This is the first broad proteomic description of Gluconacetobacter diazotrophicus, an endophytic ... more This is the first broad proteomic description of Gluconacetobacter diazotrophicus, an endophytic bacterium, responsible for the major fraction of the atmospheric nitrogen fixed in sugarcane in tropical regions. Proteomic coverage of G. diazotrophicus PAL5 was obtained by two independent approaches: 2-DE followed by MALDI-TOF or TOF-TOF MS and 1-DE followed by chromatography in a C18 column online coupled to an ESI-Q-TOF or ESI-IT mass spectrometer. The 583 identified proteins were sorted into functional categories and used to describe potential metabolic pathways for nucleotides, amino acids, carbohydrates, lipids, cofactors and energy production, according to the Enzyme Commission of Enzyme Nomenclature (EC) and Kyoto Encyclopedia of genes and genomes (KEGG) databases. The identification of such proteins and their possible insertion in conserved biochemical routes will allow comparisons between G. diazotrophicus and other bacterial species. Furthermore, the 88 proteins classified as conserved unknown or unknown constitute a potential target for functional genomic studies, aiming at the understanding of protein function and regulation of gene expression. The knowledge of metabolic fundamentals and coordination of these actions are crucial for the rational, safe and sustainable interference on crops. The entire dataset, including peptide sequence information, is available as Supporting Information and is the major contribution of this work.
Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwid... more Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signalling and metabolism to favour bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes 3 VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated, and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin, and induces actin remodelling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis. This article is protected by copyright. All rights reserved.
Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwid... more Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signalling and metabolism to favour bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes 3 VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated, and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin, and induces actin remodelling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis. This article is protected by copyright. All rights reserved.
The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to lepr... more The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to leprosy in a small subset of the infected individuals. Transmission is believed to occur mainly by exposure to bacilli present in aerosols expelled by infected individuals with high bacillary load. Mycobacterium leprae-specific DNA has been detected in the blood of asymptomatic household contacts of leprosy patients years before active disease onset, suggesting that, following infection, the bacterium reaches the lymphatic drainage and the blood of at least some individuals. The lower temperature and availability of protected microenvironments may provide the initial conditions for the survival of the bacillus in the airways and skin. A subset of skin-resident macrophages and the Schwann cells of peripheral nerves, two M. leprae permissive cells, may protect M. leprae from effector cells in the initial phase of the infection. The interaction of M. leprae with these cells induces metabolic ch...
Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence... more Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence of T6SS genes in NTUH-K2044. Position Weight Matrix (PWM), Start and End position, Strand, Score and Sequence binding for transcriptional regulators. (XLS 47 kb)
Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence... more Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence of T6SS genes in HS11286. Position Weight Matrix (PWM), Start and End position, Strand, Score and Sequence binding for transcriptional regulators. (XLS 42 kb)
Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence... more Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence of T6SS genes in Kp52.145. Position Weight Matrix (PWM), Start and End position, Strand, Score and Sequence binding for transcriptional regulators. (XLS 55 kb)
Putative promoter sequence of T6SS genes. 250 bp upstream those transcriptional start sites of T6... more Putative promoter sequence of T6SS genes. 250 bp upstream those transcriptional start sites of T6SS genes in genome of Klebsiella pneumoniae Kp52.145, HS11286 and NTUH-K2044. (TXT 11 kb)
Predicted 휎70 promoter sequences of T6SS genes. Binding sites − 10, spacer region, − 35 and Trans... more Predicted 휎70 promoter sequences of T6SS genes. Binding sites − 10, spacer region, − 35 and Transcriptional Start Site upstream the CDSs coding for the T6SS-related genes in genome of Klebsiella pneumoniae Kp52.145, HS11286 and NTUH-K2044. (XLS 98 kb)
Multiple sequence alignment of ClpV, ClpB and ClpA proteins from Klebsiella pneumoniae Kp52.145. ... more Multiple sequence alignment of ClpV, ClpB and ClpA proteins from Klebsiella pneumoniae Kp52.145. Green and yellow regions indicate the Clp_N domain; Red, blue and purple are the AAA, AAA2 and ClpB_D2-small domains, respectively. Black and blue boxes indicate the Walker A and Walker B domains; Arrows point to ATP binding site residues. "*"Identity of all aminoacid residues in the indicated column, ":"Similarity of pairs aligned, "." Low similarity of pairs aligned. (PDF 58 kb)
Identification and location of T6SS components in the genome of KP Kp52.145, HS11286 and NTUH-K20... more Identification and location of T6SS components in the genome of KP Kp52.145, HS11286 and NTUH-K2044. Table describing locus tag, previous annotation and indicated if T6SS components to belong in loci or are orphan. (XLS 284 kb)
Summary of Secret6 and VRprofile results contributing to the annotation of T6SS genes in KP. Tabl... more Summary of Secret6 and VRprofile results contributing to the annotation of T6SS genes in KP. Table describing T6SS genes in KP genomes: old and current locus-tag, genomic coordinates, gene orientation, gene size, gene product, SecreT6 and VRprofile predictions. (XLS 56 kb)
Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredo... more Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous tr...
New approaches are needed to control leprosy, but understanding of the biology of the causative a... more New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target. IMPORTANCE Leprosy remains a major probl...
Few studies investigate the major protein antigens targeted by the antibody diversity of infected... more Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with . To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole extracts. By proteomic analysis, we were able to identify the most immunogenic proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was β-tubulin. The β-tubulin gene was cloned, expressed in , and recombinant β-tubulin was obtained. Infection increased IgG reactivity against recombinant β-tubulin. A single immunization of mice with recombinant β-tubulin increased specific IgG reactivity and induced protection against infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.
Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors withou... more Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.
Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-con... more Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigates the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-HSD, is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side ch...
This is the first broad proteomic description of Gluconacetobacter diazotrophicus, an endophytic ... more This is the first broad proteomic description of Gluconacetobacter diazotrophicus, an endophytic bacterium, responsible for the major fraction of the atmospheric nitrogen fixed in sugarcane in tropical regions. Proteomic coverage of G. diazotrophicus PAL5 was obtained by two independent approaches: 2-DE followed by MALDI-TOF or TOF-TOF MS and 1-DE followed by chromatography in a C18 column online coupled to an ESI-Q-TOF or ESI-IT mass spectrometer. The 583 identified proteins were sorted into functional categories and used to describe potential metabolic pathways for nucleotides, amino acids, carbohydrates, lipids, cofactors and energy production, according to the Enzyme Commission of Enzyme Nomenclature (EC) and Kyoto Encyclopedia of genes and genomes (KEGG) databases. The identification of such proteins and their possible insertion in conserved biochemical routes will allow comparisons between G. diazotrophicus and other bacterial species. Furthermore, the 88 proteins classified as conserved unknown or unknown constitute a potential target for functional genomic studies, aiming at the understanding of protein function and regulation of gene expression. The knowledge of metabolic fundamentals and coordination of these actions are crucial for the rational, safe and sustainable interference on crops. The entire dataset, including peptide sequence information, is available as Supporting Information and is the major contribution of this work.
Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwid... more Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signalling and metabolism to favour bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes 3 VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated, and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin, and induces actin remodelling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis. This article is protected by copyright. All rights reserved.
Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwid... more Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signalling and metabolism to favour bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes 3 VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated, and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin, and induces actin remodelling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis. This article is protected by copyright. All rights reserved.
The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to lepr... more The initial infection by the obligate intracellular bacillus Mycobacterium leprae evolves to leprosy in a small subset of the infected individuals. Transmission is believed to occur mainly by exposure to bacilli present in aerosols expelled by infected individuals with high bacillary load. Mycobacterium leprae-specific DNA has been detected in the blood of asymptomatic household contacts of leprosy patients years before active disease onset, suggesting that, following infection, the bacterium reaches the lymphatic drainage and the blood of at least some individuals. The lower temperature and availability of protected microenvironments may provide the initial conditions for the survival of the bacillus in the airways and skin. A subset of skin-resident macrophages and the Schwann cells of peripheral nerves, two M. leprae permissive cells, may protect M. leprae from effector cells in the initial phase of the infection. The interaction of M. leprae with these cells induces metabolic ch...
Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence... more Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence of T6SS genes in NTUH-K2044. Position Weight Matrix (PWM), Start and End position, Strand, Score and Sequence binding for transcriptional regulators. (XLS 47 kb)
Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence... more Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence of T6SS genes in HS11286. Position Weight Matrix (PWM), Start and End position, Strand, Score and Sequence binding for transcriptional regulators. (XLS 42 kb)
Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence... more Hypothetical binding sites for transcriptional regulators predicted in putative promoter sequence of T6SS genes in Kp52.145. Position Weight Matrix (PWM), Start and End position, Strand, Score and Sequence binding for transcriptional regulators. (XLS 55 kb)
Putative promoter sequence of T6SS genes. 250 bp upstream those transcriptional start sites of T6... more Putative promoter sequence of T6SS genes. 250 bp upstream those transcriptional start sites of T6SS genes in genome of Klebsiella pneumoniae Kp52.145, HS11286 and NTUH-K2044. (TXT 11 kb)
Predicted 휎70 promoter sequences of T6SS genes. Binding sites − 10, spacer region, − 35 and Trans... more Predicted 휎70 promoter sequences of T6SS genes. Binding sites − 10, spacer region, − 35 and Transcriptional Start Site upstream the CDSs coding for the T6SS-related genes in genome of Klebsiella pneumoniae Kp52.145, HS11286 and NTUH-K2044. (XLS 98 kb)
Multiple sequence alignment of ClpV, ClpB and ClpA proteins from Klebsiella pneumoniae Kp52.145. ... more Multiple sequence alignment of ClpV, ClpB and ClpA proteins from Klebsiella pneumoniae Kp52.145. Green and yellow regions indicate the Clp_N domain; Red, blue and purple are the AAA, AAA2 and ClpB_D2-small domains, respectively. Black and blue boxes indicate the Walker A and Walker B domains; Arrows point to ATP binding site residues. "*"Identity of all aminoacid residues in the indicated column, ":"Similarity of pairs aligned, "." Low similarity of pairs aligned. (PDF 58 kb)
Identification and location of T6SS components in the genome of KP Kp52.145, HS11286 and NTUH-K20... more Identification and location of T6SS components in the genome of KP Kp52.145, HS11286 and NTUH-K2044. Table describing locus tag, previous annotation and indicated if T6SS components to belong in loci or are orphan. (XLS 284 kb)
Summary of Secret6 and VRprofile results contributing to the annotation of T6SS genes in KP. Tabl... more Summary of Secret6 and VRprofile results contributing to the annotation of T6SS genes in KP. Table describing T6SS genes in KP genomes: old and current locus-tag, genomic coordinates, gene orientation, gene size, gene product, SecreT6 and VRprofile predictions. (XLS 56 kb)
Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredo... more Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous tr...
New approaches are needed to control leprosy, but understanding of the biology of the causative a... more New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target. IMPORTANCE Leprosy remains a major probl...
Few studies investigate the major protein antigens targeted by the antibody diversity of infected... more Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with . To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole extracts. By proteomic analysis, we were able to identify the most immunogenic proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was β-tubulin. The β-tubulin gene was cloned, expressed in , and recombinant β-tubulin was obtained. Infection increased IgG reactivity against recombinant β-tubulin. A single immunization of mice with recombinant β-tubulin increased specific IgG reactivity and induced protection against infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.
Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors withou... more Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.
Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-con... more Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigates the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-HSD, is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side ch...
Uploads