RESUMEN
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. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and 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 the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, 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.
Asunto(s)
Factor H de Complemento/metabolismo , Leptospira/enzimología , Factor Tu de Elongación Peptídica/metabolismo , Plasminógeno/metabolismo , Animales , Coagulación Sanguínea , Fibrinolisina/metabolismo , Interacciones Huésped-Patógeno , Evasión Inmune , Inmunidad Innata/inmunología , Leptospira/metabolismo , Leptospira/fisiología , Lisina/metabolismo , Factor Tu de Elongación Peptídica/química , Unión Proteica , Transporte de ProteínasRESUMEN
BACKGROUND: Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins. RESULTS: The vaccine candidates LigANI and LipL32 were cloned and expressed in P. pastoris as secreted proteins. Large-scale expression resulted in a yield of 276 mg/L for LigANI and 285 mg/L for LipL32. The recombinant proteins were glycosylated and were recognized by antibodies present in the sera of patients with severe leptospirosis. CONCLUSIONS: The expression of LigANI and LipL32 in P. pastoris resulted in a significant increase in yield compared to expression in E. coli. In addition, the proteins were secreted, allowing for easy purification, and retained the antigenic characteristics of the native proteins, demonstrating their potential application as subunit vaccine candidates.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Vacunas Bacterianas/genética , Leptospira/inmunología , Leptospirosis/inmunología , Lipoproteínas/genética , Pichia/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/metabolismo , Glicosilación , Humanos , Leptospira/enzimología , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/metabolismoRESUMEN
INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. CONCLUSIONS: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases.
Asunto(s)
Electroforesis en Gel de Campo Pulsado , Leptospira/clasificación , Serotipificación/métodos , Pruebas de Aglutinación , ADN Bacteriano/análisis , Desoxirribonucleasas de Localización Especificada Tipo II/análisis , Leptospira/enzimología , Leptospira/genéticaRESUMEN
INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.
INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. CONCLUSIONS: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases.
Asunto(s)
Electroforesis en Gel de Campo Pulsado , Leptospira/clasificación , Serotipificación/métodos , Pruebas de Aglutinación , ADN Bacteriano/análisis , Desoxirribonucleasas de Localización Especificada Tipo II/análisis , Leptospira/enzimología , Leptospira/genéticaRESUMEN
Leptospirosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-Å-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL3221-272). The LipL3221-272 monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL3221-272 is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL3221-272. Ca2+ binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca2+ as no structural or stability perturbations were observed for Mg2+, Zn2+, or Cu2+. Careful examination of the crystallographic structure suggests the locations of putative regions that could mediate Ca2+ binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and laminin.
Asunto(s)
Masculino , Humanos , Animales , Leptospira/enzimología , LeptospirosisRESUMEN
Se aplicó un método de la reacción en cadena de la polimerasa para la detección temprana de Leptospiras spp. en hemocultivos procedentes de pacientes con sospecha de leptospirosis humana. Los métodos moleculares, y en particular la amplificación del ADN por la reacción en cadena de la polimerasa y sus variantes, constituyen herramientas muy útiles y específicas, utilizadas en la actualidad con esta finalidad. Se lograron identificar por la reacción en cadena de la polimerasa Leptospiras spp. en 41,3 por ciento (33/80) de los hemocultivos a los 7 d de incubación, los que también se clasificaron como positivos por los métodos convencionales. Sin embargo, hubo 20 por ciento (16/80) en los que también por este método se lograron identificar Leptospiras spp., pero por los métodos convencionales resultaron ser negativos. De los hemocultivos 38,7 por ciento (31/80) resultó negativo tanto por la reacción en cadena de la polimerasa como por los métodos convencionales. El uso del método de la reacción en cadena de la polimerasa a partir de hemocultivos, es una alternativa valiosa para la detección temprana y el diagnóstico rápido de Leptospiras spp(AU)
The PCR method for the early detection of Leptospiras spp. in hemocultures from patients suspected of human leptospiros was applied. Molecular methods and, particularly, the amplification of DNA by PCR, and its variants, are very useful and specific tools used nowdays to this end. Leptospiras spp. were identied by means of PCR in 41.3 percent(33/80) of the hemocultures at 7 days of incubation.They were also classified as positive by the conventional methods. However, it was also possible to identify Leptospiras spp. in 20 percent (16/80) by this method, but they proved to be negative by the conventional methods. Of the hemocultures, 38.7 percent (31/80) yielded negative by PCR and by the conventional methods. The use of PCR starting from hemocultures is a valuable alternative for the early detection and rapid diagnosis of Leptospiras spp(AU)
Asunto(s)
Leptospira/enzimología , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Se aplicó un método de la reacción en cadena de la polimerasa para la detección temprana de Leptospiras spp. en hemocultivos procedentes de pacientes con sospecha de leptospirosis humana. Los métodos moleculares, y en particular la amplificación del ADN por la reacción en cadena de la polimerasa y sus variantes, constituyen herramientas muy útiles y específicas, utilizadas en la actualidad con esta finalidad. Se lograron identificar por la reacción en cadena de la polimerasa Leptospiras spp. en 41,3 por ciento (33/80) de los hemocultivos a los 7 d de incubación, los que también se clasificaron como positivos por los métodos convencionales. Sin embargo, hubo 20 por ciento (16/80) en los que también por este método se lograron identificar Leptospiras spp., pero por los métodos convencionales resultaron ser negativos. De los hemocultivos 38,7 por ciento (31/80) resultó negativo tanto por la reacción en cadena de la polimerasa como por los métodos convencionales. El uso del método de la reacción en cadena de la polimerasa a partir de hemocultivos, es una alternativa valiosa para la detección temprana y el diagnóstico rápido de Leptospiras spp.
The PCR method for the early detection of Leptospiras spp. in hemocultures from patients suspected of human leptospiros was applied. Molecular methods and, particularly, the amplification of DNA by PCR, and its variants, are very useful and specific tools used nowdays to this end. Leptospiras spp. were identied by means of PCR in 41.3 percent(33/80) of the hemocultures at 7 days of incubation.They were also classified as positive by the conventional methods. However, it was also possible to identify Leptospiras spp. in 20 percent (16/80) by this method, but they proved to be negative by the conventional methods. Of the hemocultures, 38.7 percent (31/80) yielded negative by PCR and by the conventional methods. The use of PCR starting from hemocultures is a valuable alternative for the early detection and rapid diagnosis of Leptospiras spp
Asunto(s)
Leptospira/enzimología , Reacción en Cadena de la Polimerasa/métodosRESUMEN
This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/análisis , Leptospira/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Leptospira/enzimología , Análisis de Secuencia de ProteínaRESUMEN
This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.