SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

Parasitic infection caused by blood flukes of the genus Schistosoma is a major global health problem. More than 200 million people are infected. Identifying and characterizing the constituent enzymes of the parasite's biochemical pathways should reveal opportunities for developing new therapies (i.e., vaccines, drugs). Schistosomes feed on host blood, and a number of proteolytic enzymes (proteases) contribute to this process. We have identified and characterized a new protease, SmCL3 (for Schistosoma mansoni cathepsin L3), that is found within the gut tissue of the parasite. We have employed various biochemical and molecular biological methods and sequence similarity analyses to characterize SmCL3 and obtain insights into its possible functions in the parasite, as well as its evolutionary position among cathepsin L proteases in general. SmCL3 hydrolyzes major host blood proteins (serum albumin and hemoglobin) and is expressed in parasite life stages infecting the mammalian host. Enzyme substrate specificity detected by positional scanning-synthetic combinatorial library was confirmed by molecular modeling. A sequence analysis placed SmCL3 to the cluster of other cathepsins L in accordance with previous phylogenetic analyses

Digestion des protéines de l'hôte par le réseau de protéases intestinales du parasite helminthe schistosoma mansoni

En utilisant les inhibiteurs protéasiques et la technique d'interférence à l'ARN, nous avons décomposé le réseau d'endopeptidases impliquées dans la digestion intestinale de Schistosoa mansoni. L'initiation de la dégradation des protéines de l'hôte, hémoglobine et albumine, avec les protéases à cystéine ou à acide aspartique du parasite, s'est révélée ordonnée, ocasionnellement redondante et spécifique au substrat. Alors que l'inhibition de la cathepsine D a un effet majeur surla dégradation de l'hemoglibine, les protéases à cystéine semblent prédominer dans la dégradation. Une asparaginyl endopeptidase synergise avec les cathepsines B et L soit en activant les zymogènes, soit en facilitant le clivage du substrat. Ces protéases fonctionnent de façon optimale dans des compartiments acidiques de la lumière intestinale ou du gastroderme. Définir le rôle de ces enzymes permet de mieux comprendre l'intéraction des protéases au sein d'un réseau complexe, conservé au cours de l'évolution des invertébrés, des nématodes aux insectes. Cette étude fournit également des éléments intéressants quant à la détermination de protéases pouvant être utilisées comme cilbles thérapeutiques potentielles contre la schistosomiase, un problème de santé mondial.Utilizing protease inhibitors and Rna interference, we deconvulated the network of endopeptidases functioning in intestinal protein digestion in Schistosoa mansoni. We showed that initial degradation of hemoglobin and albumin by parasite cysteine and aspartic rpoteases, is ordered, occasionally redundant, and substrate specific. While inhibition of cathepsin D had a greater effect on primary cleavage of hemoglobin, inhibition of cysteine proteases predominated in albumin degradation. Nevertheless, in both cases inhibitor combinations were synergistic. An asparaginyl endopeptidase synergized with cathepsin B and L by zymogen activation or facilitating substrate cleavage ; these proteases operate optimally in two distinct acidic compartments : one formed by lamellar fusion in the gut lumen and the second in the gastrodermis. Definig the role of these enzymes provides a clearer understanding of the function of a complex protease network found throughout invertebrate evolution from nematodes to insects. It also provides insights into which of these protease are logical chemotherapeutic targets for schistosomiasis, a major global health problem.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Cell-Penetrating Peptides for Antiviral Drug Development

pharmaceutical

Flow-cytometric analysis of human monocyte subsets targeted by Mycobacterium bovis BCG before granuloma formation

Infection with Mycobacterium tuberculosis (Mtb) is characterized by an inflammatory response resulting in the formation of granulomas. These tight aggregates of immune cells play an important role in bacterial containment and in the eventual outcome of infection. Monocytes are a major component of the early immune response to Mtb and contribute to the cellular matrix of the newly forming granuloma. Therefore, defining which monocyte subset is the target of mycobacterial infection is critical. Here, we describe a flow-cytometry-based assay to analyze infectivity in vitro of monocyte subsets by Mycobacterium bovis BCG before granuloma formation. We identified CD14+CD16- monocytes as the main target of infection in peripheral blood mononuclear cells from six healthy donors. CD14+CD16+ monocytes displayed the lowest infection rates and remained uninfected in some donors. We found that a longer infection time resulted in an increase of the percentage of monocytes infected and of the number of granulomas produced. We did not observe changes in monocyte cell death or subset expansion upon infection. Future experiments with our in vitro method could help define Mtb infectivity of monocyte subsets. Our study provides a platform to investigate how early infection of different monocyte subsets may alter granuloma formation and outcomes of Mtb infection

Single Nucleotide Polymorphisms in IL17A and IL6 Are Associated with Decreased Risk for Pulmonary Tuberculosis in Southern Brazilian Population

Submitted by Sandra Infurna ([email protected]) on 2017-02-09T15:11:52Z No. of bitstreams: 1 milton2_moraes_eta_IOC_2016.pdf: 275009 bytes, checksum: 0e5da130f7534c7658222a349633047e (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-02-09T15:31:06Z (GMT) No. of bitstreams: 1 milton2_moraes_eta_IOC_2016.pdf: 275009 bytes, checksum: 0e5da130f7534c7658222a349633047e (MD5)Made available in DSpace on 2017-02-09T15:31:06Z (GMT). No. of bitstreams: 1 milton2_moraes_eta_IOC_2016.pdf: 275009 bytes, checksum: 0e5da130f7534c7658222a349633047e (MD5) Previous issue date: 2016Universidade Federal do Rio Grande do Sul. Programa de Pós-Graduação em Biologia Celular e Molecular. Porto Alegre, RS, Brasil / Fundação Estadual de Produção e Pesquisa em Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Estadual de Produção e Pesquisa em Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.University of California.Division of Infectious Disease and Vaccinology. Berkeley, CA, USA.Universidade Federal de Ciências da Saúde de Porto Alegre. Porto Alegre, RS, Brasil.Fundação Estadual de Produção e Pesquisa em Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, Brasil / Universidade Federal do Rio de Janeiro. Programa de Pós-Graduação em Clínica Médica. Rio de Janeiro, RJ, Brasil.Hospital Sanatório Partenon. Porto Alegre, RS, Brasil.Fundação Estadual de Produção e Pesquisa em Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, BrasilUniversidade Federal do Rio Grande do Sul. Programa de Pós-Graduação em Biologia Celular e Molecular. Porto Alegre, RS, Brasil / Fundação Estadual de Produção e Pesquisa em Saúde. Centro de Desenvolvimento Científico e Tecnológico. Porto Alegre, RS, Brasil / Universidade Luterana do Brasil. Canoas, RS, Brasil.In Mycobacterium tuberculosis (MTB) infection, the complex interaction of host immune system and the mycobacteria is associated with levels of cytokines production that play a major role in determining the outcome of the disease. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have been associated with tuberculosis (TB) outcome. The aim of this study was to evaluate the association between previously reported SNPs IL2-330 T>G (rs2069762); IL4-590 C>T (rs2243250); IL6-174 G>C (rs1800795); IL10-592 A>C (rs1800872); IL10-1082 G>A (rs1800896); IL17A -692 C>T (rs8193036); IL17A -197 G>A (rs2275913); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) and pulmonary TB (PTB) susceptibility. We conducted a case-control study in individuals from Southern Brazil who were recruited between February 2012 and October 2013 in a high incidence TB city. We performed a multiplex genotyping assay in 191 patients with PTB and 175 healthy subjects. Our results suggest a decreased risk for PTB development associated with the IL17A -197A allele (OR = 0.29; p = 0.04), AA genotype (OR = 0.12; p = 0.04) and A carrier (AG/AA) (OR = 0.29; p = 0.004) and IL6 -174C carrier (CC/CG) (OR = 0.46; p = 0.04). We could not properly analyze IL17A -692 C>T (rs8193036) and IFNG +874T>A due to genotypic inconsistencies and found no evidence of association for the IL2, IL4, IL10 and TNF polymorphisms and PTB. In conclusion, our results show a protective effect of IL17 and IL6 polymorphisms on PTB outcome in Southern Brazilian population

Characteristics of The Population Studied.

<p>Characteristics of The Population Studied.</p