Language：English   Publishing type：Research paper (scientific journal)  

Survival of the live attenuated Bacillus Calmette-Guérin (BCG) vaccine amidst harsh host environments is key for BCG effectiveness as it allows continuous immune response induction and protection against tuberculosis. Mycobacterial DNA binding protein 1 (MDP1), a nucleoid associated protein, is essential in BCG. However, there is limited knowledge on the extent of MDP1 gene regulation and how this influences BCG survival. Here, we demonstrate that MDP1 conditional knockdown (cKD) BCG grows slower than vector control in vitro, and dies faster upon exposure to antibiotics (bedaquiline) and oxidative stress (H2O2 and menadione). MDP1-cKD BCG also exhibited low infectivity and survival in THP-1 macrophages and mice indicating possible susceptibility to host mediated stress. Consequently, low in vivo survival resulted in reduced cytokine (IFN-gamma and TNF-alpha) production by splenocytes. Temporal transcriptome profiling showed more upregulated (81-240) than downregulated (5-175) genes in response to MDP1 suppression. Pathway analysis showed suppression of biosynthetic pathways that coincide with low in vitro growth. Notable was the deferential expression of genes involved in stress response (sigI), maintenance of DNA integrity (mutT1), REDOX balance (WhiB3), and host interactions (PE/PE_PGRS). Thus, this study shows MDP1's importance in BCG survival and highlights MDP1-dependent gene regulation suggesting its role in growth and stress adaptation.

DOI： 10.1038/s41598-023-40941-9

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<jats:title>Abstract</jats:title><jats:p>Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to <jats:italic>Mycobacterium tuberculosis</jats:italic> proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from <jats:italic>M. tuberculosis</jats:italic>. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from <jats:italic>Escherichia coli (E. coli)</jats:italic> couldn’t distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from <jats:italic>M. tuberculosis</jats:italic>. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734–0.890, <jats:italic>p</jats:italic> &lt; 0.0001) and 0.821 (95% CI 0.752–0.890, <jats:italic>p</jats:italic> &lt; 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB’s POCT by antibody-based diagnosis.</jats:p>

DOI： 10.1038/s41598-023-39436-4

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BACKGROUND: Mycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice. RESULTS: We classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs. CONCLUSIONS: The virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments.

DOI： 10.1186/s12866-023-02831-y

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INTRODUCTION: Controlling pulmonary Mycobacterium avium complex (MAC) disease is difficult because there is no way to know the clinical stage accurately. There have been few attempts to use cell-mediated immunity for diagnosing the stage. The objective of this study was to characterize cytokine profiles of CD4+T and CD19+B cells that recognize various Mycobacterium avium-associated antigens in different clinical stages of MAC. METHODS: A total of 47 MAC patients at different stages based on clinical information (14 before-treatment, 16 on-treatment, and 17 after-treatment) and 17 healthy controls were recruited. Peripheral blood mononuclear cells were cultured with specific antigens (MAV0968, 1160, 1276, and 4925), and the cytokine profiles (IFN-γ, TNF-α, IL-2, IL-10, IL-13, and IL-17) of CD4+/CD3+ and CD19+ cells were analyzed by flow cytometry. RESULTS: The response of Th1 cytokines such as IFN-γ and TNF-α against various antigens was significantly higher in both the on-treatment and after-treatment groups than in the before-treatment group and control (P < 0.01-0.0001 and P < 0.05-0.0001). An analysis of polyfunctional T cells suggested that the presence of IL-2 is closely related to the stage after the start of treatment (P = 0.0309-P < 0.0001) and is involved in memory function. Non-Th1 cytokines, such as IL-10 and IL-17, showed significantly higher responses in the before-treatment group (P < 0.0001 and P < 0.01-0.0001). These responses were not observed with purified protein derivative (PPD). CD19+B cells showed a response similar to that of CD4+T cells. CONCLUSION: There is a characteristic cytokine profile at each clinical stage of MAC.

DOI： 10.3389/fimmu.2023.1222428

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Tuberculosis (TB) is treated by chemotherapy with multiple anti-TB drugs for a long period, spanning 6 months even in a standard course. In perspective, to prevent the emergence of antimicrobial resistance, novel drugs that act synergistically or additively in combination with major anti-TB drugs and, if possible, shorten the duration of TB therapy are needed. However, their combinatorial effect cannot be predicted until the lead identification phase of the drug development. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool that enables high-throughput screening of novel drug targets. The development of anti-TB drugs promises to be accelerated by CRISPRi. This study determined whether CRISPRi could be applicable for predictive screening of the combinatorial effect between major anti-TB drugs and an inhibitor of a novel target. In the checkerboard assay, isoniazid killed Mycobacterium smegmatis synergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown of inhA, which encodes a target molecule of isoniazid. Additionally, knockdown of rpoB, which encodes a target molecule of rifampicin, increased the susceptibility to isoniazid and ethambutol, which act synergistically with rifampicin in the checkerboard assay. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A, a novel TB drug candidate, with isoniazid or rifampicin. These results demonstrate that CRISPRi is a useful tool not only for drug target exploration but also for screening the combinatorial effects of novel combinations of anti-TB drugs. This study provides a rationale for anti-TB drug development using CRISPRi.

DOI： 10.1099/mic.0.001285

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Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.

DOI： 10.1128/aac.00171-22

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Language：Japanese   Publisher：(一社)日本結核・非結核性抗酸菌症学会  

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Pathogenic mycobacteria, such as Mycobacterium tuberculosis , Mycobacterium avium subsp. hominissuis (MAH), and Mycobacterium intracellulare , cause pulmonary infections as intracellular parasites of lung macrophages and epithelial cells. Here, using histopathological examinations we found that MAH and M. intracellulare colocalized with erythrocytes in lung infection sites.

DOI： 10.1128/spectrum.02454-21

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Language：English   Publishing type：Research paper (scientific journal)  

Clinical characteristics and outcomes of multidrug chemotherapy have been used as the main prognostic factors for Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD) over the last decade; however, no useful prognostic biomarkers have been reported. The aim is to ascertain whether the serum antibody titers could include useful prognostic predictors of MAC-PD. Ninety-four patients with MAC-PD were enrolled and regularly followed up with for more than 5 years or until death. Cox proportional hazard regression and receiver operating characteristic (ROC) curve analyses were used to identify predictors of mortality in this prospective observational study. According to treatment outcomes, 85 patients completed follow-up and were classified into four groups. Seventeen patients (20%) died during follow-up (median, 10.1 years; interquartile range, 8.1 to 12.4 years). All 11 patients with MAC-PD-specific death were included in the 14 patients of the group nonresponsive to the multidrug chemotherapy. They had significantly higher anti-Mycobacterium glycolipid (MBGL) antibody titers than those in the other groups and a significantly (P < 0.0001) poorer survival prognosis. The anti-MBGL antibody titers also served as a negative prognostic factor. A cutoff score of 7, which was calculated by clinical poor prognostic characteristics and anti-MBGL antibody titers, differentiated the nonresponse group and the other groups at baseline (sensitivity, specificity, and area under the curve: 92.9%, 81.7%, and 0.95, respectively). In conclusion, anti-MBGL antibody titers were useful to assess the refractory MAC-PD. The predictions of treatment outcome and mortality become more accurate by using anti-MBGL antibody and clinical poor prognostic characteristics together. IMPORTANCE The natural history of MAC-PD is challenging to predict in immunocompetent patients at diagnosis, and the current multidrug chemotherapy options are not strong enough to eliminate mycobacteria from the lungs. Therefore, the diagnosis of MAC-PD does not necessarily lead to the decision to start chemotherapy. We have also observed refractory patients in clinical practice, who were resistant to multiple-drug chemotherapy and showed persistent excretion of MAC bacilli and progressive worsening of chest radiographic findings until death. We have reported that the measurements of anti-MBGL antibody titers helped assess refractory MAC-PD in this study. Furthermore, the predictions of treatment outcome and mortality become more accurate by using the anti-MBGL antibody in addition to clinical poor prognostic characteristics, which were older age, lower body mass index, the positive results of a smear test for acid-fast bacteria (AFB), and presence of cavitary disease.

DOI： 10.1128/spectrum.00530-22

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Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.

DOI： 10.1038/s41598-022-08228-7

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/febs.16412

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.16412

Language：English   Publisher：日本細菌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1β production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1β production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1β production during mycobacterial infection.

DOI： 10.1096/fj.202101074rr

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.202101074RR

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>DNA is basically an intracellular molecule that stores genetic information and carries instructions for growth and reproduction in all cellular organisms. However, in some bacteria, DNA has additional roles outside the cells as extracellular DNA (eDNA), which is an essential component of biofilm formation and hence antibiotic tolerance. Mycobacteria include life-threating human pathogens, most of which are slow growers. However, little is known about the nature of pathogenic mycobacteria’s eDNA. Here we found that eDNA is present in slow-growing mycobacterial pathogens, such as <italic>Mycobacterium tuberculosis</italic>, <italic>M. intracellulare</italic>, and <italic>M. avium</italic> at exponential growth phase. In contrast, eDNA is little in all tested rapid-growing mycobacteria. The physiological impact of disrupted eDNA on slow-growing mycobacteria include reduced pellicle formation, floating biofilm, and enhanced susceptibility to isoniazid and amikacin. Isolation and sequencing of eDNA revealed that it is identical to the genomic DNA in <italic>M. tuberculosis</italic> and <italic>M. intracellulare</italic>. In contrast, accumulation of phage DNA in eDNA of <italic>M. avium</italic>, suggests that the DNA released differs among mycobacterial species. Our data show important functions of eDNA necessary for biofilm formation and drug tolerance in slow-growing mycobacteria.

DOI： 10.1038/s41598-021-90156-z

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Other Link： http://www.nature.com/articles/s41598-021-90156-z

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title><sec>
<title>Background</title>
<italic>Mycobacterium intracellulare</italic> is a representative etiological agent of emerging pulmonary <italic>M. avium-intracellulare</italic> complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary <italic>M. avium-intracellulare</italic> complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for <italic>M. avium</italic>; however, the genomic characteristics of <italic>M. intracellulare</italic> remain to be elucidated.


</sec><sec>
<title>Results</title>
In this study, we performed comparative genomic analysis of 55 <italic>M. intracellulare</italic> and related strains such as <italic>M. paraintracellulare</italic> (MP), <italic>M. indicus pranii</italic> (MIP) and <italic>M. yonogonense</italic>. Based on the average nucleotide identity, the clinical <italic>M. intracellulare</italic> strains were phylogenetically grouped in two clusters: (1) the typical <italic>M. intracellulare</italic> (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., <italic>fadE3</italic>, <italic>fadE33</italic>), transporters (e.g., <italic>mce3</italic>), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. <italic>M. intracellulare</italic> was shown to be pan-genomic at the species and subspecies levels. The <italic>mce</italic> genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies.


</sec><sec>
<title>Conclusions</title>
Our data suggest that genomic diversity among <italic>M. intracellulare</italic>, <italic>M. paraintracellulare</italic>, <italic>M. indicus pranii</italic> and <italic>M. yonogonense</italic> remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as <italic>mce</italic> genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for <italic>M. intracellulare</italic> and related strains.


</sec>

DOI： 10.1186/s12866-021-02163-9

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Other Link： http://link.springer.com/article/10.1186/s12866-021-02163-9/fulltext.html

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Tuberculosis is a major threat to global health and its increased incidence in adolescents as well as onset in the elderly presents a serious problem. One strategy to control tuberculosis involves taking advantage of Bacillus Calmette-Guérin's (BCG) superior effects on childhood tuberculosis. Accordingly, here we aimed to develop a booster vaccine for adults who received the BCG vaccine during early childhood. Therefore, we first devised a system to assess the efficacy of a candidate booster vaccine. Specifically, variant strain BCG-II, a minor component of BCG-Tokyo strain, which elicits weak immunity, was administered to guinea pigs. Vaccine-induced immunity and protection against Mycobacterium tuberculosis (Mtb) infection were evaluated using skin delayed-type hypersensitivity (DTH) and Mtb colony forming unit counts in organs, respectively. Candidate booster vaccine containing the mycobacterial DNA-binding protein 1 (MDP1) as antigen and CpG oligodeoxynucleotide G9.1 as adjuvant increased T-bet expression and IFN-γ production in human peripheral blood mononuclear cells. Intradermal administration of MDP1 or MDP1 and G9.1 to unimmunized guinea pigs produced DTH on MDP1-inoculated skin. Boosting BCG-II-primed guinea pigs with this protocol effectively enhanced DTH against MDP1 and protection against Mtb infection, particularly when combined with G9.1. The candidate vaccine may contribute to efforts to prevent tuberculosis.

DOI： 10.1016/j.tube.2021.102067

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Language：Japanese   Publisher：(一社)日本結核・非結核性抗酸菌症学会  

[目的]全ゲノム解析を用いて,早期発症者と長期潜伏後発症者から分離した結核菌北京株のゲノム変異を解析した。[方法]1999年に中学校で発生した結核集団感染の接触者と二次感染者のうち2009年までに結核を発症した患者より分離した結核菌北京株6株と,別の事例で初発時と再発時の患者より分離した結核菌北京株2株の計8株の全ゲノム解析を行った。結核菌の突然変異率は,初発から1年以内に発症した早期発症者と1年以上の潜伏期を経てから発症または再燃した長期潜伏後発症者の2群間で比較した。[結果]結核菌北京株の突然変異率は,Lineage 4に属する結核菌よりも高く,結核菌北京株の高い病原性や薬剤耐性の要因である可能性が示唆された。遺伝子多型解析では,酸化的損傷に起因すると推定されている突然変異が長期潜伏後発症群のほうに多く,潜伏期間中にも薬剤耐性変異が起こる可能性が示唆された。[結論]結核菌北京株の高頻度の薬剤耐性化を防ぐためには,感染した結核菌の系統により治療法を検討する必要性も考えられた。今後,結核菌系統の特性を理解し,治療法を工夫することで結核の薬剤耐性化や重篤化を防ぐ有効な対策の構築につながることが期待される。(著者抄録)

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Mycobacterium avium complex (MAC) is the most common non-tuberculous mycobacterium (NTM) and causes different types of pulmonary diseases. While genomic and transcriptomic analysis of Mycobacterium avium 104 (M. avium 104) has been extensive, little is known about the proteomics of M. avium 104. We utilized proteomics technology to analyze the changes in the whole proteome of M. avium 104 during exponential and stationary growth phases. We found 12 dys-regulated proteins; the up-regulated protein hits in the stationary phase were involved in aminopeptidase, choline dehydrogenase, oxidoreductase, and ATP binding, while the down-regulated proteins in the stationary phase were acetyl-CoA acetyltransferase, universal stress protein, catalase peroxidase, and elongation factor (Tu). The differently expressed proteins between exponential and stationary phases were implicated in metabolism and stress response, pointing to the functional adaptation of the cells to the environment. Proteomic analysis in different growth phases could participate in understanding the course of infection, the mechanisms of virulence, the means of survival, and the possible targets for treatment.

DOI： 10.3390/molecules26020305

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Language：English  

[This corrects the article DOI: 10.1371/journal.pone.0204160.].

DOI： 10.1371/journal.pone.0256946

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>The global incidence of the human nontuberculous mycobacteria (NTM) disease is rapidly increasing. However, knowledge of gene essentiality under optimal growth conditions and conditions relevant to the natural ecology of NTM, such as hypoxia, is lacking. In this study, we utilized transposon sequencing to comprehensively identify genes essential for growth in <italic>Mycobacterium intracellulare</italic>. Of 5126 genes of <italic>M. intracellulare</italic> ATCC13950, 506 genes were identified as essential genes, of which 280 and 158 genes were shared with essential genes of <italic>M. tuberculosis</italic> and <italic>M. marinum</italic>, respectively. The shared genes included target genes of existing antituberculous drugs including SQ109, which targets the trehalose monomycolate transporter MmpL3. From 175 genes showing decreased fitness as conditionally essential under hypoxia, preferential carbohydrate metabolism including gluconeogenesis, glyoxylate cycle and succinate production was suggested under hypoxia. Virulence-associated genes including proteasome system and mycothiol redox system were also identified as conditionally essential under hypoxia, which was further supported by the higher effective suppression of bacterial growth under hypoxia compared to aerobic conditions in the presence of these inhibitors. This study has comprehensively identified functions essential for growth of <italic>M. intracellulare</italic> under conditions relevant to the host environment. These findings provide critical functional genomic information for drug discovery.

DOI： 10.1038/s41598-020-62287-2

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Other Link： http://www.nature.com/articles/s41598-020-62287-2

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>
<italic>Mycobacterium tuberculosis</italic> (<italic>Mtb</italic>) strains of Beijing lineage have caused great concern because of their rapid emergence of drug resistance and worldwide spread. DNA mutation rates that reflect evolutional adaptation to host responses and the appearance of drug resistance have not been elucidated in human-infected Beijing strains. We tracked and obtained an original <italic>Mtb</italic> isolate of Beijing lineage from the 1999 tuberculosis outbreak in Japan, as well as five other isolates that spread in humans, and two isolates from the patient caused recurrence. Three isolates were from patients who developed TB within one year after infection (rapid-progressor, RP), and the other three isolates were from those who developed TB more than one year after infection (slow-progressor, SP). We sequenced genomes of these isolates and analyzed the propensity and rate of genomic mutations. Generation time versus mutation rate curves were significantly higher for RP. The ratio of oxidative versus non-oxidation damages induced mutations was higher in SP than RP, suggesting that persistent <italic>Mtb</italic> are exposed to oxidative stress in the latent state. Our data thus demonstrates that higher mutation rates of <italic>Mtb</italic> Beijing strains during human infection is likely to account for the higher adaptability and an emergence ratio of drug resistance.

DOI： 10.1038/s41598-020-75028-2

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Other Link： http://www.nature.com/articles/s41598-020-75028-2

Language：Japanese   Publisher：(一社)日本結核・非結核性抗酸菌症学会  

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Language：Japanese   Publisher：(有)科学評論社  

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Language：Japanese   Publisher：日本バイオフィルム学会  

環境から感染する肺非結核性抗酸菌症は世界中で著しく増加しているため公衆衛生上の解決すべき課題の一つである。私たちは主な起因菌であるMycobacterium avium subsp.hominissuis(MAH)は低酸素環境にするとバイオフィルム形成が促進されることを見出した。一方で類縁菌の結核菌は酸素欠乏で休眠する。結核菌は二成分制御系のDosS、DosTが酸素濃度の低下を感知し、DosRが休眠に必要なタンパク質の発現調節を行なっている。MAHも結核菌と同様に二成分制御系が働いているとの仮説を立て、トランスクリプトーム解析を行って検証した。MAHを大気条件下または5%酸素条件下で培養後RNAを抽出し、RNAseq解析を行った。その結果、141/109遺伝子の有意な発現増加/低下が認められた。MAH 104は17セットの2成分制御系がゲノム上認められる。このうち結核菌のDosS/DosRオルソログに相当する遺伝子(MAV_RS19700/MAV_RS19705)は変化しなかった。結核菌にはないセンサーヒスチジンキナーゼ遺伝子(MAV_RS11960)の発現が増加した。この遺伝子は低酸素を感知するPASドメイン構造を有し、DosSと相同性の高いヒスチジンキナーゼを有していることから、有力な候補遺伝子であると思われた。次に、低酸素環境に適応する共通の変化があるのではないかと考え、MAH発現増加遺伝子中にDosRレギュロンのオルソログがないか検索したところ、10遺伝子が該当した。これらの遺伝子は、低酸素環境に適応して生存を維持するために必要な遺伝子であると推察された。(著者抄録)

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F
₄₂₀
)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated
<named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
var.

DOI： 10.1128/aac.01755-19

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

<title>ABSTRACT</title>
Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F₄₂₀)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (<italic>ndh</italic>) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type <italic>ndh</italic>. Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.

DOI： 10.1128/aac.01755-19

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ymthe.2019.09.023

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Nutritional status contributes to the regulation of immune responses against pathogens, and malnutrition has been considered as a risk factor for tuberculosis (TB). Mycobacterium tuberculosis (Mtb), the causative agent of TB, can modulate host lipid metabolism and induce lipid accumulation in macrophages, where the bacilli adopt a dormant phenotype. In addition, serum lipid components play dual roles in the regulation of and protection from Mtb infection. We analyzed the relationship between nutritional status and the humoral immune response in TB patients. We found that serum HDL levels are positively correlated with the serum IgA specific for Mtb antigens. Analysis of the relationship between serum nutritional parameters and clinical parameters in TB patients showed that serum albumin and CRP levels were negatively correlated before treatment. We also observed reduced serum LDL levels in TB patients following treatment. These findings may provide insight into the role of serum lipids in host immune responses against Mtb infection. Furthermore, improving the nutritional status may enhance vaccination efficacy.

DOI： 10.1371/journal.pone.0237062

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DOI： 10.1016/j.bjid.2019.07.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Macrophages are major components of tuberculosis (TB) granulomas and are responsible for host defenses against the intracellular pathogen, Mycobacterium tuberculosis. We herein showed the strong expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in TB granulomas and more rapid death of HIF-1 alpha-conditional knockout mice than wild-type (WT) mice after M. tuberculosis infection. Although interferon-gamma (IFN-gamma) is a critical host-protective cytokine against intracellular pathogens, HIF-1-deficient macrophages permitted M. tuberculosis growth even after activation with IFN-gamma. These results prompted us to investigate the role of HIF-1 alpha in host defenses against infection. We found that the expression of lactate dehydrogenase-A (LDH-A) was controlled by HIF-1 alpha in M. tuberculosisinfected macrophages IFN-gamma independently. LDH-A is an enzyme that converts pyruvate to lactate and we found that the intracellular level of pyruvate in HIF-1 alpha-deficient bone marrow-derived macrophages (BMDMs) was significantly higher than in WT BMDMs. Intracellular bacillus replication was enhanced by an increase in intracellular pyruvate concentrations, which were decreased by LDH-A. Mycobacteria in phagosomes took up exogenous pyruvate more efficiently than glucose, and used it as the feasible carbon source for intracellular growth. These results demonstrate that HIF-1 alpha prevents the hijacking of pyruvate in macrophages, making it a fundamental host-protective mechanism against M. tuberculosis.

DOI： 10.1093/intimm/dxz048

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

肺非結核性抗酸菌症は近年世界中で著しく増加している。日本など先進国において増加率が高く、環境から感染することから公衆衛生上の重要課題になっている。なかでも患者の多いMycobacterium avium subsp.hominissuis(MAH)がシャワーヘッドや浴槽内注水口に生息していることを報告し、その浴室ではバイオフィルムを形成していることを見出した。罹患率の増加を阻止するために、浴室など感染源のバイオフィルムを除去する必要がある。私たちは次に除去方法の標的を見出すためにMAHのバイオフィルム形成機構解明に取り組んだ。その結果気液界面の菌膜形成がバイオフィルムの一形態であり、低酸素(5%O2)環境が形成促進因子であることを見出した。この事実は、MAHが低酸素を感知するセンサーとその感知を菌膜形成につなげる遺伝子発現変化が起きていることを示唆する。そこで、遺伝子発現変化を解析する目的でRNAseq法を行った。ゲノム配列がわかっている3株(MAH 104、MAH OCU464、MAH OCU873)を用いて低酸素条件で培養して得られたmRNA発現量を通常の培養条件で得られたmRNA発現量と比較した。その結果、有意に発現量が変化した遺伝子は250、うち141遺伝子が増加、109遺伝子が減少した。このうちセンサー機能を持つDevSホモログ(RS11960)が増加していた。今後、このセンサー候補が実際にセンサーとして働くことを検証し、レスポンスレギュレーターやレギュロンを明らかにしたい。(著者抄録)

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One-third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single-center prospective observational study, we analyzed IgG-antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA-binding protein 1 (MDP1) and alpha-crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18-month follow-up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.

DOI： 10.1111/1348-0421.12674

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Language：English   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Mycobacterium tuberculosis (M. tuberculosis) produces a diverse range of antigenic proteins in its dormant phase. The cytokine profiles of CD4+ T cell responses, especially subsets other than Th1 type (non-Th1 type), against these latency-associated M. tuberculosis antigens such as α-crystallin (Acr), heparin-binding hemagglutinin (HBHA), and mycobacterial DNA-binding protein 1 (MDP-1) remain elusive in relation to the clinical stage of M. tuberculosis infection. In the present study, peripheral blood mononuclear cells (PBMCs) collected from different stages of M. tuberculosis-infected cases and control PBMCs were stimulated with these antigens and ESAT-6/CFP-10. Cytokine profiles of CD4+ T cells were evaluated by intracellular cytokine staining using multicolor flow cytometry. Our results demonstrate that Th1 cytokine responses were predominant after TB onset independent of the type of antigen stimulation. On the contrary, non-Th1 cytokine responses were preferentially induced by latency-associated M. tuberculosis antigens, specifically IL-10 response against Acr in latent M. tuberculosis infection. From these results, we surmise a shift in the CD4+ T cell response from mixed non-Th1 to Th1 dominant type during TB progression.

DOI： 10.3389/fimmu.2019.02807

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

The architecture of the genome influences the functions of DNA from bacteria to eukaryotes. Intrinsically disordered regions (IDR) of eukaryotic histones have pivotal roles in various processes of gene expression. IDR is rare in bacteria, but interestingly, mycobacteria produce a unique histone-like protein, MDP1 that contains a long C-terminal IDR. Here we analyzed the role of IDR in MDP1 function. By employing Mycobacterium smegmatis that inducibly expresses MDP1 or its IDR-deficient mutant, we observed that MDP1 induces IDR-dependent DNA compaction. MDP1-IDR is also responsible for the induction of growth arrest and tolerance to isoniazid, a front line tuberculosis drug that kills growing but not growth-retardated mycobacteria. We demonstrated that MDP1-deficiency and conditional knock out of MDP1 cause spreading of the M. smegmatis genome in the stationary phase. This study thus demonstrates for the first time a C-terminal region-dependent organization of the genome architecture by MDP1, implying the significance of IDR in the function of bacterial histone-like protein.

DOI： 10.1038/s41598-018-26463-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

DOI： 10.1038/s41598-018-24233-1

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A novel tuberculosis vaccine to replace BCG has long been desired. However, recent vaccine trials focused on cell-mediated immunity have failed to produce promising results. It is worth noting that most commercially available successful vaccines rely on humoral immunity. To establish a basic understanding of humoral immunity against tuberculosis, we analyzed and evaluated longitudinal levels and avidity of immunoglobulin to various tuberculosis antigens compared with bacterial and clinical parameters during treatment. We found that levels of IgG antibodies against HrpA and HBHA prior to treatment exhibited a positive correlation with bacterial burden. Analysis of changes in CRP during treatment revealed an association with high levels of specific IgG and IgA antibodies against mycobacterial antigens. Levels of CRP prior to treatment were negatively associated with IgG avidity to CFP-10 and MDP1 and IgA avidity to HrpA, while IgA avidity to MDP1 and Acr exhibited a negative correlation with CRP levels after 60 days of treatment. These results may provide insight for the development of a novel tuberculosis (TB) vaccine candidate to induce protective humoral immunity against tuberculosis.

DOI： 10.1155/2018/4928757

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Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5-10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis.

DOI： 10.1371/journal.pone.0204160

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

Background: Large-scale schistosomiasis control programs are implemented in regions with diverse social and economic environments. A key epidemiological feature of schistosomiasis is its small-scale heterogeneity. Locally profiling disease dynamics including risk factors associated with its transmission is essential for designing appropriate control programs. To determine spatial distribution of schistosomiasis and its drivers, we examined schoolchildren in Kwale, Kenya. Methodology/Principal findings: We conducted a cross-sectional study of 368 schoolchildren from six primary schools. Soil-transmitted helminths and Schistosoma mansoni eggs in stool were evaluated by the Kato-Katz method. We measured the intensity of Schistosoma haematobium infection by urine filtration. The geometrical mean intensity of S. haematobium was 3.1 eggs/10 ml urine (school range, 1.4–9.2). The hookworm geometric mean intensity was 3.2 eggs/g feces (school range, 0–17.4). Heterogeneity in the intensity of S. haematobium and hookworm infections was evident in the study area. To identify factors associated with the intensity of helminth infections, we utilized negative binomial generalized linear mixed models. The intensity of S. haematobium infection was associated with religion and socioeconomic status (SES), while that of hookworm infection was related to SES, sex, distance to river and history of anthelmintic treatment. Conclusions/Significance: Both S. haematobium and hookworm infections showed micro-geographical heterogeneities in this Kwale community. To confirm and explain our observation of high S. haematobium risk among Muslims, further extensive investigations are necessary. The observed small scale clustering of the S. haematobium and hookworm infections might imply less uniform strategies even at finer scale for efficient utilization of limited resources.

DOI： 10.1371/journal.pntd.0005872

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background
Large-scale schistosomiasis control programs are implemented in regions with diverse social and economic environments. A key epidemiological feature of schistosomiasis is its small-scale heterogeneity. Locally profiling disease dynamics including risk factors associated with its transmission is essential for designing appropriate control programs. To determine spatial distribution of schistosomiasis and its drivers, we examined schoolchildren in Kwale, Kenya.
Methodology/Principal findings
We conducted a cross-sectional study of 368 schoolchildren from six primary schools. Soiltransmitted helminths and Schistosoma mansoni eggs in stool were evaluated by the KatoKatz method. We measured the intensity of Schistosoma haematobium infection by urine filtration. The geometrical mean intensity of S. haematobium was 3.1 eggs/10 ml urine (school range, 1.4-9.2). The hookworm geometric mean intensity was 3.2 eggs/g feces (school range, 0-17.4). Heterogeneity in the intensity of S. haematobium and hookworm infections was evident in the study area. To identify factors associated with the intensity of helminth infections, we utilized negative binomial generalized linear mixed models. The intensity of S. haematobium infection was associated with religion and socioeconomic status (SES), while that of hookworm infection was related to SES, sex, distance to river and history of anthelmintic treatment.
Conclusions/Significance
Both S. haematobium and hookworm infections showed micro-geographical heterogeneities in this Kwale community. To confirm and explain our observation of high S. haematobium risk among Muslims, further extensive investigations are necessary. The observed small scale clustering of the S. haematobium and hookworm infections might imply less uniform strategies even at finer scale for efficient utilization of limited resources.

DOI： 10.1371/journal.pntd.000587

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD(+) ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.

DOI： 10.1038/s41598-017-06480-w

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Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of nontuberculous mycobacteriosis, the representative case of environment-related infectious diseases the incidence of which is increasing in industrialized countries. MAH is found in biofilm in drinking water distribution system and residential environments. We investigated the effect of gaseous and nutritional conditions, and the role of glycopeptidolipids (GPLs) on biofilm-like pellicle formation in MAH. Pellicle formation was observed under 5% oxygen in Middlebrook 7H9 broth containing 0.2% glycerol and 10% albumindextrose- catalase enrichment but not under normoxia or in nutrient- poor media. An analysis of 17 environmental isolates revealed that hypoxia (5% oxygen) preferentially enhanced pellicle formation both in plastic plates and in glass tubes, compared with hypercapnia (5% carbon dioxide). Wild-type strains (WT) developed much thicker pellicles than GPL-deficient rough mutants (RM). WT bacterial cells distributed randomly and individually in contrast to that RM cells positioned linearly in a definite order. Exogenous supplementation of GPLs thickened the pellicles of RM, resulting in a similar morphological pattern to WT. These data suggest a significant implication of eutrophication and hypoxia in biofilm-like pellicle formation, and a functional role of GPLs on development of pellicles in MAH.

DOI： 10.1038/srep41775

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The detailed epidemiology of meningococcal diseases in Japan has yet to be determined and, moreover, the healthy carriage rate is also unknown. In this study, to obtain insight into the carriage rate of Neisseria meningitidis in healthy individuals in Japan, we developed a new method to detect the N. meningitidisspecific ctrB gene, one of the genes encoding enzymes for capsule synthesis, by Loop-Mediated Isothermal Amplification (LAMP) and examined the meningococcal carriage rate by using self collected oral throat wash specimens from 836 students at a university. Examination by LAMP showed that 7 out of 836 samples were positive for N. meningitidis DNA, and the results were also verified by the nested PCR method for the meningococcus specific ggt gene. The N. meningitidis carriage rate in healthy individuals was estimated to be 0.84%. Moreover, we further confirmed by the nested-PCR-based serogroup typing method that 5 of the positive samples belonged to serogroup Y, 1 belonged to group B and 1 was unidentifiable. Considering the epidemiology for meningococcal diseases in Japan, the carriage rate and the serogroup profile seem to be consistent with low incidence of meningococcal diseases and serogroup distribution of clinical meningococcal isolates in Japan, respectively. (C) 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jiac.2015.12.016

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Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multidrug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb)grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase- expressing recombinant Mycobacterium bovis bacillus Calmette-Gueren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 mu g/ml, 0.5 mu g/ml, and 2.0-7.5 mu g/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.

DOI： 10.1371/journal.pone.0141658

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Although tuberculosis remains a major global health problem, Bacille Calmette-Guerin (BCG) is the only available vaccine. However, BCG has limited applications, and a more effective vaccine is needed. Cellular mediated immunity (CMI) is thought to be the most important immune response for protection against Mycobacterium tuberculosis (Mtb). However, the recent failure of a clinical trial for a booster BCGvaccine and increasing evidence of antibody-mediated immunity prompted us to evaluate humoral immunity to Mtb-specific antigens. Using Enzyme-Linked Immuno Spot and Enzyme-Linked ImmunoSorbentAssays, we observed less correlation of both CMI and IgG titers with patient clinical status, including serumconcentration of C reactive protein. However, IgA titers against Mtb were significantly correlated with clinical status, suggesting that specific IgA antibodies protect against Mtb proliferation. In addition, in some cases, IgA antibody titers were significantly associated with the serum concentration of total albumin, which supports the idea that humoral immunity can be influenced by the nutritional status. Based on these observations, we propose that the induction of humoral immunity should be included as an option in TB vaccine development strategies.

DOI： 10.1155/2015/527395

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We report here the first draft genome sequence of Mycobacterium kyorinense, which was described in 2009 and exhibits significant pathogenicity to humans.

DOI： 10.1128/genomeA.01062-14

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Bacterial resistance to antibiotics has become a worldwide problem. One potential alternative for bacterial control is photodynamic therapy. 5-aminolevulinic acid is a natural precursor of the photosensitizer protoporphyrin IX. Relatively little is known about the antibacterial efficacy of photodynamic therapy using the systemic administration of 5-aminolevulinic acid; a few reports have shown that 5-aminolevulinic acid exerts photodynamic effects on methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In this study, we evaluated the effectiveness of photodynamic therapy using 5-aminolevulinic acid and a 410-nm wavelength light-emitting diode in vitro and in vivo for the treatment of MRSA. We found that 5-aminolevulinic acid photodynamic therapy with the light-emitting diode had an in-vitro bactericidal effect on MRSA. In vivo, protoporphyrin IX successfully accumulated in MRSA on ulcer surfaces after intraperitoneal administration of 5-aminolevulinic acid to mice. Furthermore, 5-aminolevulinic acid photodynamic therapy accelerated wound healing and decreased bacterial counts on ulcer surfaces; in contrast, vancomycin treatment did not accelerate wound healing. Our findings indicate that 5-aminolevulinic acid photodynamic therapy may be a new treatment option for MRSA-infected wounds.

DOI： 10.1371/journal.pone.0105173

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DOI： 10.1371/journal.pntd.0003040

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Background: An increasing risk of Schistosoma mansoni infection has been observed around Lake Victoria, western Kenya since the 1970s. Understanding local transmission dynamics of schistosomiasis is crucial in curtailing increased risk of infection.
Methodology/Principal Findings: We carried out a cross sectional study on a population of 310 children from eight primary schools. Overall, a total of 238 (76.8%) children were infected with S. mansoni, while seven (2.3%) had S. haematobium. The prevalence of hookworm, Trichuris trichiura and Ascaris lumbricoides were 6.1%, 5.2% and 2.3%, respectively. Plasmodium falciparum was the only malaria parasite detected (12.0%). High local population density within a 1 km radius around houses was identified as a major independent risk factor of S. mansoni infection. A spatial cluster of high infection risk was detected around the Mbita causeway following adjustment for population density and other potential risk factors.
Conclusions/Significance: Population density was shown to be a major factor fuelling schistosome infection while individual socio-economic factors appeared not to affect the infection risk. The high-risk cluster around the Mbita causeway may be explained by the construction of an artificial pathway that may cause increased numbers of S. mansoni host snails through obstruction of the waterway. This construction may have, therefore, a significant negative impact on the health of the local population, especially school-aged children who frequently come in contact with lake water.

DOI： 10.1371/journal.pntd.0002991

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We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA (R) elute card,DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

DOI： 10.2166/wh.2013.007

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Background:An increasing risk of Schistosoma mansoni infection has been observed around Lake Victoria, western Kenya since the 1970s. Understanding local transmission dynamics of schistosomiasis is crucial in curtailing increased risk of infection.Methodology/Principal Findings:We carried out a cross sectional study on a population of 310 children from eight primary schools. Overall, a total of 238 (76.8%) children were infected with S. mansoni, while seven (2.3%) had S. haematobium. The prevalence of hookworm, Trichuris trichiura and Ascaris lumbricoides were 6.1%, 5.2% and 2.3%, respectively. Plasmodium falciparum was the only malaria parasite detected (12.0%). High local population density within a 1 km radius around houses was identified as a major independent risk factor of S. mansoni infection. A spatial cluster of high infection risk was detected around the Mbita causeway following adjustment for population density and other potential risk factors.Conclusions/Significance:Population density was shown to be a major factor fuelling schistosome infection while individual socio-economic factors appeared not to affect the infection risk. The high-risk cluster around the Mbita causeway may be explained by the construction of an artificial pathway that may cause increased numbers of S. mansoni host snails through obstruction of the waterway. This construction may have, therefore, a significant negative impact on the health of the local population, especially school-aged children who frequently come in contact with lake water. © 2014 Nagi et al.

DOI： 10.1371/journal.pntd.0002991

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BACKGROUND: A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya. METHODS: We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized. FINDINGS: Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively. INTERPRETATION: A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.

DOI： 10.1371/journal.pntd.0003040

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.5025/hansen.82.119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EUROPEAN RESPIRATORY SOC JOURNALS LTD  

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Background: Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccine, which has been inoculated to more than one billion people world-wide, has significant effect in preventing tuberculous meningitis and miliary tuberculosis (TB) in neonate and early childhood. However, BCG fails to adequately protect against pulmonary TB and reactivation of latent infections in adults. To overcome this problem, adequate booster is urgently desired in adult who received prior BCG vaccination, and appropriate animal models that substitute human cases would be highly valuable for further experimentation.
Findings: The booster effect of the synthesized CpG oligomer (Oligo-B) on aged mice which had been primarily vaccinated with BCG at the age of 4-week old. The specific Th1 type reaction, production of interferon-gamma, in response to TB antigens, purified protein derivatives (PPD) and protection against challenge with Mycobacterium tuberculosis (MTB) H(37)Rv decreased with increasing age and were not observed in 89-week old mice. In order to rejuvenate the Th1 type response against PPD and protection activity against MTB infection, Oligo-B, which is known to augment Th1 responses, was administered as a booster to 81-90-week old mice (late 50's in human equivalent) vaccinated with BCG at 4-week old. The boosting with Oligo-B increased the number of CD4(+)CD44(high) CD62L(high), central memory type T cell. Furthermore, the Oligo-B boosting rejuvenated the ability of mice to protect against infection with MTB H(37)Rv.
Conclusions: Th1-adjuvant CpG oligo DNA, such as Oligo-B, may be a promising booster when coupled with BCG priming.

DOI： 10.1186/1742-4933-10-25

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Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to beta-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to beta-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis.
IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

DOI： 10.1128/mBio.00472-12

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Development of accurate methods for predicting progression of tuberculosis (TB) from the latent state is recognized as vitally important in controlling TB, because a majority of cases develop from latent infections. Past TB that has never been treated has a higher risk of progressing than does latent Mycobacterium tuberculosis infection in patients who have previously received treatment. Antibody responses against 23 kinds of M. tuberculosis proteins in individuals with past TB who had not been medicated were evaluated. These individuals had significantly higher concentrations of antibodies against Antigen 85A and mycobacterial DNA-binding protein 1 (MDP1) than did those with active TB and uninfected controls. In addition, immunohistochemistry revealed colocalization of tubercle bacilli, antigen 85 and MDP1 inside tuberculous granuloma lesions in an asymptomatic subject, showing that M. tuberculosis in lesions expresses both antigen 85 and MDP1. Our study suggests the potential usefulness of measuring antibody responses to antigen 85A and MDP1 for assessing the risk of TB progression.

DOI： 10.1111/j.1348-0421.2012.12005.x

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Language：English   Publisher：Blackwell Publishing Asia Pty  

DOI： 10.1111/j.1348-0421.2012.12005.x

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Other Link： http://search.jamas.or.jp/link/ui/2013358036

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Although IFN-γ release assays (IγRAs) provide increased specificity over tuberculin skin tests, the early and sensitive detection of reactivation of latently infected Mycobacterium tuberculosis is required to control tuberculosis (TB). Recently, a multicolor flow cytometry has been developed to study CD4+ T cell cytokine responses (IFN-γ/IL-2/TNF-a) to purified protein derivatives (PPD) and M. tuberculosis-specific antigens (ESAT-6/CFP-10) and provided useful information regarding anti-TB immunity. However, the diagnostic relevancy remains uncertain. Here, we analyzed three additional CD4+ T cell cytokine responses (IL-10/IL-13/IL-17) to latent mycobacterial antigens (a-crystallin, methylated heparin-binding hemagglutinin [HBHA], and mycobacterial DNA-binding protein 1 [MDP-1]) as well as PPD and ESAT-6/CFP-10 in 12 IGRA+ TB cases and 8 healthy controls. No significant difference in IFN-γ response was observed between TB cases and controls, which was likely due to the high variation among the individuals. However, we found a significant increase over healthy controls in (i) the IL-2 response to HBHA in recovery stage TB cases, (ii) the number of M. tuberculosisspecific polyfunctional CD4+ T cells in on-treatment and recovery stage cases, and (iii) the IL-17 response to HBHA and MDP-1 in on-treatment and recovery stage cases. These results suggest that a combination of these T cell cytokine parameters could aid in accurate diagnosis of latent TB infection.

DOI： 10.7883/yoken.66.207

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

We report the draft genome sequence of Mycobacterium tuberculosis Beijing strain OM-V02_005, which exhibits possible hypertransmissible characteristics among the population of patients with multidrug-resistant tuberculosis in Osaka Prefecture, the largest urban area in western Japan.

DOI： 10.1128/genomeA.00608-13

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DOI： 10.7883/yoken.66.207

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We have developed a novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system. Two intraperitoneal vaccinations with rBCG expressing the Stx2 B subunit (Stx2B) resulted in an increase of protective serum IgG and mucosal IgA responses to Stx2B in BALB/c mice. When orally challenged with 103 CFU of STEC strain B2F1 (O91: H21), the immunized mice survived statistically significantly longer than the nonvaccinated mice. We suggest that intraperitoneal immunization with rBCG expressing Stx2B would be a potential vaccine strategy for STEC. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/CVI.00473-12

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DOI： 10.1128/CVI.00473-12

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We report herein the draft genome sequence of Mycobacterium intracellulare clinical strain M.i.198, which consistently exhibits hypervirulence in human patients, human macrophages in vitro, and immunocompetent mice.

DOI： 10.1128/JB.01439-12

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DOI： 10.1128/JB.01439-12

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Tuberculosis remains one of the most deadly infectious diseases worldwide and is a leading public health problem. Although isoniazid (INH) is a key drug for the treatment of tuberculosis, tolerance to INH necessitates prolonged treatment, which is a concern for effective tuberculosis chemotherapy. INH is a prodrug that is activated by the mycobacterial enzyme, KatG. Here, we show that mycobacterial DNA-binding protein 1 (MDP1), which is a histone-like protein conserved in mycobacteria, negatively regulates katG transcription and leads to phenotypic tolerance to INH in mycobacteria. Mycobacterium smegmatis deficient for MDP1 exhibited increased expression of KatG and showed enhanced INH activation compared with the wild-type strain. Expression of MDP1 was increased in the stationary phase and conferred growth phase-dependent tolerance to INH in M. smegmatis. Regulation of KatG expression is conserved between M. smegmatis and Mycobacterium tuberculosis complex. Artificial reduction of MDP1 in Mycobacterium bovis BCG was shown to lead to increased KatG expression and susceptibility to INH. These data suggest a mechanism by which phenotypic tolerance to INH is acquired in mycobacteria. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M111.333385

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Infection and transmission of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) and extensively drug-resistant M. tuberculosis (XDR-Mtb) is a serious health problem. We analyzed a total of 1,110 Mtb isolates in Osaka Prefecture and neighboring areas from April 2000 to March 2009. A total of 89 MDR-Mtb were identified, 36 (48.5%) of which were determined to be XDR-Mtb. Among the 89 MDR-Mtb isolates, 24 (27.0%) phylogenetically distributed into six clusters based on mycobacterial interspersed repetitive units-various number of tandem repeats (MIRU-VNTR) typing. Among these six clusters, the MIRU-VNTR patterns of four (OM-V02, OM-V03, OM-V04, and OM-V06) were only found for MDR-Mtb. Further analysis revealed that all isolates belonging to OM-V02 and OM-V03, and two isolates from OM-V04 were clonal. Importantly such genotypes were not observed for drug-sensitive isolates. These suggest that few but transmissible clones can transmit after acquiring multidrug resistance and colonize even in a country with a developed, well-organized healthcare system.

DOI： 10.1371/journal.pone.0042505

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DOI： 10.1074/jbc.M111.333385

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Language：Japanese   Publisher：JAPANESE SOCIETY FOR BACTERIOLOGY  

DOI： 10.3412/jsb.66.531

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Other Link： https://jlc.jst.go.jp/DN/JALC/00385387338?from=CiNii

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Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most often used vaccine worldwide and sole vaccine against tuberculosis. BCG is protective against severe form of childhood tuberculosis but less or not protective to adult pulmonary tuberculosis. Therefore, improved vaccination strategies and development of new tuberculosis vaccines are urgent demands. For those purposes, appropriate animal models that reflect human are critically useful. However, in animal models, BCG vaccination protects well against subsequent challenge of Mycobacterium tuberculosis. In this study we evaluated the duration of protective efficacy of the BCG vaccination in mice over time and found that efficacy was diminished 40 weeks after vaccination. The aged mice older than 45 weeks are protected sufficiently after the vaccination with BCG, suggesting that loss of its efficacy is not dependent on the age of mice but rather depends on the period from vaccination. The loss of protection occurred in TH1 polarized STAT6 deficient mice despite the maintenance of interferon (IFN)-gamma production activity of lymph node cells and splenic CD4 + T cells against M. tuberculosis antigens. Our data suggest that the duration from vaccination may explain the variation in BCG efficacy against adult pulmonary tuberculosis. © 2011 Elsevier Ltd.

DOI： 10.1016/j.vaccine.2011.07.051

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the most often used vaccine worldwide and sole vaccine against tuberculosis. BCG is protective against severe form of childhood tuberculosis but less or not protective to adult pulmonary tuberculosis. Therefore, improved vaccination strategies and development of new tuberculosis vaccines are urgent demands. For those purposes, appropriate animal models that reflect human are critically useful. However, in animal models, BCG vaccination protects well against subsequent challenge of Mycobacterium tuberculosis. In this study we evaluated the duration of protective efficacy of the BCG vaccination in mice over time and found that efficacy was diminished 40 weeks after vaccination. The aged mice older than 45 weeks are protected sufficiently after the vaccination with BCG, suggesting that loss of its efficacy is not dependent on the age of mice but rather depends on the period from vaccination. The loss of protection occurred in TH1 polarized STAT6 deficient mice despite the maintenance of interferon (IFN)-gamma production activity of lymph node cells and splenic CD4(+) T cells against M. tuberculosis antigens. Our data suggest that the duration from vaccination may explain the variation in BCG efficacy against adult pulmonary tuberculosis. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.vaccine.2011.07.051

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We investigated the role of mitochondrial reactive oxygen species (ROS) in the response of macrophages to lipopolysaccharide (LPS) using RAW 264.7 cells and their ρo cells lacking mitochondria. Mitochondrial density, respiratory activity and related proteins in ρo cells were significantly lower than those in RAW cells. LPS rapidly stimulated mitochondrial ROS prior to cytokine secretion, such as TNF-α and IL-6, from RAW 264.7 cells by activating the MAPK pathway, while the response was attenuated in ρo cells. Exposure of ρo cells to H2O2 partially restored the secretion of cytokines induced by LPS. These results suggest that mitochondrial density and/or the respiratory state contribute to intracellular oxidative stress, which is responsible for the stimulation of LPS-induced MAPK signaling to enhance cytokine release from macrophages. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2011.05.049

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Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+) into Fe(3+) and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m) values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guerin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4 +/- 19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

DOI： 10.1371/journal.pone.0020985

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Background: Studies have shown that DNA vaccines can induce protective immunity, which demonstrated the high potential of DNA vaccines as an alternative to inactivated vaccines. Vaccines are frequently formulated with adjuvants to improve their release, delivery and presentation to the host immune system.Methods: The H5 gene of H5N1 virus (A/Ck/Malaysia/5858/04) was cloned separately into pcDNA3.1 + vector. The immunogenicity of the cloned H5 DNA vaccine was tested on SPF chickens using two different approaches. First approach was using H5 DNA vaccine (pcDNA3.1/H5) and the second was using H5 DNA vaccine in addition to the pcDNA3.1/MDP1 vaccine. Ten days old chickens inoculated three times with two weeks intervals. The spleen and muscle samples from chickens immunized with H5 (pcDNA3.1/H5) and H5 + MDP1 (pcDNA3.1/H5 + pcDNA3.1/MDP1) vaccines were collected after sacrificing the chickens and successfully expressed H5 and MDP1 RNA transcripts. The sera of immunized chickens were collected prior to first immunization and every week after immunization
and analyzed using enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.Results: Results of competitive ELISA showed successful antibody responses two weeks post immunization. The HI test showed an increased in antibody titers during the course of experiment in group immunized with H5 and H5 + MDP1 vaccines. The result showed that the constructed DNA vaccines were able to produce detectable antibody titer in which the group immunized with H5 + MDP1 vaccine produced higher antibody comparing to H5 vaccine alone.Conclusions: This study shows for the first time the usefulness of MDP1 as a genetic adjuvant for H5 DNA vaccine. © 2010 Jalilian et al
licensee BioMed Central Ltd.

DOI： 10.1186/1479-0556-8-4

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Lipomannan (LM) and lipoarabinomannan (LAM) are phosphatidylinositol- anchored glycans present in the mycobacterial cell wall. In Mycobacterium smegmatis, the mannan core of LM/LAM constitutes a linear chain of 20-25 α1,6-mannoses elaborated by 8-9 α1,2-monomannose side branches. At least two α1,6-mannosyltransferases mediate the linear mannose chain elongation, and one branching α1,2-mannosyltransferase (encoded by MSMEG-4247) transfers monomannose branches. An MSMEG-4247 deletion mutant accumulates branchless LAM and interestingly fails to accumulate LM, suggesting an unexpected role of mannose branching for LM synthesis or maintenance. To understand the roles of MSMEG-4247-mediated branching more clearly, we analyzed the MSMEG-4247 deletion mutant in detail. Our study showed that the deletion mutant restored the synthesis of wild-type LM and LAM upon the expression of MSMEG-4247 at wild-type levels. In striking contrast, overexpression of MSMEG-4247 resulted in the accumulation of dwarfed LM/LAM, although monomannose branching was restored. The dwarfedLAMcarried a mannan chain less than half the length of wild-type LAM and was elaborated by an arabinan that was about 4 times smaller. Induced overexpression of an elongating α1,6-mannosyltransferase competed with the overexpressed branching enzyme, alleviating the dwarfing effect of the branching enzyme. In wild-type cells, LM and LAM decreased in quantity in the stationary phase, and the expression levels of branching and elongating mannosyltransferases were reduced in concert, presumably to avoid producing abnormal LM/LAM. These data suggest that the coordinated expressions of branching and elongating mannosyltransferases are critical for mannan backbone elongation. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M109.077297

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

M.tb is an intracellular pathogen which survives within the phagosomes of host macrophages by inhibiting their fusion with lysosomes. Here, it has been demonstrated that a lysosomal glycoprotein, CD63, is recruited to the majority of M.tb phagosomes, while RILP shows limited localization. This is consistent with the author&apos;s findings that CD63, but not RILP, is recruited to the phagosomes in macrophages expressing the dominant negative form of Rab7. These results suggest that M.tb phagosomes selectively fuse with endosomes and lysosomes to escape killing activity while acquiring nutrients.

DOI： 10.1111/j.1348-0421.2010.00199.x

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DOI： 10.1093/intimm/dxp126

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Mycobacterial DNA-binding protein 1 (MDP1) is a major protein antigen in mycobacteria and induces protective immunity against Mycobacterium tuberculosis infection in mice. In this study we determined murine T-cell epitopes on MDP1 with MDP1 DNA immunization in mice. We analyzed interferon-gamma production from the MDP1 DNA-immune splenocytes in response to 20-mer overlapping peptides covering MDP1 protein. We identified several CD4+ T-cell epitopes in three inbred mouse strains and one CD8+ T-cell epitope in C57BL/6 mice. These T-cell epitopes would be feasible for analysis of the role of MDP1-specific T-cells in protective immunity and for future vaccine design against M. tuberculosis infection.

DOI： 10.1016/j.vaccine.2009.10.062

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CD4+CD25+ regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4+CD25+ Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25+ cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25+ cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4+CD25- T cells alone or a combination of CD4+CD25+ and CD4+CD25- T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4+CD25+ Treg cells to CD4+CD25- effector T cells, as demonstrated by the lack of response of CD4+CD25+ T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis.Our data show that CD4+CD25+ Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection. © The Japanese Society for Immunology. 2010.

DOI： 10.1093/intimm/dxp126

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In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that LAscorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases. © 2009 Hirayama et al.

DOI： 10.1371/journal.ppat.1000643

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In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.

DOI： 10.1371/journal.ppat.1000643

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The late endosomal marker Rab7 has been long believed to be absent from the phagosome containing Mycobacterium tuberculosis (M.tb) in macrophage, but the detail kinetics remains elusive. Here, we found that Rab7 is transiently recruited to and subsequently released from M.tb phagosomes. For further understanding of the effect of Rab7 dissociation from the phagosome, we examined the localization of lysosomal markers on the phagosome in the macrophage expressing a dominant-negative Rab7. The localization of lysosomal associated membrane protein-2 (LAMP-2) on the phagosome was Rab7-independent, while that of cathepsin D was Rab7-dependent. These results agree with the localization of each lysosomal marker on M.tb phagosome at 6 h postinfection-i.e., LAMP-2, but not cathepsin D localized on the majority of M.tb phagosomes. These results suggest that the dissociation of Rab7 from M.tb phagosome is the important process in inhibition of phagolysosome biogenesis. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.06.152

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A population-based study of Mycobacterium tuberculosis isolated from homeless tuberculosis patients was performed during 2002-2004 in Osaka City, Japan. The data show that the ancient Beijing subfamily was predominant, whereas clustered isolates based on refined variable number of tandem repeats genotyping (19 loci) mainly belonged to the modern Beijing subfamily, suggesting its increased transmissibility.

DOI： 10.1016/j.tube.2009.05.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

A population-based study of Mycobacterium tuberculosis isolated from homeless tuberculosis patients was performed during 2002-2004 in Osaka City, Japan. The data show that the ancient Beijing subfamily was predominant, whereas clustered isolates based on refined variable number of tandem repeats genotyping (19 loci) mainly belonged to the modern Beijing subfamily, suggesting its increased transmissibility. (C) 2009 Elsevier Ltd. All rights reserved.

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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)).

DOI： 10.1099/ijs.0.000760-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

Medical treatment of pulmonary Mycobacterium avium complex (MAC) disease does not always provide curative effects and is frequently hampered by recurrence. This suggests the presence of a reservoir for MAC in the environment surrounding patients. We previously reported the recovery of MAC isolates from the residential bathrooms of outpatients. In the present study, to ascertain the colonizing sites and the possibility of an MAC reservoir in the bathrooms of patients, we tested the recovery and the genetic diversity of MAC isolates from 6 sites of specimens, including 2 additional sampling sites, inside the showerhead and the bathtub inlet, in the residential bathrooms of patients with pulmonary MAC disease. MAC isolates were recovered from 15 out of the 29 bathrooms (52%), including specimens from 14 bathtub inlets and 3 showerheads. Nearly half of these bathrooms (7/15) contained MAC strains that were identical or similar to their respective clinical isolates. Additionally, in 5 out of 15 bathrooms, polyclonal colonization was revealed by pulsed-field gel electrophoresis. The results imply that colonization of MAC organisms in the bathrooms of MAC patients occurs predominantly in the bathtub inlets, and there is thus a risk of infection and/or reinfection for patients via use of the bathtub and other sites in the bathroom.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

Medical treatment of pulmonary Mycobacterium avium complex (MAC) disease does not always provide curative effects and is frequently hampered by recurrence. This suggests the presence of a reservoir for MAC in the environment surrounding patients. We previously reported the recovery of MAC isolates from the residential bathrooms of outpatients. In the present study, to ascertain the colonizing sites and the possibility of an MAC reservoir in the bathrooms of patients, we tested the recovery and the genetic diversity of MAC isolates from 6 sites of specimens, including 2 additional sampling sites, inside the showerhead and the bathtub inlet, in the residential bathrooms of patients with pulmonary MAC disease. MAC isolates were recovered from 15 out of the 29 bathrooms (52%), including specimens from 14 bathtub inlets and 3 showerheads. Nearly half of these bathrooms (7/15) contained MAC strains that were identical or similar to their respective clinical isolates. Additionally, in 5 out of 15 bathrooms, polyclonal colonization was revealed by pulsed-field gel electrophoresis. The results imply that colonization of MAC organisms in the bathrooms of MAC patients occurs predominantly in the bathtub inlets, and there is thus a risk of infection and/or reinfection for patients via use of the bathtub and other sites in the bathroom.

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DOI： 10.1016/j.micpath.2008.10.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells. The Journal of Immunology, 2008, 181: 8521-8527.

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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&#039;-methyl-3&#039;-hydroxy-4&#039;-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1--&gt;3) -4

DOI： 10.1128/JB.01850-07

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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.

DOI： 10.1128/JB.01850-07

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Secretory leukocyte protease inhibitor (SLPT) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guerin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guerin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.

DOI： 10.4049/jimmunol.180.6.4032

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Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the

DOI： 10.1128/JB.00550-07

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Bacteria coordinate assembly of the cell wall as well as synthesis of cellular components depending on the growth state. The mycobacterial cell wall is dominated by mycolic acids covalently linked to sugars, such as trehalose and arabinose, and is critical for pathogenesis of mycobacteria. Transfer of mycolic acids to sugars is necessary for cell wall biogenesis and is mediated by mycolyltransferases, which have been previously identified as three antigen 85 (Ag85) complex proteins. However, the regulation mechanism which links cell wall biogenesis and the growth state has not been elucidated. Here we found that a histone-like protein has a dual concentration-dependent regulatory effect on mycolyltransferase functions of the Ag85 complex through direct binding to both the Ag85 complex and the substrate, trehalose-6-monomycolate, in the cell wall. A histone-like protein-deficient Mycobacterium smegmatis strain has an unusual crenellated cell wall structure and exhibits impaired cessation of glycolipid biosynthesis in the growth-retarded phase. Furthermore, we found that artificial alteration of the amount of the extracellular histone-like protein and the Ag85 complex changes the growth rate of mycobacteria, perhaps due to impaired down-regulation of glycolipid biosynthesis. Our results demonstrate novel regulation of cell wall assembly which has an impact on bacterial growth.

DOI： 10.1128/JB.00550-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guerin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

DOI： 10.1128/JVI.79.20.12871-12879.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Mycobacterium consists up to 7% of mycobacterial DNA-binding protein 1 (MDP1) in total cellular proteins. Host immune responses to MDP1 were studied in mice to explore the antigenic properties of this protein. Anti-MDP1 IgG was produced after infection with either bacillus Calmette-Guerin or Mycobacterium tuberculosis in C3H/HeJ mice. However, the level of Ab was remarkably low when purified MDPI was injected. MDPI is considered to be associated with DNA in nucleoid, which contains immunostimulatory CpG motif. Therefore, we examined coadministration of MDPI and DNA derived from M. tuberculosis. Consequently, this procedure significantly enhanced the production of MDP1-specific IgG. Five nanograms of DNA was enough to enhance MDP1-specific IgG production in the administration of 5 mu g of MDP1 into mice. Strong immune stimulation by such a small amount of DNA is noteworthy, because &gt; 1,000- to 100,000-fold doses of CpG DNAs are used for immune activation. A synthetic peptide-based study showed that B cell epitopes were different between mice administered MDPI alone and those given a mixture of MDP1 and DNA, suggesting that DNA alters the three-dimensional structure of MDP1. Coadministration of DNA also enhanced MDP1-specific IFN-gamma production and reduced the bacterial burden of a following challenge of M. tuberculosis, showing that MDP1 is a novel vaccine target. Finally, we found that MDP1 remarkably enhanced TLR9-dependent immune stimulation by unmethylated CpG oligo DNA in vitro. To our knowledge, MDPI is the first protein discovered that remarkably augments the CpG-mediated immune response and is a potential adjuvant for CpG DNA-based immune therapies.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Mycobacterium tuberculosis is an intracellular pathogen of tuberculosis and its pathogenicity is related to the ability to escape killing by ingested macrophages and induce delayed-type hypersensitivity (DTH). A major component of the cell wall of M. tuberculosis is trehalose 6,6'-dimycolate (TDM), which has been implicated as a pathogenetic factor. The expression of DTH and cell-mediated immunity is dependent on the macrophage-cytokine-type 1 helper T (Th1) lymphocyte axis. Cytokines, interleukin-12 (IL-12) and interferon-gamma (IFN-gamma), play a critical role in the process and IL-12-activated signal transducer and activator of transcription (STAT) 4 is required for the development of fully functional Th1 cells. To clarify host responses to mycobacterial TDM, we have analyzed footpad reaction, histopathology and cytokine profile of experimental granulomatous lesions using STAT4-deficient mice. In the present study, we have demonstrated that mycobacterial TDM selectively induces the Th1 response through the STAT4 signaling pathway, because mice lacking STAT4 protein significantly reduced to develop DTH, hypersensitivity granulomas, and Th1 cytokine responses, when compared to BALB/c mice. These results shed light on the molecular pathogenesis of mycobacterial disease. Taken together with previous studies, TDM is a pleiotropic molecule against the host and participates in the pathogenesis. (C) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.micpath.2005.03.003

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Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guerin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.

DOI： 10.1128/JVI.79.14.8716-8723.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Mycobacterium tuberculosis infects not only host macrophages but also nonprofessional phagocytes, such as alveolar epithelial cells. Glycosaminoglycans (GAGs) are considered as the component of mycobacterial adherence to epithelial cells. Here we show that extracellularly occurring mycobacterial DNA-binding protein 1 (MDP1) promotes mycobacterial infection to A549 human lung epithelial cells through hyaluronic acid ( HA). Both surface plasmon resonance analysis and enzyme-linked immunosorbent assay revealed that MDP1 bound to HA, heparin, and chondroitin sulfate. Utilizing synthetic peptides, we next defined heparin-binding site of 20 amino acids from 31 to 50 of MDP1, which is responsible for the specific DNA-binding site of MDP1. MDP1 bound to A549 cells, and exogenous DNA and HA interfered with the interaction. The binding was also abolished by treatment of A549 cells with hyaluronidase, suggesting that HA participates in the MDP1-A549 cell interaction. Adherence of bacillus Calmette-Guerin ( BCG) and M. tuberculosis to A549 cells was inhibited by addition of HA, DNA, and anti-MDP1 antibody, showing that MDP1 participates in the interaction between mycobacteria-alveolar epithelial cells. Simultaneous treatment of intratracheal BCG-infected mice with HA reduced the growth of BCG in vivo. Taken together, theses results suggest that HA participates in Mycobacterium-lung epithelium interaction and has potential for therapeutic and prophylactic interventions in mycobacterial infection.

DOI： 10.1074/jbc.M402677200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The development of a successful recombinant Mycobacterium bovis bacillus Calmette-Guerin (rBCG) vector-based vaccine for human immunodeficiency virus type 1 (HIV-1) requires the induction of high levels of HIV-1-specific immunity while at the same time maintaining immunity to tuberculosis. To examine a combined vaccination strategy for enhancement of immune responses specific for HIV-1, guinea pigs were inoculated with either a single or combination intradermal (i.d.), intrarectal (i.r.) and intranasal (i.n.) administration of rBCG-pSOV3J1 which secretes a chimeric protein of HIV-1 V3J1 peptide and alpha-antigen. Significant level of delayed-type hypersensitivity to both V3J1 peptide and tuberculin was induced in guinea pigs inoculated with human doses of rBCG-pSOV3J1 by a combination of intrarectal and intradermal routes. Guinea pigs inoculated by combined routes also had significantly higher titers of HIV-1-specific serum IgG and IgA compared with those animals immunized only intrarectally, which led to the enhanced neutralization activity against HIV-1(MN). In addition, the induction of high levels of IFNgamma and interleukin-2 (IL-2) mRNA in PBMC, splenocytes, and intraepithelial lymphocytes from the immunized animals was detected until at least 110 weeks post-inoculation. These results suggest that enhanced immune responses specific for HIV-1 are efficiently induced by combined intrarectal and intradermal immunization with rBCG-HIV, and antigen-specific Th1-type memory cells are maintained for more than 2 years in the immunized animals. Thus, inoculation with rBCG-HIV by combined routes represents an effective vaccination strategy to elicit high levels of HIV-1-specific immune responses. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0264-410X(02)00465-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400). The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.

DOI： 10.1074/jbc.M203038200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL INST INFECTIOUS DISEASES  

DNA containing an unmethylated CpG motif has a potent immunostimulatory effect on the vertebrate immune system. Because such CpG motifs are relatively common in bacterial DNA, but rare in mammalian animal and plant DNA, they may be an evolutionary adaptation augmenting innate immunity, most likely in response to pathogens that replicate within the host cells, such as viruses and intracellular bacteria. Microbial infection induces innate immunity by triggering pattern-recognition systems. The infected cells produce proinflammatory cytokines that directly combat microbial invaders and express costimulating surface molecules, which develop adaptive immunity by inducing distinct T cell differentiation. Bacterial DNA with unmethylated CpG-DNA stimulates vertebrate immature immune cells to induce maturation and to produce TNF-alpha as well as Th1-type cytokines, IL-12 and IFN-gamma. Therefore, CpG-DNA functions as an adjuvant for regulating the initiation of Th1 differentiation. The roles of immunostimulatory CpG motifs in DNA vaccine developments and in therapeutic applications have been discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Bacillus calmette-guerin (BCG)-vaccination raised dramatically the survival rates of A/J mice from infection by Plasmodium yoelii 17XL at blood-stage. The analysis of the immune response of spleen cells indicated that BCG vaccination biased the immune response toward Th1 type. Neutralization of IFN-gamma and nitric oxide abrogated the protection. The kinetics of Ab production in the course of P. yoelii 17XL infection was monitored. Surprisingly, larger amounts of parasite-specific Abs were produced in BCC-vaccinated mice than in the placebo control. The vast majority of the produced IgG against parasites in BCG-vaccinated mice was IgG2a, which was observed hardly in placebo controls. The peak of IgG2a production coincided with the clearance of infection. The naive mice transferred adoptively with IgG2a from self-cured mice survived the lethal challenge from the parasite. These data indicated that BCG vaccination protected A/J mouse from P. yoelii 17XL infection by biasing immunity toward Th1-type after parasite infection and enhancing production of IgG2a, which ultimately played a major role in protection. (C) 2000 Elsevier Science Ltd. All rights reserved.

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Pathogenic species of Mycobacterium are slowly growing intracellular bacteria. Slow growth is import-ant for the parasitism of these organisms and chronicity of the disease, but its precise mechanism has not been elucidated. Recently, we found that a novel DNA-binding protein (MDPI) was expressed (7-10% in total protein) in mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guerin, Mycobacterium tuberculosis, and Mycobacterium leprae. In this study, we observed that MDPI interfered with replication, transcription, and translation in the analysis in in vitro E. coli cell-free macromolecular biosynthesizing systems. Furthermore, MDPI inhibited the rapid growth of both Escherichia coli and Mycobacterium smegmatis, and NH2-terminal second amino acid, asparagine, was observed to be important in terms of this function. These data suggest an important role of MDPI for suppression of growth rates of mycobacteria. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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Previously, we constructed a recombinant live BCG (rBCG) secreting a 15 kDa C-terminal region of MSP-1 from Plasmodium yoelii (MSP-1(15)) and succeeded in the induction of more efficient protective immunity against parasite infection than observed with artificial adjuvants (Matsumoto S, Yukitake H, Kanbara H, Yamada T. Recombinant Mycobacterium bovis bacillus Calmette-Guerin secreting merozoite surface protein 1 (MSP-1) induces protection against rodent malaria parasite infection depending on MSP-1-stimulated interferon gamma and parasite-specific antibodies. J Exp Med 1998;188:845-54 [1]). In this study, we examined the endurance of the protective effects. The protective effect generated by rBCGMSP-1(15) was observed even 9 months after final immunization, whereas the effects of immunization by MSP-1(15) together with incomplete Freund adjuvant (IFA) were found to last only 4 months. (C) 1999 Elsevier Science Ltd. All rights reserved.

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DOI： 10.1006/mpat.1997.0159

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Language：Japanese   Publisher：Japanese Leprosy Association  

DOI： 10.5025/hansen1977.64.163

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DOI： 10.1006/bbrc.1993.2417

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(株)医学書院  

＜文献概要＞Point ・結核菌は活性化されたマクロファージや樹状細胞での殺菌に抵抗する細胞内寄生性病原体である.・結核菌抗原により誘導されたT細胞を中心とする獲得免疫は多彩で菌の排除を促進するものと許容するものがある.・菌はこのように相反する宿主免疫機構を利用するがごとくに,生体内で排除されず長期の寄生や疾患が成立する.

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Other Link： http://search.jamas.or.jp/link/ui/2019048397

Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(公社)日本栄養・食糧学会  

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Language：Japanese   Publisher：(公社)日本栄養・食糧学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(株)近代出版  

抗酸菌は、脂質に富む堅牢な細胞壁を有するがゆえに容易に自己凝集し、菌集塊を作りながら増殖する。菌集塊は結核患者やハンセン病患者の病巣や環境に生育する非結核性抗酸菌に観察される。このように抗酸菌はバイオフィルム様の増殖形態と表現型、すなわち薬剤抵抗性と長期生存能を有しており、それは疾患の成因とかかわる。(著者抄録)

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Language：Japanese   Publisher：新潟医学会  

【目的】新潟県で開発・市販されているヤスダヨーグルト(以下YYG)は、メチシリン耐性黄色ブドウ球菌(methicillin-resistant Staphylococcus aureus;以下MRSA)に対して抗菌活性を示す。本研究の目的は、YYGのMRSAに対する抗菌活性成分を同定することである。【方法】1)抗菌活性の評価:ディスク拡散法により抗菌活性を検討した。2)YYGの遠心分離成分の抗菌活性の評価:YYGを4000rpm、15分の条件で遠心分離し、得られた上清および沈澱物成分について抗菌活性を評価した。3)抗菌活性を示す乳酸菌のYYGからの単離・同定:MRSA菌液を添加したMRS寒天培地にYYGを塗布し、37℃、24時間の好気培養を行った。阻止円を形成したコロニーを採取し、MRS液体培地を用いて24時間静置培養(37℃、好気条件)した。16S rRNA遺伝子解析を行い、抗菌活性を示す乳酸菌種を同定した。4)乳酸菌が産生する抗菌活性成分の同定:抗菌活性を示す乳酸菌培養液上清に対して加熱・限外濾過・アルカリ処理・消化酵素処理の各処理を行い、抗菌活性の変化を評価した。その結果から、抗菌活性成分の同定を試みた。5)培養条件が乳酸菌増殖に与える影響の検討:好気培養と嫌気培養、および静置培養と振盪培養による乳酸菌培養を行い、各培養条件と乳酸菌増殖、培養液上清pHおよび抗菌活性との関連をMann-WhitneyのU検定で解析した。また、培養液上清pHと抗菌活性の強さを検討した。【結果】1)抗菌活性の評価:ディスク拡散法により適切に抗菌活性を半定量的に評価することが可能であった。2)YYGの遠心分離成分の抗菌活性の評価:YYGの遠心分離成分のうち、沈澱物に強い抗菌活性を認めた。また、YYGをビーズ破砕した後に遠心分離すると、沈澱物の抗菌活性は消失した。よって、抗菌活性成分はYYGに含まれる乳酸菌が産生していると推測した。3)抗菌活性を示す乳酸菌のYYGからの分離・同定:16S rRNA遺伝子解析の結果、Lactobacillus delbrueckii subsp.bulgaricus(以下LDB)がMRSAに対する抗菌活性を示すことが判明した。4)乳酸菌が産生する抗菌活性成分の同定:LDB上清はアルカリ処理により抗菌活性が減弱または消失した。さらに、過酸化水素を分解するカタラーゼをLDB培養液に添加すると、抗菌活性が著明に減弱した。5)各培養条件における乳酸菌培養液の比較:静置培養は振盪培養に比べて菌の増殖がよく(P=0.002)、また上清のpHが有意に低下し(P=0.002)、抗菌活性は強かった(P=0.002)。一方、好気培養と嫌気培養の間では、菌の増殖、上清pH、抗菌活性のいずれも有意な差を認めなかった(P=0.310、P=0.589、P=1.000)。また、LDB培養液上清pHと阻止円径との間に強い負の相関関係を認めた(相関係数=-0.875、P&lt;0.001)。【結論】YYGのMRSAに対する抗菌活性は、YYGに含まれる乳酸菌LDBが産生する乳酸および過酸化水素によるものであることが示唆された。(著者抄録)

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2017&ichushi_jid=J00990&link_issn=&doc_id=20180328060007&doc_link_id=%2Fdg3nigta%2F2017%2F013111%2F007%2F0645-0653%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdg3nigta%2F2017%2F013111%2F007%2F0645-0653%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

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Language：Japanese   Publisher：日本バイオフィルム学会  

非結核性抗酸菌症(NTM症)は世界中で増加傾向にある。なかでも日本を始めとする先進国における増加率が高い事、環境から感染すると考えられている事から公衆衛生上の重要な課題として注目されはじめた。環境中のNTMは、バイオフィルムを形成して感染にも関与していると考えられている事からNTMのバイオフィルム形成機構を理解する事はNTM症を制御する上で重要である。我々は、気液界面の菌膜形成がバイオフィルムの一形態であり、バイオフィルム形成には低酸素(5%O2)および富栄養(通常の菌培養メディウム)が必要であることを報告してきた。一方、NTMは上水道の水道管やシャワーヘッド等にもバイオフィルムを形成して感染源のひとつであると考えられている。水道管内の栄養状態は必ずしも豊富でないにもかかわらずバイオフィルムを形成していることから、低栄養条件下におけるバイオフィルム形成について検討した。【方法】抗酸菌用の平板培地にMycobacterium avium subsp.hominissuis(MAH)環境分離株(OCU806)を塗抹してマイクロコロニーを形成させ、これをプラスチック板に移して、炭素および窒素源であるglycerol、dextrose、albuminを除いた抗酸菌用液体培地(低栄養培地)に浮遊させた。プラスチック板上に形成されるバイオフィルムを走査型電子顕微鏡で観察した。【結果】富栄養下で形成されたマイクロコロニーは長い桿菌であるが、低栄養状態におかれると一部の長桿菌に穴が開いて死滅し、細胞壁が残っていた。残った菌体は細胞分裂をして1μm以下の短い菌体が多く観察された。【考察】低栄養条件では長桿菌に穴が開いて死滅したことから、MAHの一部は自己融解を起こして細胞内構成物を外に放出して残りの菌体の栄養源としていると推察される。限られた栄養状態で細胞分裂を繰り返すため、個々の菌体の大きさは小さくなっている。この事実は低栄養状態においてもMAHが力強く増殖していることを示している。(著者抄録)

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(公社)日本化学療法学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

非結核性抗酸菌(NTM)はバイオフィルムを形成して環境中に広く分布している。バイオフィルム形成はクオラムセンシングの他、栄養源などの環境因子によって影響をうける。我々は、NTMの気液界面の菌膜がバイオフィルムの一形態であり、低酸素(5%O2)環境が形成促進因子であることを報告してきた。一方、Ohjaらは、結核菌とM.bovis BCGの菌膜形成は二酸化炭素の増加によって促進すると報告している。そこで、環境中気体組成(大気、5%酸素または5%二酸化炭素条件)がMycobacterium avium subsp.hominissuis(MAH)環境分離株、M.bovis BCGのバイオフィルム形成に及ぼす影響を検討した。その結果、MAH環境分離株のバイオフィルム形成は低酸素(5%)で促進されたが、高い二酸化炭素(5%)では遅れて形成がみられた。ガラス試験管の菌膜形成では、5%二酸化炭素条件でもバイオフィルムを形成した。一方、M.bovis BCGは、低酸素条件でバイオフィルム形成が抑制され、その他の条件で厚いバイオフィルムが形成された。MAHは酸素および二酸化炭素、M.bovis BCGは二酸化炭素がバイオフィルム形成を制御していることが示唆された。また、宿主内が主な生育環境である結核菌と、環境や宿主内に広く分布するMAHでは環境(気体)センサーが異なるのかもしれない。今後これらのセンサーとバイオフィルム形成につながる反応系について検討を進めていきたい。(著者抄録)

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：English   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(有)科学評論社  

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Other Link： http://search.jamas.or.jp/link/ui/2016139494

Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：新潟医学会  

【目的】消化管手術の周術期には、黄色ブドウ球菌などの皮膚常在菌による創部感染、絶食による腸内細菌叢の攪乱に伴うBacterial translocation、抗菌薬使用に伴うmethicillin-resistant Staphylococcus aureus(MRSA)などの薬剤耐性菌の出現が起こりうる。このような状況に対して応用的プロバイオティクスによる周術期感染制御を視野に、ヨーグルト製品のもつ抗菌効果の特性を微生物学的に解析した。【方法】新潟県で開発され市販されているヤスダヨーグルト(以下、YYG)に注目して、1)抗菌活性スペクトル(被験株:methicillin-sensitive Staphylococcus aureus(MSSA)ならびにMRSA臨床分離株、大腸菌株DH5α)、2)配合菌株の異なる他の5種の市販ヨーグルト製品との抗菌活性の相違、3)YYG各配合菌種(Yc-x11およびYc-180)の示す抗菌作用、および4)抗菌活性を示す分離画分について、ディスク法による阻止円形成により検討した。【結果】YYGは大腸菌に比してMSSAに優位な抗菌活性を示した。また、YYGはMRSA臨床分離株に対しても抗菌活性を示した。他製品との比較において、YYGは最も高い抗菌活性を示した。YYG配合菌種は、通常の低塩濃度培地上では抗菌活性を示したものの高塩濃度条件下では、その効果は減弱あるいは消失した。YYGの抗菌活性は、上清ではなく沈殿画分に認めた。【結論】YYGはMRSAを含む黄色ブドウ球菌に対する直接的な抗菌活性を示した。その抗菌効果は、特有の配合菌種に依拠する抗菌活性物質によることが示唆された。ヨーグルトの中でもYYGは極めて有望な抗菌性機能食品として、周術期感染制御に貢献できる可能性がある。(著者抄録)

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Other Link： http://hdl.handle.net/10191/44215

Language：Japanese   Publisher：(株)医薬ジャーナル社  

結核は三大感染症の一角であり、代表的な再興感染症である。病原体である結核菌はアフリカの東岸に5〜10万年前に出現し、人類の出アフリカにともない世界に伝播した。通常、菌は飛沫核感染で肺を侵入門戸として生体に侵入し感染するが、すみやかに発症に至るケースは5%程度とまれである。一方、感染成立後、ヒトの免疫系は菌を生体から駆逐することはできず、感染は宿主の命が果つるまで継続する。このような無症候感染者は人類の1/3に及び、一部は再燃により結核が発症する。このため無症候感染は潜在性結核と呼ばれる。このような事実は、病原体の源泉である潜在性結核の対処が結核の制御に重要であることを示しており、潜伏感染機構の理解はそのよりどころとなるだろう。本稿では、結核菌の潜伏感染機構についての知見と、潜在性結核の解析をベースにした新しい制御法開発の可能性について述べる。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

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J-GLOBAL

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Language：Japanese   Publisher：日本バイオフィルム学会  

非結核性抗酸菌は環境中に広く分布し、バイオフィルムを形成している。このうち30種余が環境からヒトに感染し、日和見感染を起こす。これらの菌のバイオフィルムの特徴を解析することは病原性を理解する上で重要である。そこで環境から分離したMycobacterium avium subsp.hominissuis(MAH)806と標準株のMAH 104、およびglycopeptidolipid(GPL)を欠損したラフ型株(MAH817、MAH104R)を用いてバイオフィルム形成時の特徴を検討した。我々は、気液界面の菌膜形成因子の一つが低酸素であり、GPLが気液界面の菌膜形成に寄与していることを報告した。今回、菌膜や壁面を遡上する菌体は、GPLの有無に関わらず浮遊菌に比べ消毒剤に耐性を示した。走査型電子顕微鏡を用いて観察すると、GPL産生株は厚い菌塊と、ひも状の菌体外成分で菌相互に接着していた。GPL非産生株は菌体同士が密着し、ひも状菌体外成分は殆どみられなかった。これらの結果から、菌膜および遡上する菌体はバイオフィルムと言える。このバイオフィルム形成時、trehalose dimycolate(TDM)、trehalose monomycolate(TMM)の減少が4株ともに認められた。厚い菌膜を形成するMAH806は形成時のGPL産生量が増加した。MAH817にGPLを添加すると、菌膜の形成が促進されて肥厚した。これらの結果から、MAHのバイオフィルム形成はGPLが関わる菌膜形成と、GPLが関わらないバイオフィルム形成があり、どちらにもTDM、TMMが関わっていることが示唆された。(著者抄録)

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Language：Japanese   Publisher：(一社)日本呼吸器学会  

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Language：Japanese   Publisher：(一社)日本呼吸器学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：大道学館出版部  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：EUROPEAN RESPIRATORY SOC JOURNALS LTD  

Web of Science

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

私たちは非結核性抗酸菌Mycobacterium avium subsp.hominissuis(MAH)、Mycobacterium intracellulareに着目し、これらが生活環境のシャワーヘッドや浴槽内注水口に生息していること、MAHのバイオフィルムは、特有の層構造を有することを報告してきた。これらの病原性環境菌の感染を制御するためには、環境中のバイオフィルム形成を制御する因子の理解が重要である。バイオフィルム形成促進因子を明らかにする目的で、環境分離MAHを用いて抗酸菌用液体培地培養時に形成されるバイオフィルム形成能を炭素および窒素源や酸素供給を変化させて検討した。また、バイオフィルム形成に関与が知られているglycopeptidolipid(GPL)を自然発生的に欠損した株との比較を行った。その結果、環境から分離した64株中32株は低酸素条件下で気液界面に著しいバイオフィルム形成が認められた。MAH OCU806およびラフ型菌のガラス試験管気液界面のバイオフィルム検討でも同様であった。このとき、GPL欠損株のバイオフィルム形成能は低かったが、GPL産生株と異なる形態がみられた。クリスタルバイオレット法で行ったバイオフィルム量は、環境分離株MAH806は大気圧下よりも低酸素条件(5%)下で速くバイオフィルムを形成した。低栄養培地ではバイオフィルム形成は遅かった。これらの結果から低酸素条件がMAHのバイオフィルム形成促進因子であることが明らかになった。GPLはバイオフィルム形成に大きく関与するが、GPL合成能がない株でもバイオフィルムを形成することからその他の構成因子の関与が示唆された。(著者抄録)

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

結核はAIDSやマラリアと並ぶ世界三大感染症であり、2013年には860万人の発症と130万人の死亡をもたらしている。WHOは2050年までに結核の根絶を目指しているが、それには新薬に加え、効果的な新ワクチンが必要であろう。我々は結核の病原体の源泉である潜在性結核について、その機構を解析してきたが、潜在性結核の90%が終生結核を発症しないという事実は、ワクチン開発のよりどころになると考える。現行の結核ワクチンBCG(Mycobacterium bovis bacille Calmette-Guerin)は小児の粟粒結核や結核性髄膜炎に対して顕著な効果をもつ一方、結核発症の多くを占める、内因性再燃に起因する成人型肺結核に対する効果は低調である。BCGは生ワクチンで、投与後も長期にわたって宿主内で生存することから、効果は持続すると考えられてきたが、実際には投与後、時間経過とともに減衰する。我々はマウスモデルにおいてもBCGの防御効果が経時的に低下することを見出している。本稿では、結核ワクチン開発に関する現状と潜在期の抗原を利用した新しい結核ワクチンの開発に向けた私たちの取り組みについて紹介する。(著者抄録)

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Language：Japanese   Publisher：日本小児感染症学会  

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Language：English   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(有)科学評論社  

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Language：Japanese   Publisher：新潟県医師会  

結核の歴史と現状、研究動向について以下の項目で解説した。1)結核(症)の歴史的な変遷、2)結核病態の形成と脂質成分、3)蛋白性の病原因子、4)結核菌の生活環と潜在性結核、5)ワクチン開発の試み(BCGを凌駕するワクチンの作成)、6)化学療法薬の開発状況(結核菌の休眠現象への対策)。

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Language：Japanese   Publisher：日本ハンセン病学会  

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Language：Japanese   Publisher：医歯薬出版(株)  

結核菌は飛沫核感染によって肺を侵入門戸としてヒトに感染する。感染者の約5%が速やかに結核を発症するが、感染者の約95%で無症候感染が成立する。ヒト型結核菌はヒトによく適応した寄生病原体であり、感染成立後、免疫系は菌を生体から駆逐できない。無症候感染は潜在性結核と定義されるが、その理由は5〜10%で内因性再燃による病気が生じるからである。結核菌はヒトを住み処とし、ときに発症によって住み処を拡大しながら有史前より今日まで種を継いできた。現在、無症候感染者は人類の1/3にのぼる。このような事実は病原体の源泉である潜在性結核の対処なしには結核を制圧できないことを意味しており、潜伏感染メカニズムの解明はそのよりどころとなる。潜伏菌は休眠と増殖のサイクルを繰り返し、単細胞生物でありながら長期間の生命維持を実現している。本稿では結核菌の潜伏感染のメカニズムについて、これまでの知見を概説する。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

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Language：Japanese   Publisher：日本バイオフィルム学会  

結核菌は人を住みかとし、菌の旺盛な生体内増殖によって生じる結核(症)は、年間100万人以上の死をもたらす。肉芽腫は、結核に特徴的な病変であり、一般のバイオフィルムと異なり宿主由来成分によって構成されるが、菌の生存や薬剤抵抗性を促す点で&quot;バイオフィルム様&quot;の機能構造体といえる。本稿では、長期の潜伏感染後の発病や肉芽腫形成など、結核に特徴的な病態進行を背景として、結核菌のDNA結合性蛋白質Mycobacterial DNA-binding protein 1(MDP1)の発見とMDP1がヒト結核肉芽腫で発現し、結核菌の増殖制御や薬剤抵抗性を含む長期生存のメカニズムについての研究成果を紹介する。(著者抄録)

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Language：Japanese   Publisher：日本バイオフィルム学会  

非結核性抗酸菌は世界中の土壌や水系の環境中にバイオフィルムを形成して広く分布しており、ヒトに日和見感染する。なかでもMycobacterium avium、Mycobacterium intracellulareによる肺感染症は近年世界中で増加している。不思議な事にヒト-ヒト感染の報告はないので環境から生成したエアロゾルを吸入して感染している。私たちはM.avium、M.intracellulareが家庭の浴室に生息しており浴室から感染する危険性を報告してきた。そこで環境中のM.aviumが形成するバイオフィルムの生態が感染性およびエアロゾル形成に関与していると考え、その生態を走査型電子顕微鏡で観察した。本研究では抗酸菌のバイオフィルムの細胞外マトリックスの構成成分として知られているglycopeptidolipidの欠損株を用いて生態を比較した。その結果、M.aviumは膜に覆われたバイオフィルムを構築した。その膜の下に小型の桿菌がぎっしり詰まっていた。その桿菌には細胞間マトリックスがほとんど認められなかった。glycopeptidolipid欠損株には、菌を覆う膜の形成は認められなかった。M.aviumが形成するバイオフィルムの生態学的特徴から、菌体は膜状バイオフィルムに守られて、膜の内側で増殖していることが示唆された。同時に細胞間マトリックスがないので膜の一部が破綻すると内側の菌が個々に飛び出しエアロゾル形成に寄与すると思われる。(著者抄録)

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Language：Japanese   Publisher：日本ハンセン病学会  

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Language：Japanese   Publisher：日本細菌学会  

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Language：Japanese   Publisher：(一社)日本内科学会  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

結核菌は宿主に感染後、休眠状態で長期間、潜伏感染することが知られている。また、これらの潜伏感染菌は抗菌薬に対する抵抗性を有しており、結核治療を困難にしている。筆者らは、定常期以降の結核菌体内に大量に発現する分子である抗酸菌特異的ヒストン様タンパク質Mycobacterial DNA-binding protein 1(MDP1)は生体にとって必須の元素である鉄の貯蔵および解毒に関与することで宿主内での長期生存に寄与するのみならず、抗結核薬であるイソニアジド(INH)の活性化酵素の発現を抑制することにより定常期以降にみられる結核菌のINH抵抗性の獲得に関与することを明らかにしている。本稿では、この分子をターゲットとした結核治療法開発の可能性について述べる。(著者抄録)

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Language：Japanese   Publisher：(株)日本医事新報社  

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Language：Japanese   Publisher：大阪市医学会  

結核は、発症者が900万人を超える世界最大の感染症であり、その病原菌Mycobacterium tuberculosisの感染に伴う主に肺肉芽腫を形成する疾患である。全世界人類の3分の1はM.tuberculosisに感染し、無症候状態の潜在性結核と考えられている。潜在性結核の約5%は、内因性再燃に伴い結核を発症することから、潜在性結核に対処することが重要である。結核患者の多くは開発途上国のアジア(55%)とアフリカ(30%)に集中しており、先進国ではほぼ制圧されているように窺える。しかし、最近では、渡航者によって病原菌が伝播されている。さらに、多剤耐性結核菌の出現やヒト免疫不全ウイルス感染者の約26%は結核感染者であることから、今後、結核が流行する可能性がある。結核制圧のためには、活動性結核に加え潜在性結核の早期診断・早期治療が重要だと考えられる。しかし、現在の結核診断法に用いられているインターフェロン-γ産生法(クオンティフェロン)やツベルクリン反応は、潜在性結核を十分に診断できない。そこで、我々は、23種の結核菌抗原を用いて潜在性結核のバイオマーカー探索を開始した。国内で行った研究結果から潜在性結核のバイオマーカーとして4種の抗原候補を見いだし、さらに長崎大学熱帯医学研究所との共同研究によりケニアでの結核感染調査においてもその有用性を確認した。現在、潜在性結核のバイオマーカー探索が世界中で取り組まれている中、我々の研究結果は、新たな潜在性結核の診断法へと繋がる可能性が考えられる。(著者抄録)

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Other Link： http://search.jamas.or.jp/link/ui/2013189878

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Language：Japanese   Publisher：日本医学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

非結核性抗酸菌はおもに水系の自然環境や生活環境に広く分布し、ヒト、家畜、鳥、魚に病原性を示して人獣共通感染症を引き起こす。ヒトには日和見感染を起こし、その感染源は生活環境であると考えられている。近年、基礎疾患をもたないヒトが肺Mycobacterium avium complex(MAC)症を発症する例が増加しており、その感染源である生活環境中の分布が注目されている。MACは、家庭のシャワーヘッドや浴槽出水口にバイオフィルムを形成して定着していることがわかってきた。飲料水の汚染や不十分な消毒と管理は、易感染性宿主に非結核性抗酸菌症を起こして院内感染の原因となる。院内感染事例、バイオフィルムの関与、消毒についても解説した。(著者抄録)

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(一社)日本感染症学会  

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Language：Japanese   Publisher：(株)医学書院  

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Other Link： http://search.jamas.or.jp/link/ui/2008350771

Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：医歯薬出版(株)  

結核は年間約200万人が死亡する最大級の細菌感染症である。結核菌は脂質に富む細胞壁を基本バリアとして病原因子を分泌し、宿主の殺菌機構に抵抗する。通常、結核菌と宿主の戦いは宿主優勢で発病しないが&quot;よい勝負&quot;で、宿主が感染菌を駆逐することもない。その結果、無症候の感染者が現人類の1/3に達している。宿主の抵抗力の低下とともに菌が再増殖し発症するが、これを二次結核とよぶ。感染時に発病する一次結核と合わせ、感染者の約10%が結核を発症する。結核病変は基本的には慢性の肉芽腫性炎であり、肉芽腫形成、壊死、陥落、空洞形成と推移する特異性炎である。巨大な肉芽腫は組織破壊を招き個体の死を招来するが、肉芽腫形成は本来宿主の防御応答で、結核菌の増殖をおもに酸素を遮断することで停止させる、いわば&quot;苦肉の策&quot;である。本稿では結核の歴史と現状、結核病態の形成機構、現在の対策研究の動向について概説した。(著者抄録)

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(株)日本臨床社  

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Other Link： http://search.jamas.or.jp/link/ui/2007232474

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/JVI.01681-06

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Language：Japanese   Publisher：JAPANESE SOCIETY FOR BACTERIOLOGY  

DOI： 10.3412/jsb.61.345

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結核菌は人類の1/3に潜伏感染しており、時に内因性再燃を引きおこす。成人の肺結核の多くがこの機序で発症し、現在、年間約200万人の命が失われている。潜伏感染菌は通常休眠することで、菌と宿主双方の生存を確保し、且つ薬剤抵抗性を獲得する。結核菌はヒトを住処としており、ヒト以外の生物や自然環境下での生存は難しい。したがって潜伏感染菌の根絶は根本的な結核対策となる。潜伏感染菌対策を講じるために休眠機構を解かなければならない。細菌を凍らすように菌の代謝を生理的温度下においていかに抑制するのか?休眠機構は&quot;謎&quot;に包まれていたが、我々は菌の増殖を停止させる分子MDP1を同定した。加えてMDP1が接着分子として菌の潜伏感染細胞への接着/侵入を促すことを明らかにした。MDP1の活性を中心として、接着分子を標的とした新しい治療/予防法開発の可能性を紹介する。(著者抄録)

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

毎年約200万人の命を奪う結核菌は,現在も20億人に潜伏感染している.感染者の5〜10%が終生の間に結核を発病する.WHOは,今後20年間に7,000万人の発病を予測し警告している.潜伏結核菌は増殖を停止し休眠菌となる.その結果,菌と宿主双方の生存が確保される.潜伏感染菌の根絶は,結核の根本的な対策となる.なぜなら,この菌は一般に人のみをすみかとしているからだ.潜伏感染菌対策を講じるため,休眠機構を解く必要がある.細胞を凍らすように菌の代謝を生理的にいかに抑制するのか?生物学的にも非常に興味深い.結核菌の核酸およびリボゾーム結合性蛋白質の活性を中心に,休眠機構解明に迫る研究成果を紹介する(著者抄録)

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Language：Japanese   Publisher：(一社)日本結核病学会  

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Language：Japanese   Publisher：(一社)日本結核病学会  

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