A leptospirose é causada por bactérias que pertencem ao gênero Leptospira. Em um estudo realizado por nosso grupo, observou-se que as proteases secretadas por leptospiras patogênicas foram capazes de clivar a molécula C3 do Complemento e seus fragmentos C3b e iC3b, além de Fator B, C4b e C2. Neste trabalho expandimos a análise da atividade proteolítica sobre os componentes do Complexo de Ataque à Membrana (MAC): C6, C7, C8 e C9. Nós observamos que essas proteases clivam todos os componentes do MAC inclusive o complexo solúvel formado e que essas clivagens ocorrem de modo tempo-dependente. Além disso, as clivagens dessas moléculas ocorrem de modo seletivo, pois mesmo utilizando quantidades reduzidas de sobrenadantes ainda foi possível observar produtos de clivagem. A atividade proteolítica foi inibida pela 1,10fenantrolina, indicando a participação de metaloproteases. O reconhecimento de quais moléculas do MAC são clivadas por proteases de leptospiras patogênicas pode contribuir para o desenvolvimento de estratégias terapêuticas na infecção por estes patógenos. / Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. In a previous study, our group observed that the proteases secreted by Pathogenic Leptospira were capable of cleaving C3 of Complement, as well as the fragments C3b and iC3b, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Membrane Attack Complex (MAC). We observed that the protease cleaves all MAC components including soluble complex formed and that these cleavages occur in a time-dependent manner and in a selective way, since even when reduced quantities of supernatants were used, the cleavage products were still observed. The proteolytic activity was inhibited by 1,10phenanthroline, indicating the participation of metalloproteinases. The recognition that MAC molecules are cleaved by proteases of pathogenic leptospires can contribute to the development of therapeutic strategies for the infection by these pathogens.

A leptospirose é uma zoonose causada por leptospiras patogênicas que possuem estratégias para driblar a ação do sistema complemento ao interagir com diversos reguladores negativos, como a vitronectina. Neste projeto, avaliou-se a interação de vitronectina com três proteínas de membrana de Leptospira: LigA, LigB e LcpA. Verificou-se que essas interações ocorrem nas porções C-terminais das proteínas e nos domínios de ligação com heparina da vitronectina. Essas proteínas também se ligam a C9 e são capazes de impedir a formação do poro in vitro e a formação do MAC em um ensaio clássico de hemólise. Essas proteínas de Leptospira interagem com diversos reguladores negativos do sistema complemento. Ensaios de competição demonstram que estes reguladores interagem simultaneamente com as proteínas através de sítios distintos. A caracterização funcional de proteínas e a descoberta de novos mecanismos utilizados por esse patógeno para sobreviver no hospedeiro podem auxiliar nossa compreensão no que diz respeito a aspectos relacionados à clínica e à prevenção da leptospirose. / Leptospirosis is a zoonotic disease caused by pathogenic Leptospira that have strategies to escape the action of the complement system interacting with several negative regulators, such as vitronectin. In this project, we evaluated the interaction of vitronectin with three Leptospira membrane proteins: LigA, LigB and LcpA. It has been found that such interactions occur at the C-terminal portions of these proteins and the heparin binding domain of vitronectin. These proteins also bind to C9 and are capable of preventing the formation of the pore in vitro and the formation of MAC in a classical test of hemolysis. These proteins interact with various Leptospira negative regulators of the complement system. Competition assays demonstrated that both interact with these regulatory proteins through distinct sites. The functional characterization of these proteins and the discovery of new mechanisms used by this pathogen to survive in the host may help our understanding with regard to clinical aspects and prevention of leptospirosis.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. A doença representa um grave problema de saúde pública nos países tropicais subdesenvolvidos. Mais de 500.000 casos graves de leptospirose são notificados a cada ano e a taxa de mortalidade excede 10% (World Health Organization, 1999). Os roedores são o principal reservatório urbano da doença, e eliminam leptospiras viáveis no meio ambiente ao longo de toda a vida. As bactérias entram no hospedeiro por abrasões na pele ou por membranas mucosas e rapidamente se espalham pelo organismo atingindo vários órgãos. A identificação de mecanismos de invasão e de evasão imune apresentados por leptospiras patogênicas é extremamente relevante e tem sido alvo de pesquisas recentes desenvolvidas por vários grupos. Nesse contexto, a caracterização funcional de proteínas de membrana externa de Leptospira, principais alvos de interação com moléculas do hospedeiro, é de grande importância. O Fator de Elongação Tu (EF-Tu), uma proteína bacteriana abundante envolvida na síntese protéica, pertence à categoria das proteínas conhecidas como \"moonlighting\". Tais moléculas possuem a capacidade de exercer mais de uma função e, normalmente, localizam-se em diferentes compartimentos da célula. Há relatos de que EF-Tu de agentes patogênicos possa atuar como um fator de virulência. No presente trabalho, demonstrou-se que EF-Tu de Leptospira está localizado na superfície da bactéria e possui funções adicionais, sendo receptor para moléculas presentes no plasma do hospedeiro. Tal proteína interage com vários componentes da matriz extracellular e também com plasminogênio, de maneira dosedependente. Resíduos de lisina são importantes para essa interação. Plasminogênio ligado a EF-Tu é convertido em sua forma ativa, plasmina, que, por sua vez, é capaz de clivar os substratos naturais C3b e fibrinogênio. EF-Tu de Leptospira também se liga a Fator H, principal regulador da via alternativa do sistema complemento, e este mantém sua atividade funcional ao agir como co-fator de Fator I na clivagem de C3b. O potencial imunoprotetor de EF-Tu em modelo animal foi avaliado, tendo em vista o alto grau de conservação da proteína em diferentes espécies de Leptospira. EF-Tu não conferiu proteção significativa e, portanto, não deve ser considerado como um candidato vacinal contra a leptospirose. Em suma, EF-Tu de Leptospira deve contribuir para o processo de invasão e evasão ao sistema imune inato do hospedeiro, inativando o sistema complemento. Tanto quanto é do nosso conhecimento, essa é a primeira descrição de uma proteína \"moonlighting\" em Leptospira. / Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. More than 500,000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10% (World Health Organization, 1999). Rodents are the main urban reservoir of the disease, shedding viable leptospires throughout their lives in the environment. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The identification of invasion mechanisms and immune evasion strategies employed by pathogenic leptospires is of great relevance. In this context, functional characterization of leptospiral outer membrane proteins, which represent the main targets for interaction with host molecules, is extremely important. The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. In this work we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It interacts with several extracellular matrix components and also binds plasminogen in a dose-dependent manner. Lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires Factor H (FH), the main soluble regulator of the alternative pathway of the complement system. FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). Given the wide distribution of EF-Tu among Leptospira species, its immunoprotective potential was evaluated in an animal model. EF-Tu was not able to afford significant immunoprotection, and might not be considered a vaccine candidate against leptospirosis. In conclusion, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

A leptospirose é uma zoonose causada por leptospiras patogênicas. Para estabelecer a infecção, estas bactérias desenvolveram estratégias de escape ao sistema complemento. Neste trabalho demonstramos que o sobrenadante de cultura de leptospiras patogênicas é capaz de inibir as três vias do complemento. Observamos que esse sobrenadante possui atividade proteolítica sobre C3, C3b e iC3b, além do FB (via alternativa), C2 e C4b (via clássica e das lectinas). As proteínas C3, C4, C2 e FB também foram clivadas quando soro humano normal (SHN) foi utilizado como fonte de complemento. Demonstramos que as proteases atuam em conjunto com os reguladores do hospedeiro Fator I e Fator H na clivagem de C3b. As clivagens foram inibidas pela 1,10-fenantrolina, sugerindo a participação de metaloproteases. Metaloproteases de leptospira da família das termolisinas foram produzidas como proteínas recombinantes e clivaram C3 no SHN. Concluímos que proteases de leptospiras patogênicas podem desativar moléculas do complemento e são potencias alvos para novas terapias em leptospirose. / Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. To establish the infection, these bacteria have developed strategies to escape the complement system. In this work, we demonstrate that culture supernatant from pathogenic Leptospira is capable of inhibiting the three complement pathways. We observe that this supernatant possess proteolytic activity under C3, C3b and iC3b, FB (alternative pathway), C2 and C4b (classical and lectin pathways). The proteins C3, C4, C2 and FB were also cleaved when normal human serum (NHS) was used as a source of complement. We demonstrate that these proteases act together with the host regulators Factor I and Factor H in C3b cleavage. The cleavages were inhibited by 1,10-phenanthroline, suggesting the involvement of metalloproteinases. Leptospira metalloproteinases from the thermolysin family were produced as recombinant proteins and cleaved C3 in NHS. We concluded that proteases from pathogenic Leptospira can inactivate complement molecules and are potential targets for new therapies in leptospirosis.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. A doença representa um grave problema de saúde pública nos países tropicais subdesenvolvidos. Mais de 500.000 casos graves de leptospirose são notificados a cada ano e a taxa de mortalidade excede 10% (World Health Organization, 1999). Os roedores são o principal reservatório urbano da doença, e eliminam leptospiras viáveis no meio ambiente ao longo de toda a vida. As bactérias entram no hospedeiro por abrasões na pele ou por membranas mucosas e rapidamente se espalham pelo organismo atingindo vários órgãos. A identificação de mecanismos de invasão e de evasão imune apresentados por leptospiras patogênicas é extremamente relevante e tem sido alvo de pesquisas recentes desenvolvidas por vários grupos. Nesse contexto, a caracterização funcional de proteínas de membrana externa de Leptospira, principais alvos de interação com moléculas do hospedeiro, é de grande importância. O Fator de Elongação Tu (EF-Tu), uma proteína bacteriana abundante envolvida na síntese protéica, pertence à categoria das proteínas conhecidas como \"moonlighting\". Tais moléculas possuem a capacidade de exercer mais de uma função e, normalmente, localizam-se em diferentes compartimentos da célula. Há relatos de que EF-Tu de agentes patogênicos possa atuar como um fator de virulência. No presente trabalho, demonstrou-se que EF-Tu de Leptospira está localizado na superfície da bactéria e possui funções adicionais, sendo receptor para moléculas presentes no plasma do hospedeiro. Tal proteína interage com vários componentes da matriz extracellular e também com plasminogênio, de maneira dosedependente. Resíduos de lisina são importantes para essa interação. Plasminogênio ligado a EF-Tu é convertido em sua forma ativa, plasmina, que, por sua vez, é capaz de clivar os substratos naturais C3b e fibrinogênio. EF-Tu de Leptospira também se liga a Fator H, principal regulador da via alternativa do sistema complemento, e este mantém sua atividade funcional ao agir como co-fator de Fator I na clivagem de C3b. O potencial imunoprotetor de EF-Tu em modelo animal foi avaliado, tendo em vista o alto grau de conservação da proteína em diferentes espécies de Leptospira. EF-Tu não conferiu proteção significativa e, portanto, não deve ser considerado como um candidato vacinal contra a leptospirose. Em suma, EF-Tu de Leptospira deve contribuir para o processo de invasão e evasão ao sistema imune inato do hospedeiro, inativando o sistema complemento. Tanto quanto é do nosso conhecimento, essa é a primeira descrição de uma proteína \"moonlighting\" em Leptospira. / Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. More than 500,000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10% (World Health Organization, 1999). Rodents are the main urban reservoir of the disease, shedding viable leptospires throughout their lives in the environment. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The identification of invasion mechanisms and immune evasion strategies employed by pathogenic leptospires is of great relevance. In this context, functional characterization of leptospiral outer membrane proteins, which represent the main targets for interaction with host molecules, is extremely important. The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. In this work we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It interacts with several extracellular matrix components and also binds plasminogen in a dose-dependent manner. Lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires Factor H (FH), the main soluble regulator of the alternative pathway of the complement system. FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). Given the wide distribution of EF-Tu among Leptospira species, its immunoprotective potential was evaluated in an animal model. EF-Tu was not able to afford significant immunoprotection, and might not be considered a vaccine candidate against leptospirosis. In conclusion, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

A leptospirose é uma zoonose causada por leptospiras patogênicas. Para estabelecer a infecção, estas bactérias desenvolveram estratégias de escape ao sistema complemento. Neste trabalho demonstramos que o sobrenadante de cultura de leptospiras patogênicas é capaz de inibir as três vias do complemento. Observamos que esse sobrenadante possui atividade proteolítica sobre C3, C3b e iC3b, além do FB (via alternativa), C2 e C4b (via clássica e das lectinas). As proteínas C3, C4, C2 e FB também foram clivadas quando soro humano normal (SHN) foi utilizado como fonte de complemento. Demonstramos que as proteases atuam em conjunto com os reguladores do hospedeiro Fator I e Fator H na clivagem de C3b. As clivagens foram inibidas pela 1,10-fenantrolina, sugerindo a participação de metaloproteases. Metaloproteases de leptospira da família das termolisinas foram produzidas como proteínas recombinantes e clivaram C3 no SHN. Concluímos que proteases de leptospiras patogênicas podem desativar moléculas do complemento e são potencias alvos para novas terapias em leptospirose. / Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. To establish the infection, these bacteria have developed strategies to escape the complement system. In this work, we demonstrate that culture supernatant from pathogenic Leptospira is capable of inhibiting the three complement pathways. We observe that this supernatant possess proteolytic activity under C3, C3b and iC3b, FB (alternative pathway), C2 and C4b (classical and lectin pathways). The proteins C3, C4, C2 and FB were also cleaved when normal human serum (NHS) was used as a source of complement. We demonstrate that these proteases act together with the host regulators Factor I and Factor H in C3b cleavage. The cleavages were inhibited by 1,10-phenanthroline, suggesting the involvement of metalloproteinases. Leptospira metalloproteinases from the thermolysin family were produced as recombinant proteins and cleaved C3 in NHS. We concluded that proteases from pathogenic Leptospira can inactivate complement molecules and are potential targets for new therapies in leptospirosis.

No description available.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Brian V. Geisbrecht / Neutrophils are our most abundant white blood cells and the first leukocytes to infiltrate sites of infection or damaged/healing tissue. Activation of neutrophils results in the mobilization of several types of granules stored within their cytosol, such as the so-called azurophilic granules, which either fuse with the maturing endophagocytic compartment or are released into the extracellular environment. One of the most abundant component of azurophilic granules is a heme-containing enzyme called myeloperoxidase (MPO), which reduces the H₂O₂ produced by the neutrophil’s respiratory burst to generate cytotoxic hypohalous acids, most typically HOCl. While neutrophil granule enzymes are essential for our innate defenses, neutrophil-driven inflammation outside this beneficial context lies at the heart of many non-infectious human diseases. Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. Recent work found a highly conserved S. aureus protein, SPIN (for Staphylococcal Peroxidase INhibitor), that specifically binds and inhibits MPO [1]. This study was focused on characterizing the structure/function relationship for MPO inhibitors, SPIN proteins. To identify key residues for SPIN function in more detail, we examined two types of SPIN proteins using structural methods, direct binding assays, and functional assays for MPO activity: deletion mutants and SPIN proteins originating from divergent staphylococcal species. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors. In addition to the focus on targeted inhibition of MPO for its therapeutic value in treatment of a number of significant human inflammatory diseases, our investigations contributed in expanding our knowledge on infection spreading. As a first cellular host defense response, the neutrophil interaction with pathogens are of major interest. Characterization of staphylococcal immune evasion proteins is vital for understanding bacterial survival when encountering neutrophils and their bioactive constituents.

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts, including AIDS patients. Pneumocystis species have a biphasic life cycle consisting of single-nucleated trophic forms and ascus-like cysts. Both stages live within the host, and, thus, must contend with threats from the host immune system. The cyst cell wall β-glucans have been shown to stimulate immune responses in lung epithelial cells, dendritic cells and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with immune cells. In this study, the immune response to the life cycle stages of Pneumocystis murina was evaluated. Here, we report differences in the immune response of immunocompetent mice to the trophic and cystic life cycle stages of P. murina. Upon infection with purified trophic forms, wild-type adult mice developed a delayed innate and adaptive immune response compared to inoculation with the normal mixture of trophic forms and cysts. Cysts, but not trophic forms, stimulated Th1-type responses in the lungs of infected mice. Surprisingly, trophic forms are sufficient to generate protective adaptive responses, leading to clearance in immunocompetent mice. We report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised RAG2-/- mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia. Dendritic cells screen the alveolar spaces for pathogens, and are in a prime position to initiate the immune response against lung pathogens, including Pneumocystis. Our data demonstrate that trophic forms broadly dampen the ability of dendritic cells to respond to pathogen-associated molecular patterns. Bone marrow-derived dendritic cells were stimulated with trophic forms, a mixture of trophic forms and cysts, and various other inflammatory materials, including β-glucan. Trophic forms inhibited multiple components involved in antigen presentation by dendritic cells, including secretion of inflammatory cytokines and expression of MHC class II and costimulatory molecules on the cell surface. Furthermore, trophic forms suppressed or failed to induce the expression of multiple genes related to activation and maturation in dendritic cells. Dendritic cells silenced by trophic forms are unable to induce CD4+ T cell responses. These data suggest that immune evasion by trophic forms is dependent on the suppression of innate responses, and the development of adaptive immunity represents a “point of no return” at which the trophic forms are no longer able to escape clearance.

No description available.