BMC Microbiology, 2014, 01, 300─312

Bacillus anthracis genomic DNA enhances lethal toxin？induced cytotoxicity through TNF-α production

JunHo Jeon, YeonHee Kim, M Choi,K Kim,H Lee,J Jang,J Chun,G Rhie

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.

• ISBN or ISSN: 1471-2180

• 본 연구는 질병관리본부 연구개발과제(과제번호 2009-N45001-00) 연구비를 지원받아 수행되었습니다.
• This research was supported by a fund(code 2009-N45001-00) by Research of Korea Centers for Disease Control and Prevention.