Expressão heteróloga de uma trans-sialidase de Trypanosoma cruzi por Trypanosoma rangeli

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.O Trypanosoma rangeli é um parasito hemoflagelado, com capacidade de infectar animais incluindo o homem, nas Américas Central e do Sul. Sua distribuição geográfica, reservatórios e vetores são compartilhados com o Trypanosoma cruzi, agente etiológico da doença de Chagas. As Trans-sialidases (TS) de T. cruzi estão relacionadas ao processo de interação, penetração do parasito na célula hospedeira e seu escape do vacúolo fagocítico. O T. rangeli possui alguns genes da família das TS, não possuindo nenhuma atividade catalítica como perfaz algumas TS de T. cruzi (TcTS). O objetivo do presente estudo foi realizar a expressão heteróloga da TcTS de T. cruzi em T. rangeli buscando a geração de uma ferramenta para estudos da interação celular patógeno-hospedeiro. Para tanto, a partir da construção plasmidial que possui o gene da TcTS de T. cruzi foi confirmada a expressão heteróloga da TcTS de T. cruzi por T. rangeli por western blot e por microscopia de fluorescência. Ainda que preliminares, foram realizados ensaios de atividade TS nos parasitos transfectados. Os resultados confirmaram a expressão heteróloga da TcTS de T. cruzi por T. rangeli, além de apontar que o parasito transfectado possui uma TS ativa.Trypanosoma rangeli is an hemoflagellate parasite, with ability to infect animals including humans, in Central and South America. Geographical distribution, reservoirs and vectors are shared with Trypanosoma cruzi, etiological agent of Chagas disease. The trans-sialidases (TS) of T. cruzi are related to the processes of interaction, penetration of the parasite in the host cell and its evasion from parasitophorus vacuole T. rangeli revealed to have genes similar to TS family members but with no catalytic activity has been proved so far, as TS of T. cruzi (TcTS). The goal of this study was to perform the heterologous expression of TcTS of T. cruzi by T. rangeli to generate a tool for the study of host-pathogen interaction. Heterologous expression of TcTS by T. rangeli was confirmed by western blot and by fluorescence microscopy using antibodies directed to the catalytic site and terminal repeats of TcTS (SAPA). Although preliminary, TS activity tests in transfected flagellate was performed. The results confirm the heterologous expression of TcTS of T. cruzi by T. rangeli, in addition to pointing out that the transfected parasite has an active TS

Estudo da interação celular parasito-hospedeiro a partir da expressão heteróloga de trans-sialidase de Trypanosoma cruzi por Trypanosoma rangeli em ensaios in vitro e in vivo

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-graduação em Biotecnologia e Biociências, Florianópolis, 2013As trans-sialidades de Trypanosoma cruzi (TcTS) são capazes de clivar resíduos de ácidos siálicos da célula hospedeira e transferí-los à superfície do parasito, e portanto, fundamentais nessa interação. Já o Trypanosoma rangeli, considerado não patogênico para mamíferos e com ciclo desconhecido nestes hospedeiros, possui no seu genoma genes similares aos membros da família das Trans-sialidase (TS), embora sem nenhuma atividade catalítica. Uma vez que essa atividade está relacionada a pontos cruciais do ciclo do parasito patogênico e que, a ausência de atividade TS em T. rangeli é apontada como um dos motivos de sua incapacidade de infectar e sobreviver em células de mamíferos, o objetivo do presente trabalho foi estudar o envolvimento da TS a partir daexpressão heteróloga de uma TcTS ativa pelo T. rangeli. Após a transfecção e a confirmação da atividade de TS em T. rangeli transfectado (T. rangeli TcTS), ninfas de 4° e 5° estágio de Rhodnius prolixus (R. prolixus) foram infectadas por inoculação intracelômica com formas epimastigotas de T. rangeli selvagem e T. rangeli TcTS. Observamos uma diferença estatística no número de glândulas infectadas para T. rangeli selvagem (92%), e T. rangeli TcTS (16%), seis semanas p.i.. A infecção em camundongos BALB/c e C57BL/6 não revelou diferenças na de parasitemia entre parasitos selvagens e transfectados. Já nos ensaios de infecção in vitro utilizando-se células Vero, observou-se que o T.rangeli TcTS apresentou maior capacidade de infecção quando comparados ao T. rangeli selvagem, não sendo observado nenhum indício de divisão celular para esses parasitos após 120 horas de interação. Os resultados apontam que a expressão heteróloga da TcTS por T. rangeli embora não altere o tempo e o padrão da parasitemia experimental in vivo em camundongos, promoveu mudanças no comportamento da interação parasito-vetor e parasito-célula, reduzindo sua capacidade de penetração nas glândulas salivares de R. prolixus e facilitando a entrada do parasito em células in vitro. Abstract: The Trypanosoma cruzi trans-sialidases (TcTS) are able to cut and transfer sialic acid from the host cell to the parasite surface and are important molecules on this interaction. Trypanosoma rangeli is a non-pathogenic parasite to mammal, that posses an unknown life cicle in this host, and it has similar genes to TS family members in the genome, however, with no catalytic activity. Once TS are crucial molecules involved in parasite-host cell interaction in the pathogenic parasite, and the absence of TS activity is pointed as one of the reasons of its inability capacity of infecting and surviving in mammals cells, the aim of this work was to study the roll of TS in the host-parasite interaction using T. rangeli expressing an active TcTS (T. rangeli TcTS). After transfection and assessment of protein activity, 4° and 5° instar nymphs of Rhodnius prolixus were infected by intracelomic inoculation of T. rangeli wild type and T. rangeli TcTS epimastigote forms. A statistically significant difference was observed in the number of salivary gland infected for T. rangeli wild type (92%) and for T. rangeli TcTS (16%) at six weeks p.i.. Parasitemia of BALB/c mice and C57BL/6 infected by T. rangeli TcTS showed no difference from mice infected with non-transfected parasites. For in vitro infection assays with Vero cells, T. rangeli TcTS presented higher infection rates when compared with T. rangeli wild type, but no indication of intracellular division was detected for these parasites up to 120 hours. Despite not altering the parasitemia in mice, our results indicate that TcTS expressed by T. rangeli may be responsible for changes on the interaction between vector-parasite and cell-parasite by reducing the ability to penetrate on R. prolixus salivary glands and facilitating cell entry in vitro

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test

Introduction Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies. Materials and Methods The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow. Conclusions Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.The authors are thankful to Mia Jullig for assistance with Fig. 2. Callaghan Innovation and Bioresource Processing Alliance provided PhD stipends for James Daniell and Ninna Granucci respectively.info:eu-repo/semantics/publishedVersio

Fermentative Production of Volatile Metabolites Using <i>Brettanomyces bruxellensis</i> from Fruit and Vegetable By-Products

Natural sources of flavour and aroma compounds are highly sought by the modern consumer; however, traditional sources are often low-yielding, and global supply is often outstripped by consumer demand. Fermentation is a favourable route by which natural flavours and fragrances can be produced. A non-Saccharomyces yeast, Brettanomyces bruxellensis, was investigated for its fermentative potential for the production of flavour and aroma metabolites from juice industry by-products: apple pomace, carrot pomace, and orange pomace. Submerged solid-substrate fermentations were carried out using sterile by-products without nutrient supplementation. Gas chromatography–mass spectrometry was used for volatile metabolite profiling of fermented substrates. One compound of interest, phenylethyl alcohol (rose fragrance), was extracted and quantified using GC-MS at a yield of 2.68 g/kg wet carrot pomace weight. This represents a novel, natural production strategy for phenylethyl alcohol compared to the traditional steam distillation of Rosa domascus sp. petals

Fermentative Production of Volatile Metabolites Using Brettanomyces bruxellensis from Fruit and Vegetable By-Products

Natural sources of flavour and aroma compounds are highly sought by the modern consumer; however, traditional sources are often low-yielding, and global supply is often outstripped by consumer demand. Fermentation is a favourable route by which natural flavours and fragrances can be produced. A non-Saccharomyces yeast, Brettanomyces&nbsp;bruxellensis, was investigated for its fermentative potential for the production of flavour and aroma metabolites from juice industry by-products: apple pomace, carrot pomace, and orange pomace. Submerged solid-substrate fermentations were carried out using sterile by-products without nutrient supplementation. Gas chromatography&ndash;mass spectrometry was used for volatile metabolite profiling of fermented substrates. One compound of interest, phenylethyl alcohol (rose fragrance), was extracted and quantified using GC-MS at a yield of 2.68 g/kg wet carrot pomace weight. This represents a novel, natural production strategy for phenylethyl alcohol compared to the traditional steam distillation of Rosa domascus sp. petals

Identification of New Natural Sources of Flavour and Aroma Metabolites from Solid-State Fermentation of Agro-Industrial By-Products

Increasing consumer demand for natural flavours and fragrances has driven up prices and increased pressure on natural resources. A shift in consumer preference towards more sustainable and economical sources of these natural additives and away from synthetic production has encouraged research into alternative supplies of these valuable compounds. Solid-state fermentation processes support the natural production of secondary metabolites, which represents most flavour and aroma compounds, while agro-industrial by-products are a low-value waste stream with a high potential for adding value. Accordingly, four filamentous fungi species with a history of use in the production of fermented foods and food additives were tested to ferment nine different agro-industrial by-products. Hundreds of volatile compounds were produced and identified using headspace (HS) solid-phase microextraction (SPME) coupled to gas chromatography–mass spectrometry (GC–MS). Four compounds of interest, phenylacetaldehyde, methyl benzoate, 1-octen-3-ol, and phenylethyl alcohol, were extracted and quantified. Preliminary yields were encouraging compared to traditional sources. This, combined with the low-cost substrates and the high-value natural flavours and aromas produced, presents a compelling case for further optimisation of the process