Journal of Biomolecular Structure and Dynamics, 2013
Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease acti... more Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.
Objective: To compare the clinical aspects and microbiological profiles, monitored by PCR-DGGE an... more Objective: To compare the clinical aspects and microbiological profiles, monitored by PCR-DGGE analyzes, of children with and without early childhood caries (ECC). Methods: 2 - 4 years old patients (n=14), 7 with caries (ECC group) and 7 without caries (caries free group) were submitted to anamnesis, clinical examination (dmf and modified dmf indexes) and saliva collection (unstimulated whole saliva) for microbiological analyzes. Streptococcus mutans, Lactobacillus spp., Candida spp.and total microorganisms were counted through methods of plaque cultivation. The microbial diversity was characterized by PCR-DGGE. Comparisons between clinical data (qui-square test) and the microbial count (Student t-test) of groups were performed. Pearson Correlation Coefficient was used for DGGE analyzes and also for correlation between microorganisms counts and breastfeeding / use of pacifiers. Results: The habits of oral hygiene and breastfeeding did not show association with ECC. The use of pacifi...
A quantidade de água produzida pode exceder em mais de 50 vezes à de petróleo. Consequentemente, ... more A quantidade de água produzida pode exceder em mais de 50 vezes à de petróleo. Consequentemente, a água produzida representa um dos maiores custos associado às empresas, uma vez que, como estratégica de gerenciamento, normalmente é bombeado ou transportado para instalações de tratamento fora do local, ou bombeada para poços de descarte. Além disso, existem quatro principais problemas relacionados com os microrganismos para este Setor: (I) biocorrosão; (II) contaminação da água produzida que pode levar à contaminação de campos de petróleo após a reinjeção; (III) incrustação biológica, que é a formação de depósitos biológicos; e (IV) acidificação, que é a produção de ácido sulfídrico (H 2 S) por bactérias. O Grupo CNPq de "Pesquisa Aplicada Multidisciplinar e Desenvolvimento Tecnológico para Produção de Petróleo e Gás em Bacias Maduras e Campos Marginais (PCM 2)" vem pesquisando e desenvolvendo produtos com aplicação direta para este segmento, contribuindo de forma significa...
International Journal of Medical Microbiology, 2013
Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is nece... more Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis cannot be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis.
Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with... more Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and p...
Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infectio... more Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis "ping-pong" ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after ...
Advances in experimental medicine and biology, 2007
The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is ... more The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion ...
International Journal of Medical Microbiology, 2008
Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by... more Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by cleaving collagen and its denatured form gelatin, and thus enhance migration of mammalian cells. The gastrointestinal pathogen Salmonella enterica survives and grows within host macrophages and dendritic cells, and can disseminate in the host by travelling within infected host cells. Here, we report that S. enterica serovar Typhimurium activates
Journal of Biomolecular Structure and Dynamics, 2013
Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease acti... more Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.
Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the pro... more Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli.
Journal of Biomolecular Structure and Dynamics, 2013
Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease acti... more Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.
Objective: To compare the clinical aspects and microbiological profiles, monitored by PCR-DGGE an... more Objective: To compare the clinical aspects and microbiological profiles, monitored by PCR-DGGE analyzes, of children with and without early childhood caries (ECC). Methods: 2 - 4 years old patients (n=14), 7 with caries (ECC group) and 7 without caries (caries free group) were submitted to anamnesis, clinical examination (dmf and modified dmf indexes) and saliva collection (unstimulated whole saliva) for microbiological analyzes. Streptococcus mutans, Lactobacillus spp., Candida spp.and total microorganisms were counted through methods of plaque cultivation. The microbial diversity was characterized by PCR-DGGE. Comparisons between clinical data (qui-square test) and the microbial count (Student t-test) of groups were performed. Pearson Correlation Coefficient was used for DGGE analyzes and also for correlation between microorganisms counts and breastfeeding / use of pacifiers. Results: The habits of oral hygiene and breastfeeding did not show association with ECC. The use of pacifi...
A quantidade de água produzida pode exceder em mais de 50 vezes à de petróleo. Consequentemente, ... more A quantidade de água produzida pode exceder em mais de 50 vezes à de petróleo. Consequentemente, a água produzida representa um dos maiores custos associado às empresas, uma vez que, como estratégica de gerenciamento, normalmente é bombeado ou transportado para instalações de tratamento fora do local, ou bombeada para poços de descarte. Além disso, existem quatro principais problemas relacionados com os microrganismos para este Setor: (I) biocorrosão; (II) contaminação da água produzida que pode levar à contaminação de campos de petróleo após a reinjeção; (III) incrustação biológica, que é a formação de depósitos biológicos; e (IV) acidificação, que é a produção de ácido sulfídrico (H 2 S) por bactérias. O Grupo CNPq de "Pesquisa Aplicada Multidisciplinar e Desenvolvimento Tecnológico para Produção de Petróleo e Gás em Bacias Maduras e Campos Marginais (PCM 2)" vem pesquisando e desenvolvendo produtos com aplicação direta para este segmento, contribuindo de forma significa...
International Journal of Medical Microbiology, 2013
Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is nece... more Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis cannot be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis.
Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with... more Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and p...
Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infectio... more Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis "ping-pong" ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after ...
Advances in experimental medicine and biology, 2007
The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is ... more The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion ...
International Journal of Medical Microbiology, 2008
Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by... more Mammalian matrix metalloproteinases (MMPs) degrade collagen networks in extracellular matrices by cleaving collagen and its denatured form gelatin, and thus enhance migration of mammalian cells. The gastrointestinal pathogen Salmonella enterica survives and grows within host macrophages and dendritic cells, and can disseminate in the host by travelling within infected host cells. Here, we report that S. enterica serovar Typhimurium activates
Journal of Biomolecular Structure and Dynamics, 2013
Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease acti... more Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg-Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8 ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.
Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the pro... more Quorum sensing is a cell-cell signaling mechanism based on cell density and that involves the production of hormone-like molecules called autoinducers (AI). One of the most studied AIs has been termed AI-2, and its biosynthesis requires the enzyme encoded by luxS. We have previously described for the first time that Bacteroides species can produce molecules with AI-2 activity. In this study, we focus on the detection of luxS and its activity as the AI-2 synthase in Bacteroides species. The strains Bacteroides fragilis B3b and Bacteroides vulgatus ATCC 8482 were selected based on a positive phenotype for AI-2 production and the presence of a putative luxS in the genome, respectively. In order to identify the luxS gene, cloning and heterologous expression strategies were utilized. We demonstrate that both strains contain functional luxS orthologs that can complement AI-2 production in Escherichia coli.
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Papers by Leandro Lobo