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4.  Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells 
BMC Microbiology  2010;10:1.
Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells.
Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter.
Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.
PMCID: PMC2824691  PMID: 20051107
5.  NF-κB activation by Helicobacter pylori requires Akt-mediated phosphorylation of p65 
BMC Microbiology  2009;9:36.
The inflammatory response in Helicobacter pylori-infected gastric tissue is mediated by cag pathogenicity island (PAI)-dependent activation of nuclear factor-κB (NF-κB). Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is known to play a role in NF-κB activation, but little information is available on the relationship between H. pylori and PI3K/Akt signaling in gastric epithelial cells. We examined whether H. pylori activates Akt in gastric epithelial cells, the role of cag PAI in this process and the role of Akt in regulating H. pylori-induced NF-κB activation.
Phosphorylated Akt was detected in epithelial cells of H. pylori-positive gastric tissues. Although Akt was activated in MKN45 and AGS cells by coculture with cag PAI-positive H. pylori strains, a cag PAI-negative mutant showed no activation of Akt. H. pylori also induced p65 phosphorylation. PI3K inhibitor suppressed H. pylori-induced p65 phosphorylation and NF-κB transactivation, as well as interleukin-8 expression. Furthermore, transfection with a dominant-negative Akt inhibited H. pylori-induced NF-κB transactivation. Transfection with small interference RNAs for p65 and Akt also inhibited H. pylori-induced interleukin-8 expression.
The results suggest that cag PAI-positive H. pylori activates Akt in gastric epithelial cells and this may contribute to H. pylori-mediated NF-κB activation associated with mucosal inflammation and carcinogenesis.
PMCID: PMC2653507  PMID: 19216748
6.  Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17 
BMC Microbiology  2009;9:11.
Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays.
Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (σE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media.
These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.
PMCID: PMC2633007  PMID: 19146705
7.  Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells 
BMC Microbiology  2007;7:102.
Legionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines.
Infection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-κB.
Collectively, these results suggest that L. pneumophila induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells.
PMCID: PMC2213657  PMID: 18034886

Results 1-7 (7)