Nagatoshi Fujiwara

Osaka City University, Ōsaka, Ōsaka, Japan

Are you Nagatoshi Fujiwara?

Claim your profile

Publications (58)189.82 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: During the process of identifying a Gram-negative coccobacillus isolated from a human clinical specimen, we found that the isolate's 16S rRNA gene had very close sequence identity with that of a variant Porphyromonas isolated from polymicrobial infections in the central bearded dragon, a species of lizard [2]. The 16S rRNA gene sequences of the human isolate and of six isolates from lizards were nearly identical (99.9-100%). Phylogenetic analysis placed all of these isolates in a single phylogenetic cluster well separated from other species in the genus Porphyromonas. The closest species was Porphyromonas catoniae with 90.7-90.9% sequence identity, although there was less than 6% DNA similarity between the P. catoniae type strain and our representative isolates from lizards (PAGU 1787(T)) and human (PAGU 1776). These isolates could grow under anaerobic or microaerobic conditions (6% O2 atmosphere). The isolates were positive for catalase and very strong β-hemolytic activity, but did not show black or brown pigmentation. Biochemically, the isolates could be differentiated from closely related species by pyroglutamic acid arylamidase and glycine arylamidase activity, and some others. The fermentation products mainly included succinic acid and propionic acid. The major fatty acids detected in cells of the isolates were iso-C15:0, anteiso-C15:0, and 3OH-iso-C17:0. The G+C content was 43.0±0.62mol%. The species name Porphyromonas pogonae sp. nov. is proposed for these isolates with the type strain of PAGU 1787(T) (=MI 10-1288(T)=JCM 19732(T)=ATCC BAA-2643(T)). Copyright © 2014 Elsevier GmbH. All rights reserved.
    Systematic and Applied Microbiology 11/2014; · 3.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174-176 and 195-196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knockout mice (i.e. hMincle(+) mice) were established and compared with non-transgenic mice (i.e. mMincle(+) mice). We showed that macrophages derived from hMincle(+) mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle(+) mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle(+), but not mMincle(+) mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.
    Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-/- mice than in TLR2-/- and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.
    Biochimica et Biophysica Acta 03/2013; · 4.66 Impact Factor
  • Isamu Matsunaga, Shinji Meda, Noboru Nakata, Nagatoshi Fujiwara
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: Intrinsic multidrug resistance of the Mycobacterium avium-intracellulare complex presents a serious problem in the treatment of the diseases caused by these bacteria. Recently, it was shown that deletion of a polyketide synthase, Pks12, in an M. avium laboratory strain decreases this intrinsic resistance. Methods: We investigated Pks12 expression and its enzymatic activity in 9 clinical isolates of M. intracellulare, and compared their drug susceptibilities to 4 drugs. Also, we made pks12-disrupted M. bovis bacillus Calmette-Guérin (BCG) mutant and its complemented strain. Using these BCG and M. intracellulare strains, we observed intracellular accumulation of ethidium bromide (EtBr). Results: We found positive correlations between Pks12 and drug resistance for all of the antibiotics tested. The drug susceptible M. intracellulare strain showed higher EtBr accumulation. Consistent with this, EtBr was much more accumulated in pks12-disrupted BCG than wild-type or the complemented strains. Conclusions: Collectively, these results suggest that Pks12 controls the multidrug resistance in part through intracellular drug accumulation.
    Chemotherapy 11/2012; 58(5):341-348. · 2.07 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mannans are mannose polymers attached to cell wall proteins in all Candida species, including the pathogenic fungus Candida albicans. Mannans are sensed by pattern recognition receptors expressed on innate immune cells. However, the detailed structural patterns affecting immune sensing are not fully understood because mannans have a complex structure that includes α- and β-mannosyl linkages. In this study, we focused on the β-1,2-mannosides of N-linked mannan in C. albicans because this moiety is not present in the non-pathogenic yeast Saccharomyces cerevisiae. To investigate the impact of β-1,2-mannosides on immune sensing, we constructed a C. albicans ∆mnn4/∆bmt1 double deletant. Thin-layer chromatography and nuclear magnetic resonance analyses revealed that the deletant lacked β-1,2-mannosides in N-linked mannan. Mannans lacking the β-1,2-mannosides induced the production of higher levels of inflammatory cytokines, including IL-6, IL-12p40 and TNF-α, in mice dendritic cells compared to wild-type mannan. Our data show that β-1,2-mannosides in N-linked mannan reduce the production of inflammatory cytokines by dendritic cells.
    Medical mycology: official publication of the International Society for Human and Animal Mycology 10/2012; · 2.13 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The antimycobacterial activities of disulfiram (DSF) and diethyldithiocarbamate (DDC) against multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB) clinical isolates were evaluated in vitro. Both DSF and DDC exhibited potent antitubercular activities against 42 clinical isolates of M. tuberculosis, including MDR/XDR-TB strains. Moreover, DSF showed remarkable bactericidal activity ex vivo and in vivo. Therefore, DSF might be a drug repurposed for the treatment of MDR/XDR-TB.
    Antimicrobial Agents and Chemotherapy 05/2012; 56(8):4140-5. · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The acid-fastness is the most important and the most specific characteristics in mycobacteria, the mechanism of which is not clear but may be attributed to the lipid rich cell wall of this bacterium. While the exact component(s) responsible for this staining method remained unidentified, a Mycobacterium tuberculosis mutant, attenuated strain that produced shorter mycolic acids with defects in trans-cyclopropanation was shown to be acid fast negative. In this study, we examined the ultrastructure of the cell envelope (CE) of the mutant strain ΔkasB (missing a beta-ketoacyl-ACP synthase involved in mycolic acid biosynthesis), the parental CDC1551 (wild type strain) and kasB complemented strain, and compared ultrastructural differences among them with conventional transmission electron microscopy (TEM) and cryo-transmission electron microscopy (CEM). Conventional TEM revealed that there were no detectable differences in the thickness of the cell envelope among three strains (wild-type: 43.35 ± 6.13 nm; ΔkasB: 45.98 ± 11.32 nm; complement: 40.71 ± 6.3 nm). However, CEM data demonstrated that the region between the inner and outer membranes of the mutant strain, which is composed mainly of cell wall anchored mycolic acids (MA), showed a significant decrease in electron density as compared to the wild type and kasB complement strain (567.1 ± 372.7 vs. 301.4 ± 262.1, or vs. 235.2 ± 174.9, p < 0.02 or p < 0.001, respectively). These results suggested that altered MA patterns in the kasB mutant may have affected the packing of the lipid rich layer of the M. tuberculosis cell envelope, resulting in a reduced electron density of this layer as seen by CEM and loss of acid-fastness in light microscopical observation, and we propose a novel model of the cell envelope structure in tubercle bacilli.
    Tuberculosis (Edinburgh, Scotland) 04/2012; 92(4):351-7. · 2.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.
    Journal of Biological Chemistry 12/2011; 286(51):44153-61. · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The genus Enhydrobacter, first reported as a member of the family Vibrionaceae, has been placed in the family Moraxellaceae, but as a genus incertae sedis in Bergey's Manual of Systematic Bacteriology 2nd edition. During our taxonomic investigation of Enhydrobacter-like organisms, we observed that the 16S rRNA sequences of E. aerosaccus-type strain versions NCIMB 12535(T) , ATCC 27094( T) and CCUG 58314(T) were very different from the accessible data (accession no. AJ550856). Phylogenetic analysis of our 16S rRNA sequence data revealed that these organisms were located within the family Rhodospirillaceae. The genera Inquilinus, Oceanibaculum, Skermanella and Nisaea were closely related (sequence similarities were 88.3~87.0%), but Enhydrobacter could be distinguished from these genera by growth characteristics, fatty acid profiles (C(19:0) cyclo ω8c; 38.4% C(18:1) ω7c; 32.2%, and C(16:0) ; 8.9% were major components), in being non-flagellated, and differing in enzymatic activities, including trypsin and β-glucosidase. From these data, we conclude that the genus Enhydrobacter should be recognized as an independent genus of the family Rhodospirillaceae within the class Alphaproteobacteria.
    Microbiology and Immunology 12/2011; 56(1):21-6. · 1.55 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium smegmatis is a rapidly growing, non-pathogenic mycobacterium, and M. smegmatis strain mc(2)155 in particular has been used as a tool for molecular analysis of mycobacteria because of its high rate of transformation. We examined another strain, M. smegmatis J15cs, which has the advantage of surviving for six days in murine macrophages. The J15cs strain produces a rough dry colony, and we hypothesized that the long survival of the J15cs strain was correlated with its cell wall components. Therefore, the lipid compositions of these two strains were compared. The subclasses and carbon species of the mycolic acids were very similar, and the major glycolipids and phospholipids were expressed in both strains. However, apolar glycopeptidolipids were deleted only in the J15cs strain. The presence of apolar glycopeptidolipids gives the cell wall a different structure. Moreover, the apolar glycopeptidolipids were recognized by macrophages via toll-like receptor 2, but not 4. We concluded that the absence of apolar glycopeptidolipids is a definitive feature of the J15cs strain, and affects its morphology and survival in host cells.
    Tuberculosis (Edinburgh, Scotland) 11/2011; 92(2):187-92. · 2.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Novel actinobacterial strains, PAGU 1247(T), PAGU 1251 and PAGU 1252, were isolated from the skin of atopic dermatitis patients and were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were located within the family Dermacoccaceae. The most closely related species of PAGU 1247(T) in phylogenetic terms was Branchiibius hedensis Mer 29717(T), with 16S rRNA gene sequence similarity of 99.6%, although the DNA-DNA relatedness value was less than 43.9%. Some biochemical traits, such as lipase (C14) and α-galactosidase activity, could distinguish these isolates from B. hedensis. Strain PAGU 1247(T) contained iso-C(16:0) and brC(18:0) as the major fatty acids. The quinone system consisted of menaquinone MK-8(H(6) and H(4)). The G+C content of the genomic DNA was 67.6mol%. On the basis of its phenotypic properties and genetic distinctiveness, strains PAGU 1247(T), PAGU 1251 and PAGU 1252 represents a novel species of the genus Branchiibius, for which the name Branchiibius cervicis sp. nov. is proposed. The type strain is PAGU 1247(T) (=NBRC 106593(T)=DSM 24166(T)).
    Systematic and Applied Microbiology 09/2011; 34(7):503-7. · 3.29 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.
    Journal of bacteriology 08/2011; 193(20):5766-74. · 3.94 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dynamic changes in the lipid composition of the cell wall occur in pathogenic mycobacteria that are often intended for adaptation to the host environment. Dormant mycobacteria should have evolved efficient maneuvers for cohabitation, allowing the microbes to persist for years within the host. Glycerol monomycolate (GroMM) has been implicated as a specific immune target in human individuals with latent, but not active, tuberculosis, but the in vivo response to GroMM and the relevance of it to latent infection remain poorly understood. Here, we immunized guinea pigs with bacillus Calmette-Guerin (BCG) expressing high levels of GroMM and then, monitored skin reactions at the site of challenge with GroMM-containing liposome. We found that BCG-immunized guinea pigs mounted enhanced skin reactions to GroMM with prominent local infiltration by eosinophils. Consistent with this, GroMM-stimulated lymph node cells upregulated the expression of T helper (Th)2-type cytokines, such as interleukin (IL)-5 and IL-10, that could potentially counteract the microbe-eliminating Th1-type cytokine response. On the basis of these observations, we predict that the host response to GroMM produced by dormant mycobacteria would contribute to their long-term survival in the host.
    Biochemical and Biophysical Research Communications 06/2011; 409(2):304-7. · 2.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Delayed-type hypersensitivity (DTH) is marked by high levels of protein antigen-specific T cell responses in sensitized individuals. Recent evidence has revealed a distinct pathway for T cell immunity directed against glycolipid antigens, but DTH to this class of antigen has been undetermined and difficult to prove due to their insolubility in aqueous solutions. Here, glucose monomycolate (GMM), a highly hydrophobic glycolipid of the cell wall of mycobacteria, was dispersed in aqueous solutions in the form of octaarginine-modified liposomes and tested for its ability to elicit cutaneous DTH responses in bacillus Calmette-Guerin (BCG)-immunized guinea pigs. After an intradermal challenge with the GMM liposome, a significant skin induration was observed in BCG-immunized, but not mock-treated, animals. The skin reaction peaked at around 2 days with local infiltration by mononuclear cells, and therefore, the response shared basic features with the classical DTH to protein antigens. Lymph node T cells from BCG-immunized guinea pigs specifically increased IFN-γ transcription in response to the GMM liposome, and this response was completely blocked by antibodies to CD1 lipid antigen-presenting molecules. Finally, whereas the T cells increased transcription of both T helper (Th) 1-type (IFN-γ and TNF-α) and Th2-type (IL-5 and IL-10) cytokines in response to the purified protein derivative or tuberculin, their GMM-specific response was skewed to Th1-type cytokine production known to be critical for protection against tuberculosis. Thus, our study reveals a novel form of DTH with medical implications.
    Journal of Biological Chemistry 03/2011; 286(19):16800-6. · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.
    Journal of bacteriology 11/2010; 192(21):5700-8. · 3.94 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-gamma) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-gamma, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-gamma transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.
    Microbiology 03/2010; 156(Pt 6):1836-47. · 3.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 'Lysobacter enzymogenes ssp. cookii' was proposed by Christensen and Cook in 1978; however, this subspecies name has not been cited in the Approved Lists of Bacterial Names and therefore the nomenclature has not been validated. In our genetic approach to clarify the relationships of the designated type strain of 'L. enzymogenes ssp. cookii' PAGU 1119 (GenBank accession number ATCC29488) within the genus Lysobacter revealed that the strain was closely related to Lysobacter capsiciYC5194(T) (99.4%) rather than L. enzymogenesDSM2043(T) (97.2%). The value for whole genome DNA-DNA relatedness between strain PAGU 1119 and L. enzymogenes DSM 2043(T) or L. capsiciYC5194(T) was 20.7-26.1% or 60.9-62.0%, respectively. Although PAGU 1119 and L. capsiciYC5194(T) showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different, and we concluded that PAGU 1119 should be placed in a new species. We therefore propose a new species with the name Lysobacter cookii sp. nov. The type strain is PAGU 1119(T) (ATCC29488).
    FEMS Microbiology Letters 10/2009; 298(1):118-23. · 2.05 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel, non-pigmented, slow-growing mycobacterium was identified on the basis of biochemical and nucleic acid analyses, as well as growth characteristics. Three isolates were cultured from clinical samples (two from sputum and one from pus in lymph nodes) obtained from three immunocompetent patients with infections. Bacterial growth occurred at 28-42 degrees C on Middlebrook 7H11-OADC agar. The isolates showed negative results for Tween hydrolysis, nitrate reductase, semiquantitative catalase, urease activity, 3 day arylsulfatase activity, pyrazinamidase, tellurite reduction and niacin accumulation tests, but positive results for 14 day arylsulfatase activity and heat-stable catalase tests. The isolates contained alpha-, keto-, and dicarboxymycolates in their cell walls. Sequence analysis revealed that all isolates had identical, unique 16S rRNA sequences. Phylogenetic analysis of the 16S rRNA, rpoB, hsp65 and sodA gene sequences confirmed that these isolates are unique but closely related to Mycobacterium celatum. DNA-DNA hybridization of the isolates demonstrated less than 50 % reassociation with M. celatum and Mycobacterium branderi. On the basis of these findings, a novel species designated Mycobacterium kyorinense sp. nov. is proposed. The type strain is KUM 060204(T) (=JCM 15038(T)=DSM 45166(T)).
    International journal of systematic and evolutionary microbiology 07/2009; 59(Pt 6):1336-41. · 2.11 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: During the serial passage of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. 'Early-shared strains' of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (alpha, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-gamma (IFN-gamma) than did 'late-shared strains' of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-alpha in the presence of IFN-gamma was also observed by trehalose 6,6'-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that 'early-shared strains' are more potent immunostimulating agents than 'late-shared strains', which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.
    FEMS Immunology & Medical Microbiology 05/2009; 56(2):116-28. · 2.68 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.
    Journal of bacteriology 06/2008; 190(10):3613-21. · 3.94 Impact Factor

Publication Stats

1k Citations
189.82 Total Impact Points

Institutions

  • 1997–2013
    • Osaka City University
      • • Department of Bacteriology
      • • Department of Host Defense
      Ōsaka, Ōsaka, Japan
  • 2011–2012
    • Kyoto University
      • Institute for Virus Research
      Kyoto, Kyoto-fu, Japan
  • 2009
    • Aichi Gakuin University
      • Department of Microbiology
      Nagoya-shi, Aichi-ken, Japan
  • 2008
    • Japan BCG Laboratory
      Edo, Tōkyō, Japan
  • 2007
    • National Institute of Infectious Diseases, Tokyo
      Edo, Tōkyō, Japan
  • 2006
    • KAKETSUKEN
      Kumamoto, Kumamoto Prefecture, Japan
  • 2003–2004
    • Gifu University
      • Graduate School of Medicine
      Gihu, Gifu, Japan
  • 2001
    • Kitasato University
      • Department of Biosciences
      Edo, Tōkyō, Japan
  • 2000
    • Gifu University Hospital
      Gihu, Gifu, Japan