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Study on the identification of immunogens and the host immune responses to Bacillus anthracis in murine macrophages

  • 김연희 (대학원 생명과학과 세포생물학)

초록/요약

Anthrax is caused by the spore-forming bacterium Bacillus anthracis¬, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (Alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (Malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (Protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells. 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. 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. Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.

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Table of Contents
Table of Contents i
List of Figures iv
List of Abbreviations vi
Abstract viii

Ⅰ. INTRODUCTION 1

Ⅱ. MATERIALS AND METHODS
1. Materials 8
2. Cell cultures 8
3. Bacterial culture and protein extraction 9
4. Preparation of anti-spore sera 10
5. Electrophoresis and western blots 12
6. DNA constructs and expression of recombinant proteins 14
7. GP immunization and in vivo protection assays 16
8. Genomic DNA preparation 16
9. Spore preparation and infection 17
10. ODN2088 and CQ treatment upon spore infection and BAG stimulation 18
11. Real-time RT-PCR 19
12. Transfection with small interfering RNA (siRNA) 20
13. TNF-α ELISA 21
14. Reporter gene assay 21
15. Immunofluorescence microscopy 22
16. Determination of the effect of BAG and TNF- on LT-mediated cytotoxicity 23
17. Quantification of BAG after infection 24
18. In vivo challenge with LT, BAG and TNF-α inhibitor 25
19. Statistical analysis 26

Ⅲ. RESULTS (Part I)
: Identification of Bacillus anthracis immunogens by immunoproteomics

1. Immunoreactivity of B. anthracis H9401 proteins 27
2. Identification of immunoreactive proteins 29
3. Selection and expression of vaccine candidates 33
4. Protective efficacy of candidate proteins in GP 35
5. Localization of GBAA_0345 40

Ⅳ. RESULTS (Part II)
: Host innate immune response against Bacillus anthracis

1. Infection with B. anthracis spores enhances TLR9 mRNA and TNF-α expression 43
2. BAG induces TNF-α protein and mRNA expression in a dose- and time-dependent manner 45
3. BAG stimulates TNF-α production in a TLR9-dependent manner 47
4.MAPK pathways are essential for BAG-induced TNF-α production 50
5. BAG and B. anthracis spores induce NF-κB activation 52
6. Treatment with BAG or TNF-α enhances the cytotoxic activity of LT on the macrophage cell line J774A.1 55
7. Genomic DNA is detected during anthrax infection and enhances LT-mediated lethality in mice 59
Ⅴ. DISCUSSION 62
Ⅵ. REFERENCES 80
Abstract in Korean 100

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