Analysis of Fusobacterium nucleatum antigens: identification of in vivo-induced antigens and functional analysis of GroEL, a heat shock protein

Abstract

Objectives Periodontitis is one of the most predominant chronic inflammations of the oral cavity, and has been reported to be associated with systemic diseases. Among the periodontal pathogens, Fusobacterium nucleatum is one of the most predominant bacterium found in periodontitis and plays an important role for subgingival biofilm formation by mediating multiple interactions between early and late colonizers. Accumulating data have demonstrated an association between periodontal pathogens and cardiovascular diseases. And bacterial GroEL, a homologus protein of human heat shock protein 60, is represented as an additional risk factor for atherosclerosis progression. Despite the importance of F. nucleatum in human disease, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. The purpose of this study is to identify the F. nucleatum genes induced in vivo using in vivo-induced antigen technology (IVIAT) and to investigate whether GroEL of F. nucleatum induces factors that predispose to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE-/-) mice. Methods To identify specifically expressed F. nucleatum genes during infection, an immune-screening technique termed IVIAT was performed. An IPTG-inducible expression library of F. nucleatum was constructed using pET30 expression vector and F. nucleatum genomic DNA. Total 30,000 recombinant clones of a F. nucleatum genomic DNA expression library were reacted with pooled sera from 10 periodontitis patients pre-adsorbed against in vitro-grown F. nucleatum and Escherichia coli. The reproducibly reactive clones were selected and the inserted DNA sequences in expression vector were analyzed by sequencing to identify the genes. To verify in vivo-induction of genes, the expression of 10 selected genes among the IVIAT-identified genes were analyzed by real time RT-PCR after F. nucleatum was infected into a human epithelial cell line, HOK-16B cells. To investigate the role of F. nucleatum GroEL in the progression of atherosclerosis, recombinant GroEL (rGroEL) of F. nucleatum was produced and tested for its endotoxin decontamination. Invasion assay of F. nucleatum into an endothelial cells, HMEC-1, was performed and then intracellular bacteria were observed and quantified by confocal laser scanning microscopy and flow cytometry, respectively. Anti-F. nucleatum GroEL Ab in serum and F. nucleatum DNA in gingival crevicular fluid were analyzed in periodontitis patients and healthy subjects. rGroEL-treated HMEC-1 cells were analyzed for the expression of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, tissue factor (TF), and tissue factor pathway inhibitor (TFPI). rGroEL-induced foam cell formation, a hall mark of atherosclerotic lesion, was detected. And the monocyte adhesion to HMEC-1 and transendothelial migration activity were also tested. To explore the effect of F. nucleatum and rGroEL on atherosclerotic lesion progression, 10-week old ApoE-/- mice were fed high fat diet and mice were injected with live F. nucleatum (5 × 107 CFU/mouse), rGroEL (50 μg/mouse), or PBS once a week for 8 weeks. The atherosclerotic lesion in proximal aorta of the mice was detected and the risk markers (C-reactive protein (CRP), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and IL-6) of atherosclerosis were analyzed in serum. And the intracellular signaling pathway of GroEL in HMEC-1 was analyzed using specific inhibitors of MAPK and NF-κB and TLR4 siRNA knockdown system. Results For detection of in vivo-induced genes, 87 clones showed reproducibly the reactivity with pooled sera from 10 periodontitis patients. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. The expression of 10 selected in vivo-induced genes were verified to be increased in F. nucleatum after infection into HOK-16B cells, an epithelial cell line. F. nucleatum invaded the HMEC-1 cells and GroEL induced the expression of chemokines such as MCP-1 and IL-8 as well as cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin. GroEL induced the activity of TF and reduced the activity of the TFPI. Foam cell formation was induced by GroEL. GroEL-injected ApoE-/- mice showed significant atherosclerotic lesion progression compared to control mice. Serum levels of risk factors for atherosclerosis such as IL-6, CRP, and LDL were increased in GroEL-injected ApoE-/- mice compared to control mice, whereas serum levels of HDL were decreased. There were significantly higher levels of anti-F. nucleatum GroEL Ab in serum and F. nucleatum DNA in gingival crevicular fluid in periodontitis patients than those of healthy subjects. The expression levels of GroEL-induced inflammatory mediators were mediated by activation of NF-κB and MAPKs through TLR4 signaling pathway. Conclusion In vivo-induced 32 immunogenic proteins of F. nucleatum could be used to screen antibacterial and anti-inflammatory compounds using a cell infection model. The ability of F. nucleatum GroEL to induce the expression of host factors that were involved in atherosclerosis progression in endothelial cells and ApoE-deficient mice models supports the association of periodontitis and systemic infection.