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Mathieu Picardeau - One of the best experts on this subject based on the ideXlab platform.
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Pathogenic but not saprophyte Leptospira spp. degrade DNA.
2015Co-Authors: Emilia Scharrig, Mathieu Picardeau, Agostina Carestia, María F. Ferrer, Maia Cédola, Gabriela Pretre, Ricardo Drut, Mirta Schattner, Ricardo M. GómezAbstract:Representative analysis of DNA digestion by gel electrophoresis. From left to right: plasmid DNA (100 ng/μL) after incubation with PBS (negative control), DNase I (positive control), and live Leptospira interrogans serovar Copenhageni (LIC) or Leptospira Biflexa serovar Patoc (Patoc) (1x108/mL) after 60 minutes of incubation at 37°C.
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Leptospira spp. motility is not a relevant factor for NET formation.
2015Co-Authors: Emilia Scharrig, Mathieu Picardeau, Agostina Carestia, María F. Ferrer, Maia Cédola, Gabriela Pretre, Ricardo Drut, Mirta Schattner, Ricardo M. GómezAbstract:(A) Human neutrophils (2x105/mL) were incubated with Leptospira interrogans serovar Manilae (LIM) and Leptospira Biflexa serovar Patoc (Patoc) and their non-mobile mutants flaA2 and flaB (MOI = 50) for 180 min and then fixed (PF 4%) and stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green) and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (B) Quantification of NETs released by fluorometry in the same conditions as in (A).
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Pathogenicity and viability are relevant factors for NET formation.
2015Co-Authors: Emilia Scharrig, Mathieu Picardeau, Agostina Carestia, María F. Ferrer, Maia Cédola, Gabriela Pretre, Ricardo Drut, Mirta Schattner, Ricardo M. GómezAbstract:(A) Human neutrophils (2x105/mL) were incubated with live or inactivated with 4% PF or by heat Leptospira interrogans serovar Copenhageni (LIC) or Leptospira Biflexa serovar Patoc (Patoc) (MOI = 50) for 180 min and then fixed (PF 4%), stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (B) Quantification of NETs released by fluorometry in the same conditions as in (A). Bars represent standard error of the mean (SEM) of assays from ten independent assays; *p
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Construction of a library of random mutants in the spirochete Leptospira Biflexa using a mariner transposon.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Leyla Slamti, Mathieu PicardeauAbstract:In comparison to other bacterial species, genetics of leptospires are in their infancy. Recently, we developed a system for random transposon mutagenesis in the saprophyte Leptospira Biflexa and then applied this approach to the pathogen L. interrogans. Thousands of random mutants can be readily obtained in -L. -Biflexa by random insertion of Himar1 in the genome, thereby generating extensive libraries of mutants that could be screened for phenotypes affecting diverse aspects of the biology of the bacterium. This system should be particularly useful for the identification of new genes of unknown function in Leptospira spp. This chapter describes a procedure for transposition in L. Biflexa via conjugation of a plasmid delivering Himar1, isolation of mutants, and mapping of the insertion sites on the chromosome.
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Characterization of the Bat proteins in the oxidative stress response of Leptospira Biflexa.
BMC Microbiology, 2012Co-Authors: Philip Stewart, Mathieu Picardeau, James Carroll, David Dorward, Hunter Stone, Amit Sarkar, Patricia RosaAbstract:ABSTRACT: BACKGROUND: Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not been experimentally demonstrated. RESULTS: We investigated the contribution of the Bat proteins in the model organism Leptospira Biflexa for their potential contributions to growth rate, morphology and protection against oxidative challenges. A genetically engineered mutant strain in which all bat ORFs were deleted did not exhibit altered growth rate or morphology, relative to the wild-type strain. Nor could we demonstrate a protective role for the Bat proteins in coping with various oxidative stresses. Further, pre-exposing L. Biflexa to sublethal levels of reactive oxygen species did not appear to induce a general oxidative stress response, in contrast to what has been shown in other bacterial species. Differential proteomic analysis of the wild-type and mutant strains detected changes in the abundance of a single protein only - HtpG, which is encoded by the gene immediately downstream of the bat loci. CONCLUSION: The data presented here do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress as previously proposed. L. Biflexa is relatively sensitive to reactive oxygen species such as superoxide and H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe.
Hélène Louvel - One of the best experts on this subject based on the ideXlab platform.
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Heme rescues a two-component system Leptospira Biflexa mutant
BMC Microbiology, 2008Co-Authors: Hélène Louvel, Jean-michel Betton, Mathieu PicardeauAbstract:Background Heme is typically a major iron source for bacteria, but little is known about how bacteria of the Leptospira genus, composed of both saprophytic and pathogenic species, access heme. Results In this study, we analysed a two-component system of the saprophyte Leptospira Biflexa . In vitro phosphorylation and site-directed mutagenesis assays showed that Hklep is a histidine kinase which, after autophosphorylation of a conserved histidine, transfers the phosphate to an essential aspartate of the response regulator Rrlep. Hklep/Rrlep two-component system mutants were generated in L. Biflexa . The mutants could only grow in medium supplemented with hemin or δ-aminolevulinic acid (ALA). In the pathogen L. interrogans , the hklep and rrlep orthologous genes are located between hemE and hemL genes, which encode proteins involved in heme biosynthesis. The L. Biflexa hklep mutant could be complemented with a replicative plasmid harbouring the L. interrogans orthologous gene, suggesting that these two-component systems are functionally similar. By real-time quantitative reverse transcription-PCR, we also observed that this two-component system might influence the expression of heme biosynthetic genes. Conclusion These findings demonstrate that the Hklep/Rrlep regulatory system is critical for the in vitro growth of L. Biflexa , and suggest that this two-component system is involved in a complex mechanism that regulates the heme biosynthetic pathway.
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Heme rescues a two-component system Leptospira Biflexa mutant
BMC microbiology, 2008Co-Authors: Hélène Louvel, Jean-michel Betton, Mathieu PicardeauAbstract:Background Heme is typically a major iron source for bacteria, but little is known about how bacteria of the Leptospira genus, composed of both saprophytic and pathogenic species, access heme.
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Current Protocols in Microbiology - Genetic manipulation of Leptospira Biflexa.
Current protocols in microbiology, 2007Co-Authors: Hélène Louvel, Mathieu PicardeauAbstract:The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira Biflexa and L. interrogans, respectively. A major factor contributing to our ignorance of spirochetal biology has been the lack of methods available for genetic analysis of these organisms. In recent years, an E. coli-L. Biflexa shuttle vector has been constructed and a system for targeted mutagenesis and random transposon mutagenesis of the saprophyte L. Biflexa has been developed. These studies enable the use of L. Biflexa as a model bacterium among spirochetes. Keywords: spirochetes; Leptospira; electrotransformation; genetics; mutagenesis; transposon; allelic exchange
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genetic manipulation of Leptospira Biflexa
Current protocols in microbiology, 2007Co-Authors: Hélène Louvel, Mathieu PicardeauAbstract:The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira Biflexa and L. interrogans, respectively. A major factor contributing to our ignorance of spirochetal biology has been the lack of methods available for genetic analysis of these organisms. In recent years, an E. coli-L. Biflexa shuttle vector has been constructed and a system for targeted mutagenesis and random transposon mutagenesis of the saprophyte L. Biflexa has been developed. These studies enable the use of L. Biflexa as a model bacterium among spirochetes. Keywords: spirochetes; Leptospira; electrotransformation; genetics; mutagenesis; transposon; allelic exchange
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Genetic manipulation of Leptospira Biflexa.
Current protocols in microbiology, 2007Co-Authors: Hélène Louvel, Mathieu PicardeauAbstract:The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira Biflexa and L. interrogans, respectively. A major factor contributing to our ignorance of spirochetal biology has been the lack of methods available for genetic analysis of these organisms. In recent years, an E. coli-L. Biflexa shuttle vector has been constructed and a system for targeted mutagenesis and random transposon mutagenesis of the saprophyte L. Biflexa has been developed. These studies enable the use of L. Biflexa as a model bacterium among spirochetes.
Yoshio Araki - One of the best experts on this subject based on the ideXlab platform.
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occurrence of 3 β d manp 1 4 β d manp 1 n units in the antigenic polysaccharides from Leptospira Biflexa serovar patoc strain patoc i
Carbohydrate Research, 2000Co-Authors: Kouki Matsuo, Emiko Isogai, Yoshio ArakiAbstract:Abstract In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1∼3) from cells of Leptospira Biflexa serovar patoc strain Patoc I ( L. Biflexa patoc Patoc I) by the hot phenol–water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti- L. Biflexa serum and also cross-reacted in similar extents toward most of the other Leptospiral antisera tested. Such immunoreactions were specifically inhibited by a β-(1→4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1 H and 13 C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit →3)-β- d -Man p -(1→4)-β- d -Man p -(1→ in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. Biflexa polysaccharides.
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Occurrence of [→3)-β-d-Manp-(1→4)-β-d-Manp-(1→]n units in the antigenic polysaccharides from Leptospira Biflexa serovar patoc strain Patoc I
Carbohydrate research, 2000Co-Authors: Kouki Matsuo, Emiko Isogai, Yoshio ArakiAbstract:Abstract In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1∼3) from cells of Leptospira Biflexa serovar patoc strain Patoc I ( L. Biflexa patoc Patoc I) by the hot phenol–water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti- L. Biflexa serum and also cross-reacted in similar extents toward most of the other Leptospiral antisera tested. Such immunoreactions were specifically inhibited by a β-(1→4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1 H and 13 C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit →3)-β- d -Man p -(1→4)-β- d -Man p -(1→ in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. Biflexa polysaccharides.
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Control of Immunologically Crossreactive Leptospiral Infection by Administration of Lipopolysaccharides from a Nonpathogenic Strain of Leptospira Biflexa
Microbiology and immunology, 2000Co-Authors: Kouki Matsuo, Emiko Isogai, Yoshio ArakiAbstract:In our previous paper (Matsuo, K., Isogai, E., and Araki, Y., Carbohydr. Res., 328: 517-524, 2000), antigenic polysaccharides obtained from the lipopolysaccharide (LPS) fraction of a nonpathogenic Leptospira, Leptospira Biflexa patoc Patoc I, are shown to be broadly crossreactable with most rabbit antisera elicited by immunization with various pathogenic leptospires. The result led us to test a protective effect of the same LPS in a hamster model system by heterologously challenging with a pathogenic Leptospira, L. interrogans manilae UP-MMG. Firstly, a similarity in the antigenic epitopes of L. Biflexa and L. interrogans was confirmed by the following assays. In the microscopic agglutination test (MAT), a hamster antiserum elicited by immunization with the L. Biflexa-LPS preparation was shown to agglutinate cells of L. interrogans. Contrarily, in the enzyme-linked immunosorbent assay (ELISA), the L. Biflexa-LPS preparation was shown to crossreact with a hamster antiserum elicited by immunization with whole cells of L. interrogans. These results suggest that the same or closely related antigens may be present on the cell surfaces of both L. Biflexa patoc Patoc I and L. interrogans manilae UP-MMG. Furthermore, in a protective assay, the prior administration of a L. Biflexa-LPS preparation resulted in raising a protective response in hamsters against challenge by L. interrogans without any side effect. The protective effect was strongly dependent on the dose amounts and/or administration times of L. Biflexa-LPS. Thus, L. Biflexa-LPS preparations can use as a potent vaccine against leptospirosis caused by various leptospires.
Kouki Matsuo - One of the best experts on this subject based on the ideXlab platform.
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occurrence of 3 β d manp 1 4 β d manp 1 n units in the antigenic polysaccharides from Leptospira Biflexa serovar patoc strain patoc i
Carbohydrate Research, 2000Co-Authors: Kouki Matsuo, Emiko Isogai, Yoshio ArakiAbstract:Abstract In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1∼3) from cells of Leptospira Biflexa serovar patoc strain Patoc I ( L. Biflexa patoc Patoc I) by the hot phenol–water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti- L. Biflexa serum and also cross-reacted in similar extents toward most of the other Leptospiral antisera tested. Such immunoreactions were specifically inhibited by a β-(1→4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1 H and 13 C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit →3)-β- d -Man p -(1→4)-β- d -Man p -(1→ in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. Biflexa polysaccharides.
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Occurrence of [→3)-β-d-Manp-(1→4)-β-d-Manp-(1→]n units in the antigenic polysaccharides from Leptospira Biflexa serovar patoc strain Patoc I
Carbohydrate research, 2000Co-Authors: Kouki Matsuo, Emiko Isogai, Yoshio ArakiAbstract:Abstract In this study, we isolated three kinds of antigenic polysaccharide components (tentatively designed as AP-1∼3) from cells of Leptospira Biflexa serovar patoc strain Patoc I ( L. Biflexa patoc Patoc I) by the hot phenol–water procedure, followed by treatment with mild acid and column chromatography. Two of them (AP-1 and AP-2) were recovered from the phenol-soluble fraction whereas another (AP-3) was recovered from the aqueous fraction. All of them reacted toward an anti- L. Biflexa serum and also cross-reacted in similar extents toward most of the other Leptospiral antisera tested. Such immunoreactions were specifically inhibited by a β-(1→4)-linked mannobiose, but were not by any mono- and oligosaccharide tested. From their structural analyses including 1 H and 13 C NMR spectrometry, Smith degradation and methylation analysis, it was revealed that all of these antigenic polysaccharides had the same disaccharide unit →3)-β- d -Man p -(1→4)-β- d -Man p -(1→ in their major polysaccharide parts, but they differed in the acyl substituents. Therefore it is most likely that such mannobiose unit is a candidate for the antigenic epitopes of L. Biflexa polysaccharides.
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Control of Immunologically Crossreactive Leptospiral Infection by Administration of Lipopolysaccharides from a Nonpathogenic Strain of Leptospira Biflexa
Microbiology and immunology, 2000Co-Authors: Kouki Matsuo, Emiko Isogai, Yoshio ArakiAbstract:In our previous paper (Matsuo, K., Isogai, E., and Araki, Y., Carbohydr. Res., 328: 517-524, 2000), antigenic polysaccharides obtained from the lipopolysaccharide (LPS) fraction of a nonpathogenic Leptospira, Leptospira Biflexa patoc Patoc I, are shown to be broadly crossreactable with most rabbit antisera elicited by immunization with various pathogenic leptospires. The result led us to test a protective effect of the same LPS in a hamster model system by heterologously challenging with a pathogenic Leptospira, L. interrogans manilae UP-MMG. Firstly, a similarity in the antigenic epitopes of L. Biflexa and L. interrogans was confirmed by the following assays. In the microscopic agglutination test (MAT), a hamster antiserum elicited by immunization with the L. Biflexa-LPS preparation was shown to agglutinate cells of L. interrogans. Contrarily, in the enzyme-linked immunosorbent assay (ELISA), the L. Biflexa-LPS preparation was shown to crossreact with a hamster antiserum elicited by immunization with whole cells of L. interrogans. These results suggest that the same or closely related antigens may be present on the cell surfaces of both L. Biflexa patoc Patoc I and L. interrogans manilae UP-MMG. Furthermore, in a protective assay, the prior administration of a L. Biflexa-LPS preparation resulted in raising a protective response in hamsters against challenge by L. interrogans without any side effect. The protective effect was strongly dependent on the dose amounts and/or administration times of L. Biflexa-LPS. Thus, L. Biflexa-LPS preparations can use as a potent vaccine against leptospirosis caused by various leptospires.
Daniela V. Rial - One of the best experts on this subject based on the ideXlab platform.
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Cloning and characterization of the Type I Baeyer–Villiger monooxygenase from Leptospira Biflexa
AMB Express, 2017Co-Authors: Romina D. Ceccoli, Dario A. Bianchi, Michael J. Fink, Marko D. Mihovilovic, Daniela V. RialAbstract:Baeyer–Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer–Villiger monooxygenase from Leptospira Biflexa . This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer–Villiger monooxygenase from L. Biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.
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Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira Biflexa.
AMB Express, 2017Co-Authors: Romina D. Ceccoli, Dario A. Bianchi, Michael J. Fink, Marko D. Mihovilovic, Daniela V. RialAbstract:Baeyer–Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer–Villiger monooxygenase from Leptospira Biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer–Villiger monooxygenase from L. Biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.