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Futoshi Nakazawa - One of the best experts on this subject based on the ideXlab platform.
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establishment of a species specific primer pair for detecting veillonella infantium based on the 70 kda heat shock Protein gene DnaK
Anaerobe, 2018Co-Authors: Izumi Mashima, Frank A. Scannapieco, Ariadna Adisattya Djais, Elaine M Haase, Maiko Otomo, Masato Saitoh, Futoshi NakazawaAbstract:Abstract Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock Protein (DnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect DnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.
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identification of veillonella tobetsuensis in tongue biofilm by using a species specific primer pair
Anaerobe, 2013Co-Authors: Izumi Mashima, Futoshi NakazawaAbstract:Abstract Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae have been reported to be isolated from human oral cavities. The recently detected Veillonellatobetsuensis in human tongue biofilms was proposed as a novel Veillonella sp. In this study, to determine the distribution and frequency of V. tobetsuensis, we established a method for the detection and identification of V. tobetsuensis by using polymerase chain reaction (PCR) using a species-specific primer pair. The primer pair for V. tobetsuensis was designed on the basis of the nucleotide sequence of the 70-kDa heat shock Protein (DnaK) gene of V. tobetsuensis JCM 17976T (=ATCC BAA-2400T). The primer pair generated a specific PCR product for V. tobetsuensis but not for other oral Veillonella spp. With the PCR procedure using the primer pair, we could detect less than 10 ng of genomic DNA extracted from V. tobetsuensis. Thus, the PCR method using this primer pair is suitable for the specific detection and identification of V. tobetsuensis. The distribution and frequency of V. tobetsuensis were investigated by PCR using this species-specific primer pair. V. tobetsuensis was detected in 5 of 27 subjects. V. tobetsuensis was recovered from 19% (5/27) of subjects with other Veillonella species. And, prevalence of V. tobetsuensis ranged from 7.6% to 20.0% in these subjects. V. tobetsuensis is likely to coexist with other Veillonella spp. in tongue biofilm. In this study, the species-specific PCR primer pair for V. tobetsuensis was designed using partial sequences of the DnaK gene. This is the first report using a species-specific primer pair for PCR to determine the distribution and frequency of V. tobetsuensis in tongue biofilm.
Izumi Mashima - One of the best experts on this subject based on the ideXlab platform.
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establishment of a species specific primer pair for detecting veillonella infantium based on the 70 kda heat shock Protein gene DnaK
Anaerobe, 2018Co-Authors: Izumi Mashima, Frank A. Scannapieco, Ariadna Adisattya Djais, Elaine M Haase, Maiko Otomo, Masato Saitoh, Futoshi NakazawaAbstract:Abstract Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock Protein (DnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect DnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.
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identification of veillonella tobetsuensis in tongue biofilm by using a species specific primer pair
Anaerobe, 2013Co-Authors: Izumi Mashima, Futoshi NakazawaAbstract:Abstract Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae have been reported to be isolated from human oral cavities. The recently detected Veillonellatobetsuensis in human tongue biofilms was proposed as a novel Veillonella sp. In this study, to determine the distribution and frequency of V. tobetsuensis, we established a method for the detection and identification of V. tobetsuensis by using polymerase chain reaction (PCR) using a species-specific primer pair. The primer pair for V. tobetsuensis was designed on the basis of the nucleotide sequence of the 70-kDa heat shock Protein (DnaK) gene of V. tobetsuensis JCM 17976T (=ATCC BAA-2400T). The primer pair generated a specific PCR product for V. tobetsuensis but not for other oral Veillonella spp. With the PCR procedure using the primer pair, we could detect less than 10 ng of genomic DNA extracted from V. tobetsuensis. Thus, the PCR method using this primer pair is suitable for the specific detection and identification of V. tobetsuensis. The distribution and frequency of V. tobetsuensis were investigated by PCR using this species-specific primer pair. V. tobetsuensis was detected in 5 of 27 subjects. V. tobetsuensis was recovered from 19% (5/27) of subjects with other Veillonella species. And, prevalence of V. tobetsuensis ranged from 7.6% to 20.0% in these subjects. V. tobetsuensis is likely to coexist with other Veillonella spp. in tongue biofilm. In this study, the species-specific PCR primer pair for V. tobetsuensis was designed using partial sequences of the DnaK gene. This is the first report using a species-specific primer pair for PCR to determine the distribution and frequency of V. tobetsuensis in tongue biofilm.
Jeanpierre Liautard - One of the best experts on this subject based on the ideXlab platform.
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induction of DnaK through its native heat shock promoter is necessary for intramacrophagic replication of brucella suis
Infection and Immunity, 2002Co-Authors: Stephan Kohler, Euloge Ekaza, Jeanyves Paquet, Karl Walravens, Jacques Teyssier, Jacques Godfroid, Jeanpierre LiautardAbstract:The heat shock Protein DnaK is essential for intramacrophagic replication of Brucella suis. The replacement of the stress-inducible, native DnaK promoter of B. suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK. In contrast to a DnaK null mutant, this strain grew at 37°C, with a thermal cutoff at 39°C. However, the constitutive DnaK mutant, which showed high sensitivity to H2O2-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of DnaK is a crucial event in the intracellular replication of B. suis.
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cloning and characterization of the brucella ovis heat shock Protein DnaK functionally expressed in escherichia coli
Journal of Bacteriology, 1992Co-Authors: M F M Cellier, Jacques Teyssier, Jeanpierre Liautard, M Nicolas, Jacques Marti, Sri J WidadaAbstract:The Brucella ovis DnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the DnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant Protein and a B. ovis native Protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis Protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli DnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK Protein could behave as a functional heat shock Protein in E. coli. Images
Jacques Teyssier - One of the best experts on this subject based on the ideXlab platform.
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induction of DnaK through its native heat shock promoter is necessary for intramacrophagic replication of brucella suis
Infection and Immunity, 2002Co-Authors: Stephan Kohler, Euloge Ekaza, Jeanyves Paquet, Karl Walravens, Jacques Teyssier, Jacques Godfroid, Jeanpierre LiautardAbstract:The heat shock Protein DnaK is essential for intramacrophagic replication of Brucella suis. The replacement of the stress-inducible, native DnaK promoter of B. suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK. In contrast to a DnaK null mutant, this strain grew at 37°C, with a thermal cutoff at 39°C. However, the constitutive DnaK mutant, which showed high sensitivity to H2O2-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of DnaK is a crucial event in the intracellular replication of B. suis.
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cloning and characterization of the brucella ovis heat shock Protein DnaK functionally expressed in escherichia coli
Journal of Bacteriology, 1992Co-Authors: M F M Cellier, Jacques Teyssier, Jeanpierre Liautard, M Nicolas, Jacques Marti, Sri J WidadaAbstract:The Brucella ovis DnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the DnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant Protein and a B. ovis native Protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis Protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli DnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK Protein could behave as a functional heat shock Protein in E. coli. Images
Laszlo Otvos - One of the best experts on this subject based on the ideXlab platform.
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Synergy Between Proline-Rich Antimicrobial Peptides and Small Molecule Antibiotics Against Selected Gram-Negative Pathogens in vitro and in vivo
Frontiers Media S.A., 2018Co-Authors: Laszlo Otvos, Eszter Ostorhazi, Dora Szabo, Steven D. Zumbrun, Lynda L. Miller, Stephanie A. Halasohoris, Puvi D. Desai, Sharon Int M. VeldtAbstract:As monotherapy, modified proline-rich antimicrobial peptides (PrAMPs) protect animals from experimental bacteremia in a dose-dependent manner. We evaluated the in vitro synergy of a modified PrAMP, A3-APO, a dimer, previously shown to inhibit the 70 kDa bacterial heat shock Protein DnaK, with imipenem or colistin against two antibiotic-resistant pathogens; a carbapenemase-expressing Klebsiella pneumoniae strain K97/09 and Acinetobacter baumannii (ATCC BAA-1605). Combining antimicrobials resulted in synergy for PrAMP/colistin combination against both K. pneumoniae and A. baumannii (ΣFIC = 0.08 both) and additive activity for the A3-APO/imipenem combination against K. pneumoniae (ΣFIC = 0.53). Chex1-Arg20, (designated as ARV-1502 in preclinical development), the single chain PrAMP monomer of A3-APO, showed synergy with meropenem against a carbapenem-resistant uropathogenic Escherichia coli strain (ΣFIC = 0.38). In a murine bacteremia model using K97/09, A3-APO at 1 mg/kg demonstrated improved survival when co-administered with standard (10 mg/kg) or subtherapeutic (1 mg/kg) doses of colistin at 36 h (p < 0.05). Surprisingly, the survival benefit of A3-APO was augmented when the A3-APO dose was decreased by 50% to 0.5 mg/kg (p < 0.02) in conjunction with a subtherapeutic colistin dose (1 mg/kg). ARV-1502, as monotherapy demonstrated prolonged (>24 h) activity in a mouse Escherichia coli infection assay. Co-treatment with ARV-1502 and subtherapeutic doses of ceftazidime (150 mg/kg) was studied in a mouse model of melioidosis. ARV-1502 provided a 50% improvement in long-term (62 days) survival, but only at the lowest of 3 administered doses; survival advantage was demonstrated at 2.5 mg/kg but not at 5 or 10 mg/kg. The mortality benefit of combination therapies was not routinely accompanied by a parallel decline in blood or tissue bacterial counts in surviving animals, suggesting that the anti-infective activity of the host defense peptides (HDP) is broader than simply bacterial eradication. In fact, the hormetic effect observed in either animal models suggest that low dose HDP treatment may change the dominant mode of action in experimental bacteremia
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Data_Sheet_1_Synergy Between Proline-Rich Antimicrobial Peptides and Small Molecule Antibiotics Against Selected Gram-Negative Pathogens in vitro and in vivo.pdf
2018Co-Authors: Laszlo Otvos, Eszter Ostorhazi, Dora Szabo, Steven D. Zumbrun, Lynda L. Miller, Stephanie A. Halasohoris, Puvi D. Desai, Sharon Int M. Veldt, Carl N. KrausAbstract:As monotherapy, modified proline-rich antimicrobial peptides (PrAMPs) protect animals from experimental bacteremia in a dose-dependent manner. We evaluated the in vitro synergy of a modified PrAMP, A3-APO, a dimer, previously shown to inhibit the 70 kDa bacterial heat shock Protein DnaK, with imipenem or colistin against two antibiotic-resistant pathogens; a carbapenemase-expressing Klebsiella pneumoniae strain K97/09 and Acinetobacter baumannii (ATCC BAA-1605). Combining antimicrobials resulted in synergy for PrAMP/colistin combination against both K. pneumoniae and A. baumannii (ΣFIC = 0.08 both) and additive activity for the A3-APO/imipenem combination against K. pneumoniae (ΣFIC = 0.53). Chex1-Arg20, (designated as ARV-1502 in preclinical development), the single chain PrAMP monomer of A3-APO, showed synergy with meropenem against a carbapenem-resistant uropathogenic Escherichia coli strain (ΣFIC = 0.38). In a murine bacteremia model using K97/09, A3-APO at 1 mg/kg demonstrated improved survival when co-administered with standard (10 mg/kg) or subtherapeutic (1 mg/kg) doses of colistin at 36 h (p < 0.05). Surprisingly, the survival benefit of A3-APO was augmented when the A3-APO dose was decreased by 50% to 0.5 mg/kg (p < 0.02) in conjunction with a subtherapeutic colistin dose (1 mg/kg). ARV-1502, as monotherapy demonstrated prolonged (>24 h) activity in a mouse Escherichia coli infection assay. Co-treatment with ARV-1502 and subtherapeutic doses of ceftazidime (150 mg/kg) was studied in a mouse model of melioidosis. ARV-1502 provided a 50% improvement in long-term (62 days) survival, but only at the lowest of 3 administered doses; survival advantage was demonstrated at 2.5 mg/kg but not at 5 or 10 mg/kg. The mortality benefit of combination therapies was not routinely accompanied by a parallel decline in blood or tissue bacterial counts in surviving animals, suggesting that the anti-infective activity of the host defense peptides (HDP) is broader than simply bacterial eradication. In fact, the hormetic effect observed in either animal models suggest that low dose HDP treatment may change the dominant mode of action in experimental bacteremia.
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the antibacterial peptide pyrrhocoricin inhibits the atpase actions of DnaK and prevents chaperone assisted Protein folding
Biochemistry, 2001Co-Authors: Goran Kragol, Sandor Lovas, Gyorgyi Varadi, Barry A Condie, Ralf Hoffmann, Laszlo OtvosAbstract:Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock Protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of l-amino acids diminished the ATPase activity of recombinant DnaK. The inactive d-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5‘-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded Proteins, was studied by assaying the alkaline phosphatase and β-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with l-pyrrhocoricin or drosocin. d-Pyrrhocoricin, magainin 2, or buf...
