Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

Semiconductor nanoparticles, such as quantum dots (QDs), were used to carry out experiments in vivo and ex vivo with Trypanosoma cruzi. However, questions have been raised regarding the nanotoxicity of QDs in living cells, microorganisms, tissues and whole animals. The objective of this paper was to conduct a QD nanotoxicity study on living T. cruzi protozoa using analytical methods. This was accomplished using in vitro experiments to test the interference of the QDs on parasite development, morphology and viability. Our results show that after 72 h, a 200 μM cadmium telluride (CdTe) QD solution induced important morphological alterations in T. cruzi, such as DNA damage, plasma membrane blebbing and mitochondrial swelling. Flow cytometry assays showed no damage to the plasma membrane when incubated with 200 μM CdTe QDs for up to 72 h (propidium iodide cells), giving no evidence of classical necrosis. Parasites incubated with 2 μM CdTe QDs still proliferated after seven days. In summary, a low concentration of CdTe QDs (2 μM) is optimal for bioimaging, whereas a high concentration (200 μM CdTe) could be toxic to cells. Taken together, our data indicate that 2 μM QD can be used for the successful long-term study of the parasite-vector interaction in real time.15816

In vitro Trypanocidal Activity, Genomic Analysis of Isolates, and in vivo Transcription of Type VI Secretion System of Serratia marcescens Belonging to the Microbiota of Rhodnius prolixus Digestive Tract

Serratia marcescens is a bacterium with the ability to colonize several niches, including some eukaryotic hosts. S. marcescens have been recently found in the gut of hematophagous insects that act as parasite vectors, such as Anopheles, Rhodnius, and Triatoma. While some S. marcescens strains have been reported as symbiotic or pathogenic to other insects, the role of S. marcescens populations from the gut microbiota of Rhodnius prolixus, a vector of Chagas’ disease, remains unknown. Bacterial colonies from R. prolixus gut were isolated on BHI agar. After BOX-PCR fingerprinting, the genomic sequences of two isolates RPA1 and RPH1 were compared to others S. marcescens from the NCBI database in other to estimate their evolutionary divergence. The in vitro trypanolytic activity of these two bacterial isolates against Trypanosoma cruzi (DM28c clone and Y strain) was assessed by microscopy. In addition, the gene expression of type VI secretion system (T6SS) was detected in vivo by RT-PCR. Comparative genomics of RPA1 and RPH1 revealed, besides plasmid presence and genomic islands, genes related to motility, attachment, and quorum sensing in both genomes while genes for urea hydrolysis and type II secretion system (T2SS) were found only in the RPA1 genome. The in vitro trypanolytic activity of both S. marcescens strains was stronger in their stationary phases of growth than in their exponential ones, with 65–70 and 85–90% of epimastigotes (Dm28c clone and Y strain, respectively) being lysed after incubation with RPA1 or RPH1 in stationary phase. Although T6SS transcripts were detected in guts up to 40 days after feeding (DAF), R. prolixus morbidity or mortality did not appear to be affected. In this report, we made available two trypanolytic S. marcescens strains from R. prolixus gut to the scientific community together with their genomic sequences. Here, we describe their genomic features with the purpose of bringing new insights into the S. marcescens adaptations for colonization of the specific niche of triatomine guts. This study provides the basis for a better understanding of the role of S. marcescens in the microbiota of R. prolixus gut as a potential antagonist of T. cruzi in this complex system

Imunidade humoral de Rhodnius prolixus: impacto sobre a microbiota e desenvolvimento de Trypanosoma cruzi e Trypanosoma rangeli

Made available in DSpace on 2016-03-18T12:16:56Z (GMT). No. of bitstreams: 2 cecilia_vieira_ioc_dout_2015.pdf: 5478933 bytes, checksum: 82e22f21a3f655c7a35e09359316e42f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-02-23Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Rhodnius prolixus é um dos principais insetos vetores de Trypanosoma cruzi e de Trypanosoma rangeli na América Latina. A produção de peptídeos antimicrobianos (AMPs) no trato digestivo ou corpo gorduroso do inseto é vital para evitar a proliferação de microrganismos patogênicos além de manter a homeostasia da microbiota nativa. O presente trabalho focou na modulação da imunidade humoral do intestino médio de R. prolixus desafiados oralmente com a bactéria Gram-positiva Staphylococcus aureus e Gram-negativa Escherichia coli, além de seus tripanosomatídeos naturais T. rangeli e T. cruzi, considerando a influência do desenvolvimento dos parasitas sobre a microbiota intestinal. Em condições normais, a região anterior do intestino médio houve maior abundância de transcritos de genes de lisozimas (lis) e defensinas (def), enquanto na posterior, do gene da prolixicina (prol). Insetos alimentados com bactérias Gramnegativas apresentaram maior quantidade de transcritos de defC e prol, enquanto a ingestão de bactérias Gram-positivas induziu a expressão de defA e defB no intestino médio A infecção por T. rangeli cepa Macias diminuiu a atividade fenoloxidásica, os níveis de expressão de lisozimas e prolixicina, ao mesmo tempo em que induziu aumento de atividade antibacteriana e dos níveis de defensina C no tubo digestivo do inseto, também modificando a composição de bactérias nativas. Além disso, foi verificado que as diferentes cepas de T. cruzi Dm 28c e Y modulam a resposta imune e a microbiota no intestino médio de R. prolixus de forma variável. T. cruzi Dm 28c induziu um aumento na expressão de genes de defensina C e uma diminuição da expressão de genes de prolixicina, reduzindo drasticamente a população bacteriana cultivável. Em contraste, T. cruzi Y não foi capaz de induzir a expressão de AMPs no intestino médio nem de reduzir consideravelmente a microbiota neste mesmo órgão. Nossos resultados sugerem que R. prolixus em resposta a ingestão de microrganismos, modula diferencialmente a expressão de AMPs pelas células epiteliais do intestino que acarreta a redução da microbiota e um favorecimento do desenvolvimento de T. rangeli e T. cruzi, dependendo do genótipo do parasita.Rhodnius prolixus is a major vector of Trypanosoma rangeli and Trypanosoma cruzi, in Latin America. The production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus orally challenged with Gram positive and Gram negative bacteria as well with T. rangeli Macias strain, T. cruzi Dm 28c and Y strain, considering the influence of the parasite s on the intestinal microbiota. Our results showed that the anterior midgut contents of control insects contain a higher inducible antibacterial activity and AMPs transcript abundance than those of the posterior midgut. Insects orally fed with Gram-negative bacteria presented higher amount of defC and prol transcripts, while the ingestion of Gram-positive induced defA and defB expression in the midgut. T. rangeli Macias strain successfully colonized R. prolixus midgut through a decreasing in PO activities, prolixicin and lysozyme levels, while at the same time in duced an increase in antibacterial activity and upregulated defC levels in the insect anterior midgut. rangeli infection also diminishes the amount of cultivable gut bacteria as well modif ied the composition of indigenous microo rganisms. Furthermore, different T. cruzi strains present distinct profile s of immune system and microbiota modulation in R. prolixus midgut, where T. cruzi Dm 28c was able to induce an increase in defensin C genes and a depression in prolixicin genes, whi le drastically reduce the cultivable bacteria population. In the other hand T. cruzi Y was not competent to induce AMPs expression in the gut or considerably reduce the microbiota in the anterior midgut. Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria and tripanosomatids with patterns that are distinct and dependent upon the species of the invading pathogen. Besides, t he trypanosome ability to induce immune peptides in epithelial cells seems to favor its development in the insect digestive tract by decreasing intestinal microbiota , depending on the parasite genotype

Modulation of Rab5 and Rab7 Recruitment to Phagosomes by Phosphatidylinositol 3-Kinase.

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation

In vitro Trypanocidal Activity, Genomic Analysis of Isolates, and in vivo Transcription of Type VI Secretion System of Serratia marcescens Belonging to the Microbiota of Rhodnius prolixus Digestive Tract

Submitted by Sandra Infurna ([email protected]) on 2019-04-29T17:31:35Z No. of bitstreams: 1 FabioFaria_Mota_etal_IOC_2019.pdf: 4237942 bytes, checksum: d580927bcebfc83c0dcafba666d7274b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-04-29T17:41:25Z (GMT) No. of bitstreams: 1 FabioFaria_Mota_etal_IOC_2019.pdf: 4237942 bytes, checksum: d580927bcebfc83c0dcafba666d7274b (MD5)Made available in DSpace on 2019-04-29T17:41:25Z (GMT). No. of bitstreams: 1 FabioFaria_Mota_etal_IOC_2019.pdf: 4237942 bytes, checksum: d580927bcebfc83c0dcafba666d7274b (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Computacional e Sistemas. Rio de Janeiro, RJ. Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ. Brasil.Universidade Federal Fluminense. Instituto Biomédico. Departamento de Microbiologia e Parasitologia. Laboratório de Enteropatógenos, Microbiologia Veterinária e de Alimentos. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Instituto Nacional de Ciência e Tecnologia em Inovação em Doenças Negligenciadas. Laboratório de Modelagem de Sistemas Biológicos. Rio de Janeiro, RJ. Brasil.Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ. Brasil.Serratia marcescens is a bacterium with the ability to colonize several niches, including some eukaryotic hosts. S. marcescens have been recently found in the gut of hematophagous insects that act as parasite vectors, such as Anopheles, Rhodnius, and Triatoma. While some S. marcescens strains have been reported as symbiotic or pathogenic to other insects, the role of S. marcescens populations from the gut microbiota of Rhodnius prolixus, a vector of Chagas' disease, remains unknown. Bacterial colonies from R. prolixus gut were isolated on BHI agar. After BOX-PCR fingerprinting, the genomic sequences of two isolates RPA1 and RPH1 were compared to others S. marcescens from the NCBI database in other to estimate their evolutionary divergence. The in vitro trypanolytic activity of these two bacterial isolates against Trypanosoma cruzi (DM28c clone and Y strain) was assessed by microscopy. In addition, the gene expression of type VI secretion system (T6SS) was detected in vivo by RT-PCR. Comparative genomics of RPA1 and RPH1 revealed, besides plasmid presence and genomic islands, genes related to motility, attachment, and quorum sensing in both genomes while genes for urea hydrolysis and type II secretion system (T2SS) were found only in the RPA1 genome. The in vitro trypanolytic activity of both S. marcescens strains was stronger in their stationary phases of growth than in their exponential ones, with 65-70 and 85-90% of epimastigotes (Dm28c clone and Y strain, respectively) being lysed after incubation with RPA1 or RPH1 in stationary phase. Although T6SS transcripts were detected in guts up to 40 days after feeding (DAF), R. prolixus morbidity or mortality did not appear to be affected. In this report, we made available two trypanolytic S. marcescens strains from R. prolixus gut to the scientific community together with their genomic sequences. Here, we describe their genomic features with the purpose of bringing new insights into the S. marcescens adaptations for colonization of the specific niche of triatomine guts. This study provides the basis for a better understanding of the role of S. marcescens in the microbiota of R. prolixus gut as a potential antagonist of T. cruzi in this complex system

Schistosoma mansoni: a method for inducing resistance to praziquantel using infected Biomphalaria glabrata snails

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50% of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap

Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut

Made available in DSpace on 2015-05-04T17:07:31Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) cecilia_vieiraetal_IOC-2104.pdf: 1062015 bytes, checksum: e8dde228f0bdd6c4004641893450e0cb (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense (UFF).Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil.Universidade Federal Fluminense (UFF).Instituto de Biologia. Departamento de Biologia Geral. Laboratório de Biologia de Insetos. Niterói, RJ, Brasil / Swansea University. College of Science. Swansea, Wales, UK.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Entomologia Molecular (INCT-EM). Departamento de Entomologia Molecular, Rio de Janeiro, RJ, Brasil.Background The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. Methods In the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding. Results Our results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated. Conclusion Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection

Review CdTe and CdSe Quantum Dots Cytotoxicity: A Comparative Study on Microorganisms

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