title: Bacillus anthracis genomic DNA enhances lethal toxin-induced cytotoxicity through TNF-α production.
abstract: BACKGROUND
  Bacillus anthracis is the etiological agent of anthrax. Lethal toxin (LT) produced by B. anthracis is a well-known key virulence factor for anthrax because of its strong cytotoxic activity. However, little is known about the role of B. anthracis genomic DNA (BAG) in anthrax pathogenesis.
  
  
  RESULTS
  We examined the effect of BAG on TNF-α production and LT-mediated cytotoxicity during B. anthracis spore infection in mouse macrophage cell lines (RAW264.7 cells and J774A.1) and BALB/c mice. Infection of RAW264.7 cells with B. anthracis spores induced TNF-α expression in a multiplicity of infection (MOI)-dependent manner, and this enhancement was attenuated by the toll-like receptor (TLR) 9 inhibitor oligodeoxynucleotide (ODN)2088. BAG led to TNF-α expression in a dose- and time-dependent manner when applied to RAW264.7 cells. TNF-α expression induced by BAG was reduced by either pretreatment with TLR9 inhibitors (ODN2088 and chloroquine (CQ)) or transfection with TLR9 siRNA. Furthermore, BAG-induced TNF-α production in TLR9(+/+) macrophages was completely abrogated in TLR9(-/-) macrophages. BAG enhanced the phosphorylation of mitogen-activated protein kinases (MAPK), and BAG-induced TNF-α expression was attenuated by pretreatment with MAPK inhibitors. A reporter gene assay and confocal microscopy demonstrated that BAG increased NF-κB activation, which is responsible for TNF-α expression. Treatment with BAG alone showed no cytotoxic activity on the macrophage cell line J774A.1, whereas LT-mediated cytotoxicity was enhanced by treatment with BAG or TNF-α. Enhanced LT-induced lethality was also confirmed by BAG administration in mice. Furthermore, LT plus BAG-mediated lethality was significantly recovered by administration of Infliximab, an anti-TNF-α monoclonal antibody.
  
  
  CONCLUSIONS
  Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. sasujun@hanmail.net.
authors.forename: Jun Ho
authors.initials: JH
authors.lastname: Jeon
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. hanikim@hanmail.net.|School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Republic of Korea. hanikim@hanmail.net.
authors.forename: Yeon Hee
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. mk6214@hanmail.net.
authors.forename: Min Kyung
authors.initials: MK
authors.lastname: Choi
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. kkaml3@hanmail.net.
authors.forename: Kyung Ae
authors.initials: KA
authors.lastname: Kim
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. haeruili@hotmail.com.
authors.forename: Hae-Ri
authors.initials: HR
authors.lastname: Lee
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. zzang9575@empas.com.
authors.forename: Jeyoun
authors.initials: J
authors.lastname: Jang
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. eul62@naver.com.
authors.forename: Yu-Ri
authors.initials: YR
authors.lastname: Kim
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. hg1117@korea.kr.
authors.forename: Jeong-Hoon
authors.initials: JH
authors.lastname: Chun
authors.affiliation: College of Veterinary medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju, 561-765, Republic of Korea. vetvirus@chonbuk.ac.kr.
authors.forename: Seong Kug
authors.initials: SK
authors.lastname: Eo
authors.affiliation: School of Life Sciences and Biotechnology, Korea University, Seoul, 136-701, Republic of Korea. tskim@korea.ac.kr.
authors.forename: Tae Sung
authors.initials: TS
authors.lastname: Kim
authors.affiliation: Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 187 Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk, 361-951, Republic of Korea. gerhie@nih.go.kr.
authors.forename: Gi-Eun
authors.initials: GE
authors.lastname: Rhie
mesh_terms: D000818:Animals; D000881:Anthrax / Q000473:pathology*; D000942:Antigens, Bacterial / Q000633:toxicity*; D001408:Bacillus anthracis / Q000472:pathogenicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D002460:Cell Line; D004269:DNA, Bacterial / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D008807:Mice, Inbred BALB C; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000633:toxicity*
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D014409:Tumor Necrosis Factor-alpha; C030325:anthrax toxin