Blastocystis is one of the most common fecal parasites in Brazil, but the diversity of subtypes remains unknown in the country. This study aimed to determine the distribution of Blastocystis STs in an urban community in Duque de Caxias, Rio de Janeiro, Brazil. METHODS: A total of 64 stool samples positive for Blastocystis in Pavlova's medium were subtyped by PCR and sequenced using primers targeting the small subunit rRNA gene, in addition to phylogenetic analysis and subtype-specific PCR using sequence-tagged-site (STS) primers. RESULTS: Endolimax nana (14%), Entamoeba complex (10.5%), Taenia sp. (0.6%), Trichuris trichiura (1.3%) and Enterobius vermicularis (1.3%) were detected in Blastocystis-positive samples. Of the 64 samples tested by PCR/DNA sequencing, 55 were identified as ST1 (42%), ST3 (49%), ST2 (7%) and ST4 (2%), and the presence of mixed ST (ST1 + ST3) infection was detected in nine samples (14%). CONCLUSIONS: DNA sequencing and phylogenetic analysis of Brazilian Blastocystis isolates identified four different subtypes. To our knowledge, this study provided the first genetic characterization of Blastocystis subtypes in an urban area of Rio de Janeiro, Brazil. We also identified ST4 for the first time in Brazil. Further studies are necessary to determine the distribution of STs across human populations in Rio de Janeiro.


Assuntos
Infecções por Blastocystis/epidemiologia , Infecções por Blastocystis/parasitologia , Blastocystis/isolamento & purificação , Variação Genética , Filogenia , População Urbana , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Blastocystis/classificação , Blastocystis/genética , Brasil/epidemiologia , Criança , Pré-Escolar , Primers do DNA , DNA de Protozoário/genética , DNA Ribossômico/genética , Fezes/parasitologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Análise de Sequência de DNA , Adulto Jovem
10.
Protist ; 168(3): 326-334, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28472733

RESUMO

Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors.


Assuntos
Quelantes/metabolismo , Leishmania braziliensis/fisiologia , Leishmania infantum/fisiologia , Metaloendopeptidases/metabolismo , Polissacarídeos/metabolismo , Psychodidae/parasitologia , Animais , Metais/metabolismo
12.
Mem Inst Oswaldo Cruz ; 112(1): 31-43, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27925020

RESUMO

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites' physiology.


Assuntos
Cisteína/efeitos dos fármacos , Euglenozoários/efeitos dos fármacos , Heterópteros/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Animais , Western Blotting , Cisteína/metabolismo , Dipeptídeos , Euglenozoários/enzimologia , Euglenozoários/ultraestrutura , Citometria de Fluxo , Dose Letal Mediana , Microscopia Eletrônica , Glândulas Salivares/parasitologia
13.
Parasitology ; 144(2): 117-123, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27869056

RESUMO

Leishmaniasis is a neglected disease, which needs improvements in drug development, mainly due to the toxicity, parasite resistance and low compliance of patients to treatment. Therefore, the development of new chemotherapeutic compounds is an urgent need. This opinion article will briefly highlight the feasible use of calpain inhibitors as leading compounds to search for new therapeutic options to treat leishmaniasis. The milestone of this approach is to take advantage on the myriad of inhibitors developed against calpains, some of which are in advanced clinical trials. The deregulated activity of these enzymes is associated with several pathologies, such as strokes, diabetes and Parkinson's disease, to name a few. In Leishmania, calpain upregulation has been associated to drug resistance and virulence. Whereas the difficulties in developing new drugs for neglected diseases are more economical than biotechnological, repurposing approach with compounds already approved for clinical use by the regulatory agencies can be an interesting shortcut to a successful chemotherapeutic treatment for leishmaniasis.


Assuntos
Antiprotozoários/farmacologia , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Leishmania/metabolismo , Leishmaniose/tratamento farmacológico , Animais , Antiprotozoários/química , Humanos , Leishmania/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico
14.
Mem Inst Oswaldo Cruz ; 110(8): 956-65, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26602872

RESUMO

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.


Assuntos
Biodiversidade , DNA de Protozoário/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Kinetoplastida/genética , Filogenia , RNA de Protozoário/genética , Biomarcadores , Biologia Computacional , Código de Barras de DNA Taxonômico/tendências , Bases de Dados Genéticas , Meio Ambiente , Kinetoplastida/classificação , Kinetoplastida/citologia , Metagenômica/tendências , RNA Ribossômico 18S/genética
15.
BMC Microbiol ; 15: 188, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26415499

RESUMO

BACKGROUND: Angomonas deanei is a trypanosomatid parasite of insects that has a bacterial endosymbiont, which supplies amino acids and other nutrients to its host. Bacterium loss induced by antibiotic treatment of the protozoan leads to an aposymbiotic strain with increased need for amino acids and results in increased production of extracellular peptidases. In this work, a more detailed examination of A. deanei was conducted to determine the effects of endosymbiont loss on the host calpain-like proteins (CALPs), followed by testing of different calpain inhibitors on parasite proliferation. RESULTS: Western blotting showed the presence of different protein bands reactive to antibodies against calpain from Drosophila melanogaster (anti-Dm-calpain), lobster calpain (anti-CDPIIb) and cytoskeleton-associated calpain from Trypanosoma brucei (anti-CAP5.5), suggesting a possible modulation of CALPs influenced by the endosymbiont. In the cell-free culture supernatant of A. deanei wild type and aposymbiotic strains, a protein of 80 kDa cross-reacted with the anti-Dm-calpain antibody; however, no cross-reactivity was found with anti-CAP5.5 and anti-CDPIIb antibodies. A search in A. deanei genome for homologues of D. melanogaster calpain, T. brucei CAP5.5 and lobster CDPIIb calpain revealed the presence of hits with at least one calpain conserved domain and also with theoretical molecular mass consistent with the recognition by each antibody. No significant hit was observed in the endosymbiont genome, indicating that calpain molecules might be absent from the symbiont. Flow cytometry analysis of cells treated with the anti-calpain antibodies showed that a larger amount of reactive epitopes was located intracellularly. The reversible calpain inhibitor MDL28170 displayed a much higher efficacy in diminishing the growth of both strains compared to the non-competitive calpain inhibitor PD150606, while the irreversible calpain inhibitor V only marginally diminished the proliferation. CONCLUSIONS: Altogether, these results indicate that distinct calpain-like molecules are expressed by A. deanei, with a possible modulation in the expression influenced by the endosymbiont. In addition, treatment with MDL28170 affects the growth rate of both strains, as previously determined in the human pathogenic species Leishmania amazonensis and Trypanosoma cruzi, with whom A. deanei shares immunological and biochemical relationships.


Assuntos
Bactérias/crescimento & desenvolvimento , Calpaína/antagonistas & inibidores , Calpaína/biossíntese , Glicoproteínas/metabolismo , Simbiose , Trypanosomatina/crescimento & desenvolvimento , Trypanosomatina/microbiologia , Calpaína/genética , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/genética
17.
PLoS One ; 10(5): e0124832, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25938232

RESUMO

Several studies indicate that the activity of cruzipain, the main lysosomal cysteine peptidase of Trypanosoma cruzi, contributes to parasite infectivity. In addition, the parasitic invasion process of mammalian host cells is described to be dependent on the activation of the host TGF-ß signaling pathway by T. cruzi. Here, we tested the hypothesis that cruzipain could be an important activator of latent TGF-ß and thereby trigger TGF-ß-mediated events crucial for the development of Chagas disease. We found that live epimastigotes of T. cruzi, parasite lysates and purified cruzipain were able to activate latent TGF-ß in vitro. This activation could be inhibited by the cysteine peptidase inhibitor Z-Phe-Ala-FMK. Moreover, transfected parasites overexpressing chagasin, a potent endogenous cruzipain inhibitor, prevented latent TGF-ß activation. We also observed that T. cruzi invasion, as well as parasite intracellular growth, were inhibited by the administration of Z-Phe-Ala-FMK or anti-TGF-ß neutralizing antibody to Vero cell cultures. We further demonstrated that addition of purified cruzipain enhanced the invasive activity of trypomastigotes and that this effect could be completely inhibited by addition of a neutralizing anti-TGF-ß antibody. Taken together, these results demonstrate that the activities of cruzipain and TGF-ß in the process of cell invasion are functionally linked. Our data suggest that cruzipain inhibition is an interesting chemotherapeutic approach for Chagas disease not only because of its trypanocidal activity, but also due to the inhibitory effect on TGF-ß activation.


Assuntos
Cisteína Endopeptidases/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Anticorpos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Cetonas , Parasitos/efeitos dos fármacos , Proteínas de Protozoários/farmacologia , Transfecção , Fator de Crescimento Transformador beta/imunologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Células Vero
18.
Int J Parasitol Drugs Drug Resist ; 4(3): 210-3, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25516829

RESUMO

Pentavalent antimonials have saved the lives of thousands of Leishmania-infected patients more than seventy years but, unfortunately, they are highly toxic and require parenteral delivery. Therefore, the search for safer and orally delivered alternative is a need. This paper describes the antileishmanial properties of PMIC4, a novel hydroxyethylpiperazine analogue. PMIC4 showed potent activity against intracellular amastigotes of Leishmania amazonensis, with IC50 of 1.8 µM and selectivity index higher than 100-fold, calculated in relation to the toxicity on the host cell. Following laboratory animal welfare policies, we analyzed the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties and calculated the Lipinski's rule of five of PMIC4 before proceeding to in vivo tests. PMIC4 satisfied Lipinski's rule of five and presented high probability of human intestinal absorption, suggesting a good chance of druglikeness and oral bioavailability. For in vivo studies, PMIC4 was administered via intralesional injection (3.4 mg/kg/day, three times a week) or orally (34.0 mg/kg/day, five times a week) to L. amazonensis-infected BALB/c mice throughout the 98 day experiments. At the end of the treatment period, serum markers of toxicity were measured. When administered orally, PMIC4 controlled the lesions in L. amazonensis-infected BALB/c mice without altering serological markers of toxicity. These results demonstrate that PMIC4 is a promising molecular scaffold, orally effective against experimental leishmaniasis.

20.
PLoS Negl Trop Dis ; 6(12): e1958, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23272264

RESUMO

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain. CONCLUSIONS/SIGNIFICANCE: These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.


Assuntos
Cisteína Endopeptidases/metabolismo , Rhodnius/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Adesão Celular , Trato Gastrointestinal/parasitologia , Interações Hospedeiro-Parasita , Proteínas de Protozoários
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