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

HIV Aspartyl Peptidase Inhibitors Interfere with Cellular Proliferation, Ultrastructure and Macrophage Infection of Leishmania amazonensis

Submitted by Sandra Infurna ([email protected]) on 2019-01-08T13:43:09Z No. of bitstreams: 1 Ellenf_Altoe_etal_IOC_2009.pdf: 1452755 bytes, checksum: 77127a59920cef6bca71296107f6ec63 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-01-08T13:51:34Z (GMT) No. of bitstreams: 1 Ellenf_Altoe_etal_IOC_2009.pdf: 1452755 bytes, checksum: 77127a59920cef6bca71296107f6ec63 (MD5)Made available in DSpace on 2019-01-08T13:51:34Z (GMT). No. of bitstreams: 1 Ellenf_Altoe_etal_IOC_2009.pdf: 1452755 bytes, checksum: 77127a59920cef6bca71296107f6ec63 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Microbiologia Prof. Paulo de Góes. Departamento de Microbiologia Geral,. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Microbiologia Prof. Paulo de Góes. Departamento de Microbiologia Geral,. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Microbiologia Prof. Paulo de Góes. Departamento de Microbiologia Geral,. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis

Trypsin-Like Serine Proteases in Lutzomyia longipalpis – Expression, Activity and Possible Modulation by Leishmania infantum chagasi

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil. Methodology and Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation. Conclusions and Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi

The ubiquitous gp63-like metalloprotease from lower trypanosomatids: in the search for a function

Plant and insect trypanosomatids constitute the "lower trypanosomatids", which have been used routinely as laboratory models for biochemical and molecular studies because they are easily cultured under axenic conditions, and they contain homologues of virulence factors from the classic human trypanosomatid pathogens. Among the molecular factors that contribute to Leishmania spp. virulence and pathogenesis, the major surface protease, alternatively called MSP, PSP, leishmanolysin, EC 3.4.24.36 and gp63, is the most abundant surface protein of Leishmania promastigotes. A myriad of functions have been described for the gp63 from Leishmania spp. when the metacyclic promastigote is inside the mammalian host. However, less is known about the functions performed by this molecule in the invertebrate vector. Intriguingly, gp63 is predominantly expressed in the insect stage of Leishmania, and in all insect and plant trypanosomatids examined so far. The gp63 homologues found in lower trypanosomatids seem to play essential roles in the nutrition as well as in the interaction with the insect epithelial cells. Since excellent reviews were produced in the last decade regarding the roles played by proteases in the vertebrate hosts, we focused in the recent developments in our understanding of the biochemistry and cell biology of gp63-like proteins in lower trypanosomatids.Tripanossomatídeos de insetos e de plantas são informalmente denominados de "tripanossomatídeos inferiores". Estes microrganismos são utilizados rotineiramente como modelos para estudos de bioquímica e de biologia molecular porque são facilmente cultivados sob condições axênicas e porque possuem homólogos aos fatores de virulência encontrados nos tripanossomatídeos que são patógenos humanos clássicos. Dentre os fatores moleculares que contribuem para a virulência e patogênese de Leishmania spp. destaca-se a principal protease de superfície, também conhecida como MSP, PSP, leishmanolisina, EC 3.4.24.36 e gp63, que é a proteína de superfície mais abundante encontrada nas formas promastigotas de Leishmania. Diversas funções foram descritas para a gp63 de Leishmania no hospedeiro vertebrado. Entretanto, pouco é conhecido sobre as funções desempenhadas por essa molécula no inseto vetor. Curiosamente, a gp63 é predominantemente expressa na forma evolutiva de Leishmania encontrada no inseto, e em todos os tripanossomatídeos de insetos e plantas analisados até o presente momento. Os homólogos da gp63 presentes nos tripanossomatídeos inferiores desempenham um papel essencial na nutrição assim como na interação com as células epiteliais do inseto. Uma vez que revisões de excelente qualidade foram produzidas na última década sobre a função de proteases nos hospedeiros vertebrados, nesta revisão nós abordamos os recentes progressos sobre os aspectos bioquímicos e as prováveis funções biológicas desempenhadas pelas proteínas homólogas à gp63 nos tripanossomatídeos inferiores

Miltefosine-Lopinavir Combination Therapy Against Leishmania infantum Infection: In vitro and in vivo Approaches

Submitted by Sandra Infurna ([email protected]) on 2019-09-05T13:19:01Z No. of bitstreams: 1 KarinaRebello_ClaudiaMLevy_etal_IOC_2019.pdf: 1447207 bytes, checksum: 953ae8b6393c6500175c5aca485c5210 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-09-05T13:29:54Z (GMT) No. of bitstreams: 1 KarinaRebello_ClaudiaMLevy_etal_IOC_2019.pdf: 1447207 bytes, checksum: 953ae8b6393c6500175c5aca485c5210 (MD5)Made available in DSpace on 2019-09-05T13:29:54Z (GMT). No. of bitstreams: 1 KarinaRebello_ClaudiaMLevy_etal_IOC_2019.pdf: 1447207 bytes, checksum: 953ae8b6393c6500175c5aca485c5210 (MD5) 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 Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Laboratório de Síntese de Substâncias no Combate a Doenças Tropicais. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Farmanguinhos. Laboratório de Síntese de Substâncias no Combate a Doenças Tropicais. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica de Tripanosomatídeos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ. Brasil.Concurrently, leishmaniasis and AIDS are global public health issues and the overlap between these diseases adds additional treats to the management of co-infected patients. Lopinavir (LPV) has a well characterized anti-HIV and leishmanicidal action, and to analyze its combined action with miltefosine (MFS) could help to envisage strategies to the management of co-infected patients. Here, we evaluate the interaction between LPV and MFS against Leishmania infantum infection by in vitro and in vivo approaches. The effect of the compounds alone or in association was assessed for 72 h in mouse peritoneal macrophages infected with L. infantum by the determination of the IC50s and FICIs. Subsequently, mice were orally treated twice daily during 5 days with the compounds alone or in association and evaluated after 30 days. The in vitro assays revealed an IC50 of 0.24 μM and 9.89 μM of MFS and LPV, respectively, and an additive effect of the compounds (FICI 1.28). The in vivo assays revealed that LPV alone reduced the parasite load in the spleen and liver by 52 and 40%, respectively. The combined treatment of infected BALB/c mice revealed that the compounds alone required at least two times higher doses than when administered in association to virtually eliminate the parasite. Mice plasma biochemical parameters assessed revealed that the combined therapy did not present any relevant hepatotoxicity. In conclusion, the association of MFS with LPV allowed a reduction in each compound concentration to achieve the same outcome in the treatment of visceral leishmaniasis. Although a pronounced synergistic effect was not evidenced, it does not discard that such combination could be useful in humans co-infected with HIV and Leishmania parasites

First Draft Genome of the Trypanosomatid through MinION Oxford Nanopore Technology and Illumina Sequencing.

Here, we present first draft genome sequence of the trypanosomatid . This parasite was isolated repeatedly in the black blowfly, , and it forms a phylogenetically distinct clade in the Trypanosomatidae family

Western Blot CZP

Western blot assay for cruzipain observatio

in vitro

Raw data from T. cruzi infection assay