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  • 1
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    Online Resource
    Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus)
    American Society for Microbiology ; 2016
    In:  Applied and Environmental Microbiology Vol. 82, No. 15 ( 2016-08), p. 4482-4491
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 82, No. 15 ( 2016-08), p. 4482-4491
    Abstract: Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060 , orf065 , and orf086 . The information obtained in this study should be useful for further development of safe and efficient phage therapy. IMPORTANCE Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00090-16
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
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    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 2
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    Characterization of the Enzymatic Component of Clostridium perfringens Iota-Toxin
    American Society for Microbiology ; 2000
    In:  Journal of Bacteriology Vol. 182, No. 8 ( 2000-04-15), p. 2096-2103
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 182, No. 8 ( 2000-04-15), p. 2096-2103
    Abstract: The iota a component (i a ) of Clostridium perfringens ADP ribosylates nonmuscle β/γ actin and skeletal muscle α-actin. Replacement of Arg-295 in i a with alanine led to a complete loss of NAD + -glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities ( 〈 0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity ( 〈 0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities ( 〈 0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD + to i a , that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i a on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i a are dependent on ARTase activity.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.182.8.2096-2103.2000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2000
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    SSG: 12
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  • 3
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    Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids
    American Society for Microbiology ; 2012
    In:  Applied and Environmental Microbiology Vol. 78, No. 9 ( 2012-05), p. 3193-3202
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 78, No. 9 ( 2012-05), p. 3193-3202
    Abstract: A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene ( neo r ), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium . The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium , could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C 20:3n-6 ) and eicosatetraenoic acid (C 20:4n-3 ), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C 20:4n-6 ) and eicosapentaenoic acid (C 20:5n-3 ), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.07129-11
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
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    detail.hit.zdb_id: 1478346-0
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  • 4
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    Online Resource
    Inducible Nitric Oxide Synthase Is a Key Host Factor for Toxoplasma GRA15-Dependent Disruption of the Gamma Interferon-Induced Antiparasitic Human Response
    American Society for Microbiology ; 2018
    In:  mBio Vol. 9, No. 5 ( 2018-11-07)
    Details
    In: mBio, American Society for Microbiology, Vol. 9, No. 5 ( 2018-11-07)
    Abstract: Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti- Toxoplasma host factor in mice, here we show that iNOS in humans is a pro- Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host’s NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti- Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor. IMPORTANCE Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma -infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.01738-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 2557172-2
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  • 5
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    Online Resource
    Genome Sequences of 12 Mycobacteriophages Recovered from Archival Stocks in Japan
    American Society for Microbiology ; 2018
    In:  Genome Announcements Vol. 6, No. 25 ( 2018-06-21)
    Details
    In: Genome Announcements, American Society for Microbiology, Vol. 6, No. 25 ( 2018-06-21)
    Abstract: Using Mycobacterium smegmatis mc 2 155, 12 siphoviruses were recovered from long-term archival stocks stored in Japan. Their genome sequences were 46.0 to 61.3 kbp with 63 to 68% G+C contents, which allowed them to be categorized within cluster W and subclusters A1, A2, B3, A7, I1, and K4.
    Type of Medium: Online Resource
    ISSN: 2169-8287
    URL: Article
    DOI: 10.1128/genomeA.00472-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 2968655-6
    detail.hit.zdb_id: 2704277-7
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  • 6
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    Increase of Eicosapentaenoic Acid in Thraustochytrids through Thraustochytrid Ubiquitin Promoter-Driven Expression of a Fatty Acid Δ5 Desaturase Gene
    American Society for Microbiology ; 2011
    In:  Applied and Environmental Microbiology Vol. 77, No. 11 ( 2011-06), p. 3870-3876
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 77, No. 11 ( 2011-06), p. 3870-3876
    Abstract: Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C 16:0 ), n − 6 docosapentaenoic acid (DPA) (C 22:5 n − 6 ), and docosahexaenoic acid (DHA) (C 22:6 n − 3 ), with eicosapentaenoic acid (EPA) (C 20:5 n − 3 ) and arachidonic acid (AA) (C 20:4 n − 6 ) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C 20:4 n − 3 ) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C 20:3 n − 6 ) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02664-10
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
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    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 7
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    Online Resource
    Binding and Internalization of Clostridium botulinum C2 Toxin
    American Society for Microbiology ; 2009
    In:  Infection and Immunity Vol. 77, No. 11 ( 2009-11), p. 5139-5148
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 77, No. 11 ( 2009-11), p. 5139-5148
    Abstract: Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and a binding component (C2II). The activated binding component (C2IIa) forms heptamers, and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the binding and internalization of C2IIa in cells. The C2IIa monomer formed oligomers on lipid rafts in membranes of MDCK cells. Methyl-beta-cyclodextrin inhibited the binding of C2IIa and the rounding of the cells induced by C2I plus C2IIa. C2I was localized to the rafts in the presence, but not the absence, of C2IIa. Surface plasmon resonance analysis revealed that C2I bound to the oligomer of C2IIa, but not the monomer of C2IIa. C2I and C2IIa were rapidly internalized in the cells. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited the internalization of C2IIa in the cells and the rounding activity in the presence of C2I plus C2IIa. Incubation of the cells with C2I plus C2IIa resulted in the activation of PI3K and in phosphorylation of phosphoinositide-dependent kinase 1 and protein kinase B/Akt (Akt), but that with C2IIa alone did not. Akt inhibitor X, an Akt phosphorylation inhibitor, inhibited the rounding activity but not the internalization of C2IIa. The results suggest that the binding of C2I to the oligomer of C2IIa on rafts triggers the activation of the PI3K-Akt signaling pathway and, in turn, the initiation of endocytosis.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00638-09
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2009
    detail.hit.zdb_id: 1483247-1
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  • 8
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    Bacterial rRNA-Targeted Reverse Transcription-PCR Used To Identify Pathogens Responsible for Fever with Neutropenia
    American Society for Microbiology ; 2010
    In:  Journal of Clinical Microbiology Vol. 48, No. 5 ( 2010-05), p. 1624-1628
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 48, No. 5 ( 2010-05), p. 1624-1628
    Abstract: The purpose of this study was to evaluate the clinical utility of bacterial rRNA-targeted reverse transcription-quantitative PCR (BrRNA RT-qPCR) assays for identifying the bacterial pathogens that cause fever with neutropenia in pediatric cancer patients, by comparing the bacterial detection rate of this technique with that of blood culture. One milliliter of blood was collected from pediatric patients who developed fever with neutropenia following cancer chemotherapy. BrRNA RT-qPCR was performed using 16 primer sets, each designed for a specific type of bacteria. The entire BrRNA RT-qPCR procedure took less than 5 h. Blood culture was performed at the same time, following the standard institutional procedure. Blood from 13 patients was collected during 23 febrile neutropenic episodes. Of these samples, bacteria were identified in 16 by BrRNA RT-qPCR (69.6%) and in 4 by blood culture (17.4%, P 〈 0.001). In all 4 blood culture-positive samples, BrRNA RT-qPCR detected the same type of bacteria as that identified by culture. In 9 samples, more than 4 types of bacteria were identified simultaneously by BrRNA RT-qPCR, most of which were anaerobic bacteria known to be part of the gut flora. We conclude that BrRNA RT-qPCR could be useful in the diagnosis of fever with neutropenia, given its high bacterial detection rate, short turnaround time, and the small blood sample required compared with the standard blood culture techniques. Our findings also indicate that anaerobic intestinal bacteria, which are difficult to detect by standard culture techniques, may be responsible for some cases of febrile neutropenia.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.01724-09
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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