KHT5 마커를 사용한 Bacillus cereus 그룹에서 Bacillus anthracis의 구별

김형태,김성주,채영규 한국미생물학회 2003 미생물학회지 Vol.39 No.1

탄저균은 그람양성 아포형성세균으로 탄저를 일으키는 원인균이다. Bacillus cereus그룹에 속하는 22종을 포함하여 Bacillus 속의 29종에서 탄저균을 검증할 수 있는 DNA 마커를 개발하고 이를 이용하여 B. cereus 그룹에서 탄저균만을 구분하였다. 한국산 탄저균 경주로부터 709 bp마커(KHTS)를 확보하였다. KHTS분절로부터 얻어진 internal primer set의 PCR 산물은 B. cereus 그룹의 다른 종으로부터 탄저균만을 구별하였다. Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. In order to develop a DNA marker specific for Bacillus anthracis and to discriminate this species from Bacillus cereus group, we applied the randomly amplified polymorphic DNA (RAPD)-PCR technique to a collection of 29 strains of the genus Bacillus, including 22 species of the B. cereus group. A 709-bp RAPD marker (KHT5) specific for B. anthracis was obtained from B. anthracis BAK. The PCR product of internal primer set from the KHT5 fragment distinguished B. anthracis from the other species of the B. cereus group.

Proteomic Analysis of the Oxidative Stress Response Induced by Low-Dose Hydrogen Peroxide in Bacillus anthracis

( Sang Hoon Kim ),( Se Kye Kim ),( Kyoung Hwa Jung ),( Yun Ki Kim ),( Hyun Chul Hwang ),( Sam Gon Ryu ),( Young Gyu Chai ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.6

Anthrax is a bacterial disease caused by the aerobic sporeforming bacterium Bacillus anthracis, which is an important pathogen owing to its ability to be used as a terror agent. B. anthracis spores can escape phagocytosis and initiate the germination process even in antimicrobial conditions, such as oxidative stress. To analyze the oxidative stress response in B. anthracis and thereby learn how to prevent antimicrobial resistance, we performed protein expression profiling of B. anthracis strain HY1 treated with 0.3 mM hydrogen peroxide using a comparative proteomics-based approach. The results showed a total of 60 differentially expressed proteins; among them, 17 showed differences in expression over time. We observed time-dependent changes in the production of metabolic and repair/protection signaling proteins. These results will be useful for uncovering the metabolic pathways and protection mechanisms of the oxidative response in B. anthracis.

유전자 재조합 방법을 이용한 Bacillus anthracis 방어항원에 관한 연구

오희복,박영미아,성원근,장현아 국립보건연구원 1998 국립보건원보 Vol.35 No.-

Yeast내에서 탄저병 원인균인 Bacillus anthracis의 치사독소인 Lethal Factor 단백질 발현

황혜현,김정목,최경재,정회일,한성환,구본성,윤문영,Hwang Hyehyun,Kim Joungmok,Choi Kyoung-Jae,Chung Hoeil,Han Sung-Hwan,Koo Bon-Sung,Yoon Moon-Young 한국미생물학회 2005 미생물학회지 Vol.41 No.4

Bacillus anthracis는 탄저병의 병원체이다. 탄저병의 독소는 Bacillus anthracis가 가진 세가지 독소로 이루어져 있다. protective antigen (PA), lethal factor (LF)그리고 edema factor (EF)로 구성되어 있다. PA는 세포수용체와 결합하여 활성화 과정을 거친 후 LF 흑은 EF를 세포질 안으로 이동시켜 주는 역할을 한다. LF는 금속이온 $(Zn^{2+})$ 의존적 단백질 가수분해 효소로써 탄저병에 감염된 동물들의 치사독소로 작용하게 된다. 따라서 LF에 대한 특성 분석 및 억제재 개발에 관한 연구는 탄저치료제 개발에 매우 중요한 과정이라 할 수 있다. 본 연구에서는 탄저독소의 치료제 개발을 위해 선행되어야 하는 LF 고처리량 활성검증방법 및 저해제 선별에 더 높은 효율을 가지기 위해 이러한 시스템 방법 등을 이용하여 세포내 검정방법의 기초 자료를 마련하고자 하였다. 이를 위하여 yeast를 숙주로 한 LF 발현 vector의 구축과, 구축한 발현 시스템을 yeast에 형질전환 하여 plasmid의 안정성 및 LF유전자의 발현을 확인하였다. 본 연구는 LF유전자의 발현을 진핵세포 내에서 처음으로 시도했으며, 세포내 검증 시스템 도입의 기초적 자료를 제공하였다. Yeast내에서의 LF의 발현은 탄저병의 저해제 선별이나 활성측정검증을 생체 내에서 용이하게 할 수 있다는 가능성을 나타냈다. Anthrax is an infectious disease caused by the gram-positive bacterium, Bacillus anthracis. Anthrax toxin is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF onto the cytosol. LF is a zinc-dependent metalloprotease, which is a critical virulence factor in cytotoxicity of infected animals. Therefore, it is of interest to develop its potent inhibitors for the neutralization of anthrax toxin. The first step to identify the inhibitors is the development of a rapid, sensitive, and simple assay method with a high-throughput ability. Much efforts have been concentrated on the preparation of powerful assays and on the screening of inhibitors using these system. In the present study, we have tried to construct anthrax lethal factor in yeast expression system to prepare cell-based high-throughput assay system. Here, we have shown the results covering the construction of a new vector system, subcloning of LF gene, and the expression of target gene. Our results are first trial to express LF gene in eukaryote and provide the basic steps in design of cell-based assay system.

Structural insights into the dimer-tetramer transition of FabI from <i>Bacillus anthracis</i>

Kim, Hyun Tae,Kim, Sulhee,Na, Byeong Kwan,Chung, Jiwoung,Hwang, Eunha,Hwang, Kwang Yeon Elsevier 2017 Biochemical and biophysical research communication Vol.493 No.1

<P><B>Abstract</B></P> <P>Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless of the organism, are composed of homo-tetramers, except for two structures: <I>Bacillus cereus</I> and <I>Staphylococcus aureus</I> FabI (<I>bc</I>FabI and <I>sa</I>FabI, respectively), which have been reported as dimers. However, the reason for the existence of the dimeric form in these organisms and the biological meaning of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal structure of a dimeric form of <I>Bacillus anthracis</I> FabI (<I>ba</I>FabI) and the crystal structures of tetrameric forms of <I>ba</I>FabI in the apo state and in complex with NAD<SUP>+</SUP> and with NAD<SUP>+</SUP>-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that <I>ba</I>FabI with a His<SUB>6</SUB>-tag at its C-terminus exists as a dimer, whereas untagged-<I>ba</I>FabI exists as a tetramer. The His<SUB>6</SUB>-tag may block the dimer-tetramer transition, since <I>ba</I>FabI has relatively short-length amino acids (<SUP>255</SUP>LG<SUP>256</SUP>) after the 3<SUB>10</SUB>-helix η7 compared to those of FabI of other organisms. The dimeric form of <I>ba</I>FabI is catalytically inactive, because the α-helix α5 occupies the NADH-binding site. During the process of dimer-tetramer transition, this α5 helix rotates about 55° toward the tetramer interface and the active site is established. Therefore, tetramerization of <I>ba</I>FabI is required for cofactor binding and catalytic activity.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Bacillus anthracis</I> FabI (<I>ba</I>FabI) dimer high-resolution crystal structure was reported. </LI> <LI> We obtained snapshots of <I>ba</I>FabI tetramer states (apo, NAD<SUP>+</SUP>-; NAD<SUP>+</SUP>-triclosan-bound). </LI> <LI> The C-terminal His<SUB>6</SUB>-tag may block the dimer-tetramer transition. </LI> <LI> Dimeric <I>ba</I>FabI is catalytically inactive as α-helix α5 occupies NADH-binding site. </LI> <LI> Tetramerization of <I>ba</I>FabI is required for cofactor binding and catalytic activity. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Characterization of Bacillus anthracis proteases through protein-protein interaction: an in silico study of anthrax pathogenicity

Amrita Banerjee,Pradeep Kumar Das Mohapatra,Shilpee Pal,Keshab Chandra Mondal,Bikash Ranjan Pati,Arnab Sen,Tanmay Paul 셀메드 세포교정의약학회 2014 TANG Vol.4 No.1

Anthrax is the deadly disease for human being caused by Bacillus anthracis. Instantaneous research work on the mode of infection of the organism revealed that different proteases are involved in different steps of pathogenesis. Present study reports the in silico characterization and the detection of pathogenic proteases involved in anthrax infection through protein-protein interaction. A total of 13 acid, 9 neutral, and 1 alkaline protease of Bacillus anthracis were selected for analysing the physicochemical parameter, the protein superfamily and family search, multiple sequence alignment, phylogenetic tree construction, protein-protein interactions and motif finding. Among the 13 acid proteases, 10 were found as extracellular enzymes that interact with immune inhibitor A (InhA) and help the organism to cross the blood brain barrier during the process of infection. Multiple sequence alignment of above acid proteases revealed the position 368, 489, and 498-contained 100% conserved amino acids which could be used to deactivate the protease. Among the groups analyzed, only acid protease were found to interact with InhA, which indicated that metalloproteases of acid protease group have the capability to develop pathogenesis during B. anthracis infection. Deactivation of conserved amino acid position of germination protease can stop the sporulation and germination of B anthracis cell. The detailed interaction study of neutral and alkaline proteases could also be helpful to design the interaction network for the better understanding of anthrax disease.

Enhancement of the Immune Responses of Mice to Bacillus anthracis Protective Antigen by CIA07 Combined with Alum

Kim, Su-Hee,Park, Shin-Ae,Kim, Hye-Kyeong,Cho, Yang-Je,Kim, Kwang-Sung,Kim, Yeon-Hee,Chun, Jeong-Hoon,Lee, Na-Gyong 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.11

Anthrax is an acute zoonotic disease caused by infection with Bacillus anthracis. B. anthracis spores are highly resistant to environmental degradation and are used as a biological weapon. In this study, we investigated the adjuvant activity of CIA07 to anthrax protective antigen (PA). A/J mice were immunized intraperitoneally once, or twice with a 4-week interval, with recombinant PA alone or combined with alum, CpG1826, or CIA07 as adjuvant, and serum anti-PA IgG antibody responses were measured 4 weeks after each immunization. All three adjuvants significantly enhanced anti-PA IgG antibody titer 4 weeks after the priming and boosting immunizations, and alum gave the highest titer. In order to evaluate the adjuvant activity of CIA07 in the presence of alum, Balb/c mice were immunized 3 times at 1-week intervals with PA in combination with alum, CIA07 or alum plus CIA07, and the immune responses were assessed 2 weeks after the third immunization. The serum anti-PA IgG antibody titer of the CIA07-treated group was l4-fold higher than the group given PA alone, and the coadministration of CIA07 with alum further increased the titer 3.5-fold (P < 0.05). The toxin neutralizing activity of the sera from the mice given the combination of CIA07 and alum was 109-times higher than the animals given PA alone. The mice given CIA07 plus alum also showed a marked increase in the number of IFN-$\gamma$-, IL-2-, and IL-4-producing $CD4^+$ T cells among their splenocytes. These data suggest the potential of CIA07 in combination with alum as an adjuvant for the development of a potent anthrax vaccine.

Repetitive Sequence-based Genomic Fingerprinting을 이용한 Bacillus anthracis의 분자 분류 및 동정

김원용 ( Won Yong Kim ),송미옥 ( Mi Ok Song ),조명신 ( Myung Shin Cho ),김기정 ( Ki Jung Kim ),정상인 ( Sang In Chung ) 한국수의공중보건학회 2004 예방수의학회지 Vol.28 No.3

Enhancement of the Immune Responses of Mice to Bacillus anthracis Protective Antigen by CIA07 Combined with Alum

김수희,박신애,김혜경,조양제,김광성,김연희,천정훈,이나경 대한약학회 2008 Archives of Pharmacal Research Vol.31 No.11

Anthrax is an acute zoonotic disease caused by infection with Bacillus anthracis. B. anthracis spores are highly resistant to environmental degradation and are used as a biological weapon. In this study, we investigated the adjuvant activity of CIA07 to anthrax protective antigen (PA). A/J mice were immunized intraperitoneally once, or twice with a 4-week interval, with recombinant PA alone or combined with alum, CpG1826, or CIA07 as adjuvant, and serum anti-PA IgG antibody responses were measured 4 weeks after each immunization. All three adjuvants significantly enhanced anti-PA IgG antibody titer 4 weeks after the priming and boosting immunizations, and alum gave the highest titer. In order to evaluate the adjuvant activity of CIA07 in the presence of alum, Balb/c mice were immunized 3 times at 1- week intervals with PA in combination with alum, CIA07 or alum plus CIA07, and the immune responses were assessed 2 weeks after the third immunization. The serum anti-PA IgG antibody titer of the CIA07-treated group was 14-fold higher than the group given PA alone, and the coadministration of CIA07 with alum further increased the titer 3.5-fold (P < 0.05). The toxin neutralizing activity of the sera from the mice given the combination of CIA07 and alum was 109-times higher than the animals given PA alone. The mice given CIA07 plus alum also showed a marked increase in the number of IFN- γ-, IL-2-, and IL-4-producing CD4+ T cells among their splenocytes. These data suggest the potential of CIA07 in combination with alum as an adjuvant for the development of a potent anthrax vaccine.

Muramyl dipeptide potentiates a <i>Bacillus anthracis</i> poly-γ-<small>D</small>-glutamic acid capsule surrogate that induces maturation and activation of mouse dendritic cells

Jeon, Jun Ho,Park, Deok-Bum,Woo, Sun-Je,Lee, Hae-Ri,Park, Ok-Kyu,Park, Jungchan,Rhie, Gi-eun Elsevier 2018 Cytokine Vol.110 No.-

<P><B>Abstract</B></P> <P>Poly-γ-<SMALL>D</SMALL>-glutamic acid (PGA) of anthrax is an important pathogenic factor due to its anti-phagocytic activity. Additionally, PGA has the ability to activate mouse macrophages for the secretion of cytokines through Toll-like receptor (TLR) 2. Peptidoglycan (PGN), a major bacterial cell-wall component, induces inflammatory responses in the host. We assessed whether PGA can induce maturation and cytokine expression in immature mouse dendritic cells (DCs) in the existence of muramyl dipeptide (MDP), the minimum motif of PGN with immunostimulatory activity. Stimulation of immature DCs with PGA or MDP alone augmented expression of costimulatory molecules and MHC class II proteins, which are all cell surface markers indicative of maturation. The observed effects were further enhanced by costimulation of PGA and MDP. PGA alone was sufficient to induce expression of TNF-α, IL-6, MCP-1, and MIP1-α, whereas MDP alone did not under the same conditions. Treatment with MDP enhanced PGA-induced expression of the tested inflammatory mediators; however, the synergistic effect found for PGA and MDP was not observed in TLR2- or nucleotide-binding oligomerization domain (NOD) 2–knockout DCs. Additionally, MDP augmented PGA-induced MAP kinases and NF-κB activation, which is crucial for expression of cytokines. Furthermore, MAP kinase and NF-κB inhibitors attenuated MDP enhancement of PGA-induced cytokine production. In addition, co-culture of splenocytes and PGA/MDP-matured DCs induced higher expression of IL-2 and IFN-γ compared to that of splenocytes and PGA-matured DCs. Collectively, our results suggest that PGA and MDP cooperatively induce inflammatory responses in mouse DCs through TLR2 and NOD2 via MAP kinase and NF-κB pathways, subsequently leading to lymphocyte activation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> MDP enhances PGA-induced maturation of mouse DCs. </LI> <LI> MDP enhances production of inflammatory mediators by PGA. </LI> <LI> TLR2, NOD2, MAP kinase, and NF-κB signaling pathways are involved in PGA/MDP-induced cytokine production. </LI> <LI> MDP increases lymphocyte activation induced by PGA-matured DCs. </LI> </UL> </P>