13 research outputs found
Autophagic Balance Between Mammals and Protozoa: A Molecular, Biochemical and Morphological Review of Apicomplexa and Trypanosomatidae Infections
MDL28170, a Calpain Inhibitor, Affects Trypanosoma cruzi Metacyclogenesis, Ultrastructure and Attachment to Rhodnius prolixus Midgut
BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption. CONCLUSIONS/SIGNIFICANCE: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi
Expressão gênica e localização celular de calpaÃnas em Trypanosoma cruzi
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Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilAs calpaÃnas são cisteÃno-peptidases intracelulares dependentes de cálcio classicamente citosólicas envolvidas em diversas funções moduladores da célula, como transdução de sinais, divisão celular, apoptose e diferenciação. Embora melhor caracterizadas em mamÃferos, as calpaÃnas já foram descritas em insetos, nematódeos, plantas, fungos, protozoários e bactérias. No Trypanosoma cruzi, como nos demais tripanossomatÃdeos, já foi descrita a presença de uma ampla e diversa famÃlia de calpaÃnas em seu genoma, mas pouco se sabe a respeito das suas funções especÃficas. Portanto, o presente trabalho teve como objetivo buscar as sequências de calpaÃnas do T. cruzi em seu genoma no intuito de classificá-las, assim como avaliar a expressão gênica das sequências de maior similaridade com as calpaÃnas de mamÃferos. Além disso, um anticorpo gerado contra uma sequência peptÃdica conservada entre as calpaÃnas selecionadas foi utilizado em ensaios de identificação, expressão e localização celular. Nesse contexto, as análises in silico identificaram ao todo 55 sequências relacionadas à s calpaÃnas no genoma do T. cruzi. Em seguida, através de alinhamentos múltiplos e análise de domÃnios conservados, foi possÃvel classificar as calpaÃnas em quatro grupos distintos de acordo com a presença de domÃnios conservados caracterÃsticos desta famÃlia multigênica. Dos quatro grupos identificados, foi selecionado para aprofundamento do estudo aquele que possuÃa o maior número de domÃnios conservados e continha o domÃnio que alberga o sÃtio catalÃtico das calpaÃnas (conservado ou não)
A análise de expressão gênica de um total de dezesseis genes selecionados por qPCR revelou a presença de seis genes com expressão aumentada nas formas clinicamente relevantes (amastigotas e tripomastigotas) em relação as formas epimastigotas, e cinco genes nesta última forma. Em paralelo, o anticorpo anti-calpaÃnas se mostrou reativo em ensaios de Western Blotting, citometria de fluxo e microscopia de eletrônica de transmissão. Os ensaios de Western Blotting revelaram uma calpaÃna especÃfica em amastigotas e outra em tripomastigotas, além de outras sete de massas moleculares variadas presentes nas três formas do parasito. Os resultados de citometria de fluxo indicam maior marcação intracelular do anticorpo nas formas amastigotas e tripomastigotas em relação aos epimastigotas. Por fim, as análises ultraestruturais revelaram a presença das calpaÃnas em todo citoplasma, nas membranas de vesÃculas, na membrana plasmática, e no flagelo das diferentes formas do parasito. O estudo comparativo da expressão das calpaÃnas nas formas evolutivas do T. cruzi, assim como a determinação de sua localização celular, podem ajudar a determinar as funções gerais desta famÃlia multigênica no parasitoCalpains comprise
a family of calcium dependent cy
steine peptidases
commonly
present in cytoplasm implicated in a broad range of cellular modular functions such as signal
transduction, cellular division, apoptosis and differentiation processes. Although well
-
characterized in mammals, these peptidases have
also been described in
insects, nematodes,
plants, fungi, protozoa and bacteria
.
In
Trypanosoma cruzi
, as in other trypanosomatids, the
presence of an extensive and diverse family
of
calpain
s had already been described
in its
genome
. However,
the specific
functions
of these molecules are still unclear. In this context,
the present study aimed to search calpain sequences in
T. cruzi
genome to classify the genes
and to evaluate mRNA expression levels of the most conserved calpain sequences
. In
addition, an a
ntibody against
T. cruzi
calpain was produced
from a conserved sequence of
trypanosomatid calpains to evaluate these proteases levels, also determining their
ultrastructural localization. At first,
in silico
analysis revealed a total of 55 calpain sequence
s
in
T. cruzi
genome
.
Through multiple alignments and phylogenetic analysis of conserved
domains in the
se
sequences, the calpains
were sorted
into four distinct groups characterized
by the presence of classical domains of this multigenic
family. After this
in silico
analysis, we
decided to scrutiny the group that has the highest number of conserved domains and presents
domain II, which contains the catalytic active site (either altered or conserved), in order to
analyze mRNA and protein e
xpression patterns in the different
T. cruzi
forms.
The
comparison of calpain mRNA abundance
of
sixteen
genes
by qPCR in
the three
distinct
parasite
forms revealed
six
genes with
increased
expression
in the clinical relevant
forms (amastigotes and trypomas
tigotes) and
five
in the invertebrate form. S
imultaneously
,
the anti
-
tritryp
-
calpain antibody was capable of recognizing reactive molecules in Western
Blotting, flow citometry and transmission electron microscopy analysis. Altogether, Western
Blotting anal
ysis revealed seven calpains with different molecular masses present in the three
forms of the parasite, while one specific calpain was detected either in amastigotes or
tripomastigotes. Also, flow citometry results showed a higher intracellular expression
in
amastigotes and trypomastigotes in comparison with epimastigotes. Finally, ultrastructural
analysis revealed the presence of theses proteases in the cytoplasm, vesicular membranes,
plasma membranes and flagellum of the three life cycle forms. The compa
rative study of
calpain gene expression in the distinct
T. cruzi
forms, as well as the cellular localization of
these molecules could be useful approaches to find out the calpain main functions in this
parasite
Differences in Charge Distribution in Leishmania tarentolae Leishmanolysin Result in a Reduced Enzymatic Activity
Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite
Identificação de homólogos das calpaÃnas em Trypanosoma cruzi e avaliação do efeito do MDL28170, um inibidor de calpaÃnas, sobre o parasito
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Previous issue date: 2010Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, BrasilAs calpaÃnas constituem uma famÃlia de cisteÃna-peptidases neutras dependentes de cálcio presentes numa ampla variedade de organismos. Em virtude da relevância fisiológica dessa proteases, inibidores de calpaÃnas já vêm sendo desenvolvidos para o tratamento de doenças humanas e microbianas. Estudos recentes vêm relatando a presença de diversas proteÃnas relacionadas à s calpaÃnas em tripanossomatÃdeos, mas pouco se sabe a respeito das funções especÃficas dessas proteÃnas nesses micro-organismos. Nesse contexto, uma vez que os fármacos atualmente disponÃveis para o tratamento da doença de Chagas apresentam sérios efeitos colaterais e podem ser ineficazes, inibidores proteolÃticos poderiam ser uma alternativa no tratamento desta doença. Portanto, o presente trabalho investiga a presença de moléculas similares à s calpaÃnas no Trypanosoma cruzi e o efeito inibidor do inibidor III de calpaÃnas (MDL28170) sobre a proliferação, viabilidade, diferenciação, ultraestrutura e interação das diferentes formas do parasito com células hospedeiras em ensaios in vitro. Nossos resultados revelam a reatividade cruzada de anticorpos produzidos contra calpaÃnas já bem caracterizadas de Drosophila melanogaster, Homarus americanus e.
Trypanosoma brucei contra moléculas de superfÃcie do T. cruzi, conforme demonstrado por imunofluorescência e citometria de fluxo. Em ensaios de Western Blotting foi possÃvel observar que o anticorpo anti-DM-calpaÃna foi capaz de reagir contra uma proteÃna de 80 kDa. Pesquisas realizadas no GenBank demonstraram a presença de 4 seqüências nomólogas à calpaÃna de D. melanogaster no genoma do T. cruzi. Essas 4 seqüências foram identificadas como cisteÃna-paptidases de massa molecular predita em torno de 80kDa. Nos ensaios de inibição com o MDL28170 foi possÃvel observar a redução da proliferação das formas epimastigotas ao longo de 5 dias de cultura; e a redução significativa da viabilidade das formas tripomastigotas sanguÃneas dos parasitos tratados com 25 µM do composto. O inibidor, adicionado nas concentrações de 6,25 à 25 µM, também foi capaz de diminuir de forma dose- e tempo-dependente o número de macrófagos parasitados e o número de parasitas interiorizados nos ensaios de interação com macrófagos peritoneais murinos. O MDL28170 em concentração a partir de 12,5 µM teve ainda um efeito inibitório significativo sobre a adesão de formas epimastigotas ao epitélio intestinal de Rhodnius prolixus; assim como os anticorpos anti-calpaÃnas foram capazes de inibir significativamente a interação com o inseto vetor. Por fim, foi possÃvel observar uma redução significativa no processo de diferenciação por metaciclogênese em meio TAU e alterações ultraestruturais em reservossomos, Golgi e membrana plasmática quando formas epimastigotas do T. cruzi foram tratadas com MDL28170..
Embora mais estudos sejam necessários para melhor caracterizar moléculas similares à s calpaÃnas no T. cruzi, o nosso trabalho acrescenta novos conhecimentos sobre as possÃveis funções dessas moléculas e sobre a possibilidade de utilização de inibidores de calpaÃnas como uma alternativa promissora para o desenvolvimento de compostos mais potentes e seletivos para o tratamento da doença de Chagas.The calpains are a family of cysteine ​​peptidases neutral calcium dependent present in a wide variety of organisms. Due to the physiological relevance of such proteases, calpain inhibitors have already been developed for the treatment of human diseases and microbial. Recent studies have reported the presence of several proteins related to calpain in trypanosomatids, but little is known about the specific functions of these proteins in these micro-organisms. In this context, since the drugs currently available for the treatment of Chagas' disease have serious side effects and can be ineffective, proteolytic inhibitors could be an alternative in treatment of this disease. Therefore, the present study investigates the presence of molecules similar to calpain in Trypanosoma cruzi and the inhibitory effect of calpain inhibitor III (MDL28170) on proliferation, viability, differentiation, ultrastructure and interaction of the different forms of the parasite with host cells in vitro tests . Our results reveal the cross-reactivity of antibodies produced against calpains already well-characterized Drosophila melanogaster, and Homarus americanus.
Trypanosoma brucei against surface molecules of T. cruzi, as demonstrated by immunofluorescence and flow cytometry. In assays Western Blotting was observed that the anti-DM-calpain was able to react against a protein of 80 kDa. Surveys conducted in GenBank showed the presence of 4 nomólogas the calpain sequences of D. melanogaster genome of T. cruzi. These four sequences were identified as cysteine ​​paptidases predicted molecular mass of around 80 kDa. In inhibition assays with MDL28170 was possible to observe a reduction in the proliferation of epimastigotes over 5 days of culture, a significant reduction in the viability of blood trypomastigotes of parasites treated with 25 mM of the compound. The inhibitor added at concentrations of 6.25 to 25 mM, was also able to decrease in a dose-and time-dependent number of infected macrophages and the number of parasites internalized interaction assays with murine peritoneal macrophages. The MDL28170 in concentration from 12.5 mM still had a significant inhibitory effect on the adhesion of epimastigotes to the intestinal epithelium of Rhodnius prolixus, as well as anti-calpain were able to significantly inhibit the interaction with the insect vector. Finally, we observed a significant reduction in the differentiation process by metacyclogenesis amid TAU and ultrastructural changes in reservossomos, Golgi and the plasma membrane when epimastigotes of T. cruzi were treated with MDL28170 ..
Although more studies are needed to better characterize molecules similar to calpains in T. cruzi, our work adds new insights into the possible roles of these molecules and the possibility of using inhibitors of calpain as a promising alternative for the development of more potent and selective compounds for the treatment of Chagas' disease
The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?
Calpains are calcium-dependent cysteine peptidases that were originally described in mammals and, thereafter, their homologues were identified in almost all known living organisms. The deregulated activity of these peptidases is associated with several pathologies and, consequently, huge efforts have been made to identify selective inhibitors. Trypanosomatids, responsible for life-threatening human diseases, possess a large and diverse family of calpain sequences in their genomes. Considering that the current therapy to treat trypanosomatid diseases is limited to a handful of drugs that suffer from unacceptable toxicity, tough administration routes, like parenteral, and increasing treatment failures, a repurposed approach with calpain inhibitors could be a shortcut to successful chemotherapy. However, there is a general lack of knowledge about calpain functions in these parasites and, currently, the proteolytic activity of these proteins is still an open question. Here, we highlight the current research and perspectives on trypanosomatid calpains, overview calpain description in these organisms, and explore the potential of targeting the calpain system as a therapeutic strategy. This review gathers the current knowledge about this fascinating family of peptidases as well as insights into the puzzle: are we unable to measure calpain activity in trypanosomatids, or are the functions of these proteins devoid of proteolytic activity in these parasites
Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis
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Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ, Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Laboratório de Imunofarmacologia Parasitária. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ, Brasil.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
Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor
Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets