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Acanthamoeba culbertsoni is an opportunistic free-living amoeba that is causative of granulomatous amoebic encephalitis (GAE), a chronic and often fatal central nervous system (CNS) disease that is most prevalent in immune compromised individuals. One hallmark of this disease is the formation of granulomas within the CNS, which are commonly absent in immune compromised individuals. Granulomas are usually composed of amoebae, microglia (CNS macrophages), macrophages, T cells, B cells, and neutrophils. Previous studies have demonstrated that microglia respond to Acanthamoeba by producing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1α, and IL-1β. In addition, activated microglia and macrophages have been demonstrated to be cytolytic (i.e., amoebicidal) to Acanthamoeba. Furthermore, previous studies also indicated that Acanthamoeba secrete a myriad of factors including proteases. The role of these proteases during GAE has not been fully elucidated; however, it is thought that these factors may aid in the chronic persistence of Acanthamoeba within the CNS by modulating the host immune response. Using two-dimensional (iso-dalt) gel electrophoresis, we demonstrated that A. culbertsoni secrete factors that degrade culture medium proteins. Initial gelatin zymography studies demonstrated that propagation of A. culbertsoni in medium with high iron content leads to augmentation of protease activity. Gelatin zymography in concert with protease inhibitors demonstrated that A. culbertsoni secrete proteases predominantly of the serine protease class. Using an in vitro co-culture model, we demonstrated that co-culture of A. culbertsoni with mouse microglial-like cells (BV-2 cells) results in the augmentation of A. culbertsoni serine protease activity and stimulation of pro-inflammatory cytokine and chemokine protein expression by microglial-like cells. However, the A. culbertsoni-elicited proteases were shown to degrade microglial-like cell elicited cytokines and chemokines. Collectively, our results suggest that A. culbertsoni- secreted serine proteases may play a role in A. culbertsoni CNS immune evasion by increasing A. culbertsoni CNS dissemination via the diminution of granuloma formation and by dampening microglial-dependent cytokine response.

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No. of bitstreams: 1 Dissertação - Alessandra Alves Drumond Siqueira.pdf: 694552 bytes, checksum: 063f14c557446b37809da884d1b0d0a9 (MD5) Previous issue date: 2004-03-29 / Não Informada / The proteases are enzymes which occupy a pivotal position with respect to their application in medical and commercial fields. Collagenases are detached among several kind of proteases, used in pharmaceutical and cosmetician industry, in processes such as smooth the skin, oral hygiene, disinfect suppurative wounds, cicatrizing processes, burning and as a trombolytic agent. In order to select protease producing yeasts, this study had as objectives to partially characterize the enzyme as for pH and stability temperature and to verify the effect of the enzyme in the degradation of collagen. 50 samples of yeasts isolated from different substrates, obtained from the culture collection DPUA, were analysed. Samples were cultured in Malte extract supplemented with 1,0 % gelatin at 30 ºC in shaking conditions (140 rpm). The qualitative proteolytic activity was determined in agar gelatin-milk medium and the quantitative activity was determined using azocasein 1,0 % as substrate. The best culture conditions to protease production were established for a species, being studied the following parameters: characteristics of pathogenicity, size and age of inoculate, best growth temperature, coal and nitrogen sources. The best stability conditions of protease were assessed for pH (6-12) and temperature (25 º, 37 º, 40 º, 50 º, 60 º, 70 º and 80 ºC) and the effect of the enzyme in the degradation of collagen. Among the identified species, Trichosporon pullulans was selected, because it showed the highest levels of proteolytic activity in qualitative (halo = 27 mm) and quantitative (420 U/mL) assays. The assays to determine the characteristics of pathogenicity of this species showed positive growth at 37ºC, weak positive ureasic activity and there was no phospholipasic activity, the highest proteolytic activity (246,66 U/mL) detected with inoculate corresponded to 10% of the volume of medium and stock of culture growth for 24 hours. Trials to verify the effect of temperature (25 ºC to 50 ºC) on growth and protease production demonstrated that the higher biomass (3,46 mg/mL) and proteolytic activity (513,33 U/mL) were obtained at 30 ºC. Sucrose 1,0 % was the best coal source yielding higher biomass (6,18 mg/mL) and proteasic activity (580 U/mL) and as nitrogen source detached peptone 0,5 % showing the higher values of proteolytic activity (755,55 U/mL) and biomass (7,55 mg/mL). The collagenolytic activity was determined in solid medium using dissolvable collagen as the only coal source. T. pullulans degraded collagen, producing a 28 mm halo. This work demonstrated that protease, with collagenolytic activity; produced by T. pullulans, can be used as therapeutic agent further detailed studies. / As proteases são enzimas que ocupam uma posição central com respeito à aplicação na área médico e industrial. Entre os vários tipos de proteases, utilizadas na indústria farmacêutica e de cosméticos, destacam-se as colagenases utilizadas para amaciamento da pele, higiene oral, na limpeza de feridas purulentas, em processos de cicatrização, queimaduras e como agente anti-trombolítico. Realizou-se este estudo com o objetivo de selecionar leveduras produtoras de proteases, avaliar as melhores condições de crescimento da espécie produtora de protease, caracterizar parcialmente a enzima quanto o pH e temperatura na estabilidade e verificar o efeito desta enzima na degradação do colágeno. Foram analisadas 50 amostras de leveduras isoladas de diferentes substratos, provenientes da coleção de cultura DPUA. As amostras foram cultivadas em extrato de Malte suplementado com gelatina 1,0 % a 30 ºC, sob agitação (140 rpm) por 72 horas. A atividade proteolítica qualitativa foi determinada em meio sólido ágar gelatina-leite e a quantitativa utilizando como substrato azocaseína 1 % p/v. Foram estabelecidas, para a espécie selecionada, as melhores condições de cultivo para produção da protease, foram estudados: características de patogenicidade, tamanho e idade do inóculo, temperatura ótima de crescimento, fontes de carbono e nitrogênio. Foram avaliadas as melhores condições de estabilidade da protease pH (6-12) e temperatura (25 º, 37 º, 40 º, 50 º, 60 º, 70 º e 80 ºC) e também, o efeito da enzima na degradação do colágeno. A determinação da atividade colagenolítica foi realizada em meio sólido utilizando colágeno solúvel como única fonte de carbono. Selecionou-se, entre as espécies identificadas, Trichosporon pullulans (DPUA), por apresentar o maior valor de atividade proteolítica em ensaio qualitativo (halo = 27 mm) e em ensaio quantitativo (420 U/mL). Os experimentos realizados para detectar as características de patogenicidade desta espécie demonstraram crescimento positivo a 37 ºC, atividade ureásica positiva e não foi detectada atividade fosfolipásica; a maior atividade proteolítica (246,66 U/mL) foi detectada com inóculo que correspondeu a 10 % do volume de meio e cultura estoque de 24 horas de crescimento. Os ensaios realizados para verificar o efeito da temperatura no crescimento e na produção da protease demonstraram que a maior biomassa (3,46 mg/mL) e atividade proteolítica (513,33 U/mL) foram obtidas a 30 ºC. A melhor fonte de carbono foi sacarose 1,0 % produzindo maior crescimento (6,18 mg/mL) e maior atividade proteásica (580 U/mL) e como fonte de nitrogênio destacou-se a peptona 0,5 % expressando os maiores valores de atividade proteolítica (755,55 U/mL) e biomassa de 7,55 mg/mL. Os resultados obtidos neste estudo sugerem que a protease produzida por T. pullulans classifica-se como protease alcalina (pH ótimo 8,0). E maior estabilidade térmica da protease foi observada a 37 ºC onde a enzima manteve 100 % de sua atividade durante 40 minutos de incubação. T. pullulans degradou o colágeno produzindo halo de 28 mm. Esse estudo demonstrou que a protease apresenta atividade colagenolítica e poderá ser utilizada como agente terapêutico

Metarhizium anisopliae é um fungo filamentoso entomopatogênico muito versátil que infecta, aproximadamente, 300 espécies de artrópodes, e também é adaptado à vida na rizosfera de plantas, sendo de extrema importância para o controle biológico de pragas na agricultura e pecuária. De acordo com o comportamento promíscuo desse fungo, pesquisadores têm identificado um grande número de genes relacionados à interação com o hospedeiro, e que são regulados de acordo com a sua indução. Em sua maioria, são genes que codificam para enzimas hidrolíticas. O número e a diversidade desses genes podem ser a chave para a habilidade desse patógeno em infectar uma larga variedade de artrópodes, podendo expressar genes diferentemente para cada tipo de hospedeiro. M. anisopliae secreta, entre outros, complexos quitinolítico e proteolítico para a degradação da quitina e proteínas presentes na cutícula do hospedeiro.Desse modo, o estudo da regulação dessas enzimas é de fundamental importância para o entendimento do processo de infecção; sendo assim, através desse trabalho foi observada e analisada a especialização na expressão de proteínas, especialmente de quitinases e proteases, secretadas por uma linhagem selvagem de M. anisopliae, na presença de cutículas de diferentes artrópodes, particularmente, de Dysdercus peruvianus, Boophilus microplus, Anticarsia gemmatalis e de quitina cristalina, através de ensaios de detecção enzimática e eletroforese uni e bidimensional. Em todos os experimentos, variando-se as condições de fonte de carbono e tempo de cultivo, a secreção de proteínas se mostrou altamente diferenciada, demonstrando comportamento diferencial do fungo a vários hospedeiros, o que seria um sinal da versatilidade do entomopatógeno, aqui estudado, para a capacidade de infectar centenas de hospedeiros. / Metarhizium anisopliae is a very versatile entomopathogenic filamentous fungus that infects, approximately, 300 species of arthropods, and is also adapted to the life in the rhizosphere of plants, being of extreme importance for the biological control of pests in agriculture and pecuary. In accordance with this promiscuous behavior of this fungus, researchers have identified a great number of genes related to the interaction with the host, and that they are regulated in accordance with its induction. In their majority, these genes encode for hydrolytic enzymes. The diversity of these genes can be the key for the ability of this pathogen in infect a wide variety of arthropods, being able to differently express genes for each type of host. M. anisopliae secretes chitinolytic and proteolytic complexes for the degradation of the chitin and proteins present in the cuticle of the host. In this way, the study of the regulation of these enzymes is of up fundamental importance for the understanding of the infection process. Through this research, the specialization in the expression of proteins, especially chitinases and proteases, secreted by a wild strain of M. anisopliae, in the presence of cuticles of different arthropods, particularly, of Dysdercus peruvianus, Boophilus microplus, Anticarsia gemmatalis and crystalline chitin, was observed and analyzed through enzymatic assays and one- and two-dimensional electrophoresis. In all the experiments, the conditions of the carbon source and time of culture, the protein secretion showed highly differentiated, demonstrating distinguishing fungus behavior to various hosts, which would be a sign of the versatility of this entomopathogen for the capacity of infecting hundreds of hosts.

Proteases play a key role in the cascade of tumor-associated proteolysis leading to extracellular matrix degradation, stromal invasion and blood vessel recruitment and inroad. Protease systems are widely described as implicated in the formation of new blood vessels. Until now, only few datas are available concerning their role in lymphangiogenesis. We successfully transposed the aorta ring assay to a mouse lymphatic thoracic duct assay. By immunochemistry and transmission electron microscopy, we characterized the outgrowing cells as being lymphatic cells that organize into microvessels containing a lumen and that conserved lymphatic endothelial cell features. This quantifiable model responds to several well-known lymphangiogenic factors as the VEGF-C but not to specific angiogenic factors. This model is so suitable to screen growth factors and inhibitors as well as conditioned media. Plasminogen activator inhibitor-1 is a component of the plasminogen cascade and, though it was critical for angiogenesis, it comes out that it is dispensable for lymphatic outgrowth. In sharp contrast, synthetic and physiological inhibitors of matrix metalloproteases inhibit lymphangiogenesis, and thoracic duct rings derived from MMP-2- but not MMP-9-deficient mice showed an impaired lymphatic cell outgrowth. These data identify MMP2 as an important player in lymphangiogenesis and was confirmed by an in vivo experiments. Proteases are thus also implicated in lymphangiogenesis and the lymphatic ring assay seems to be helpful to discover novel genes and mechanisms that underly the lymphangiogenesis process, including by comparing with angiogenesis in a similar system.

L'asthme est une maladie inflammatoire des voies aériennes dans laquelle différentes cellules et éléments cellulaires interagissent. L'asthme provoque des épisodes d'oppression thoracique et de toux, en particulier la nuit ou au petit matin. Ces épisodes sont généralement associés à une obstruction réversible des voies aériennes soit spontanément soit sous l'effet d'un traitement. Les principales caractéristiques de l'asthme sont une hyperréactivité bronchique, une inflammation à prédominance éosinophiles ainsi qu'un remodelage des bronches. Toutes ces caractéristiques structurelles suggèrent un rôle pour différentes protéases et notamment celles de la familles des adamlysines. En effet, les ADAM et ADAMTS protéases sont caractérisées par les domaines disintégrine et métalloprotéase qui leur confèrent les propriétés des molécules d'adhésion et des protéases. Dans ce travail, nous avons étudié ces protéases potentiellement intéressantes dans l'asthme.

Rapid, sensitive and quantitative assays for proteases are of great significance for drug development and in diagnosis of diseases. Herein, we describe work towards a novel assay for the multiplexed detection of proteases using ICP-MS. Protease substrates were synthesized containing a diethylenetriaminepentaaceticacid(DTPA) ligand to chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each substrate. A biotin label was appended to the C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic activities can then be determined by detecting the lanthanide signal of the peptide cleavage products by ICP-MS. Substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-amide, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide and DTPA-Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for calpain-1, caspase-3 and MMP-9 They were loaded with terbium, holmium and praseodymium respectively. As a proof-of-concept, α-chymotrypsin assays were carried out using DTPA-Asp-Leu-Leu-Val-Tyr-Asp-Lys(Biotin) loaded with lutetium, as a substrate. Calpain-1 assays were also performed. Parallel assays with commercially available fluorogenic substrates for both the enzymes were performed for comparison.

Rapid, sensitive and quantitative assays for proteases are of great significance for drug development and in diagnosis of diseases. Herein, we describe work towards a novel assay for the multiplexed detection of proteases using ICP-MS. Protease substrates were synthesized containing a diethylenetriaminepentaaceticacid(DTPA) ligand to chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each substrate. A biotin label was appended to the C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic activities can then be determined by detecting the lanthanide signal of the peptide cleavage products by ICP-MS. Substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-amide, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide and DTPA-Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for calpain-1, caspase-3 and MMP-9 They were loaded with terbium, holmium and praseodymium respectively. As a proof-of-concept, α-chymotrypsin assays were carried out using DTPA-Asp-Leu-Leu-Val-Tyr-Asp-Lys(Biotin) loaded with lutetium, as a substrate. Calpain-1 assays were also performed. Parallel assays with commercially available fluorogenic substrates for both the enzymes were performed for comparison.

This work is included in a largest project focused on the improvement of transgenic rice plants resistance against pests and phytopathogens. Our goal is the obtention of an improvement of the rice resistance against the lepidopterean Chilo suppressalis, the most important rice pest. The chosen strategy was the expression of the mpi gene (Maize Proteinase Inhibitor, coding for a proteinase inhibitor) on rice, by both biolistic and Agrobacterium transformation methods. The proteinase inhibitors are part of the natural defence system of plants against insect predation. Expression of this gene, and accumulation of the MPI protein, is stable through successive generations of rice transgenic lines. We have demonstrated that constitutive expression of the mpi gene confers resistance towards Chilo suppressalis larvae infestation in transgenic rice. Bioassays performed with transgenic rice plants expressing the mpi gene constitutively that were infested with Chilo larvae, showed that ingestion of the MPI lead to a significant reduction of the larvae weight with respect to the control larvae group (as far as 60 %). It is also important to point out that larvae fed on transgenic MPI plants showed a significant delay in their development, preventing the achievement of the L4 larval stage in most of them.The mpi promoter is functional in rice and confers wound-inducibility of gus reporter gene. The 2K fragment of mpi promoter confers a more rapid and intense induction than shortest C1 fragment. Moreover, 2K fragment of mpi promoter doesn't confer expression of gus reporter gene in the rice seed endosperm.On the other hand, the maize mpi gene is correctly expressed in rice plants under control of his own regulatory regions. The observed expression levels allow obtaining similar effects on Chilo larvae than those obtained with constitutive mpi expression.Besides, we fixed the optimal pH of digestive proteinase activities of Chilo suppressalis and Cacyreus marshalli (Geranium pest) as alkaline (pH 10.5 and 10 respectively). In Chilo digestive system participate serine proteinases activities of trypsin and chimotryspsin type, and also carboxy and aspartic proteinases. We also observed an adaptation effect on Chilo digestive system of larvae feed on transgenic rice plants, with an increase of 40% of total proteolytic activities in comparison to the control larvae activities. Nevertheless, this increase is not effective to overcome the MPI inhibitor effect.The analysis of AFP (Antifungal Protein) allows to conclude that this protein shows an elevated antifungal activity against F. verticillioides and M. grisea at low nM concentrations. Besides, AFP shows activity against the oomycete Phytophthora infestans at low mM concentrations. The presence of AFP protein leads to important morphological abnormalities of these phytopathogens. We have not detect toxic effects of AFP protein towards rice protoplasts at high AFP concentrations. The topic application of AFP protein on rice plants confers protection to Magnaporthe grisea infection.

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