Pseudomonas aeruginosa

P. aeruginosa is an opportunistic pathogen causing pneumonia in cystic fibrosis and hospitalized patients [49, 50]. A study by Ichikawa et al. [51] investigated the host response of the human lung carcinoma A549 epithelial cell upon exposure to P. aeruginosa strain PAK and an isogenic strain lacking expression of type IV pili. Since type IV pili are essential for adherence, this study dissected the gene expression in host cells infected with a P. aeruginosa strain which is able to adhere to epithelial cells and a strain which can not. Using high-density DNA microarrays consisting of 1506 human cDNA clones, gene expression analysis was carried out. A total of 22 genes were at least two-fold differentially expressed, including several inflammatory genes. Sixteen genes were found to be at least two-fold differentially expressed if the pilA- strain and pilA+ strains were compared. In addition, 11 genes were expressed upon infection with both the pilA- and pilA+ strains. The response upon pilA+ strain infection showed similarities to the profiles found upon infection with cagPAI+ H. pylori and plasmidless Y. enterocolitica.

Motile P. aeruginosa phenotypes characterized by the presence of flagella are essential in the establishment of acute infection, while mucoid P. aeruginosa phe-notypes characterized by the production of the polysaccharide alginate are critical in the development of chronic infections. To compare the host gene expression upon infection with motile and mucoid P. aeruginosa strains, Calu3 human airway epithelial cells were infected with different alginate and flagellin mutants [52].

Subsequently, gene expression profiling was performed using arrays comprising 14 239 human genes. The pattern of gene expression induced by flagellin+ strains includes innate host defense genes, proinflammatory cytokines, and chemokines representing the typical set of NF-jB- and TNF-regulated genes. In contrast, mucoid P. aeruginosa strains led to an overall attenuation of host response, and do not induce NF-jB activity, which is in line with the finding that almost no inflammatory response genes are induced by mucoid P. aeruginosa. The flagellin-mediat-ed host response is mediated by TLR-5. While alginate is known to signal through TLR-2 and TLR-4 in macrophages [53], alginate does not elicit a proinflammatory response in epithelial cells [54] because epithelial cells may lack components of TLR signaling.

Another study highlighted the role of type III secretion systems of P. aeruginosa for modulation of gene expression [55]. P. aeruginosa PA103, a nonmotile strain deficient for synthesis of complete flagellar filament, secretes ExoU and ExoT [56]. ExoU is a potent cytotoxin whose host cell targets and mechanism of action are not yet known [57, 58, 59]. ExoT is a bifunctional protein possessing an N-termi-nal GTPase-activating domain with GAP activity for RhoA, Rac, and Cdc42 and a C-terminal ADP-ribosyltransferase domain [60, 61]. Microarray analysis (a total of 9243 total cDNA elements) of a human tracheal epithelium cell line exposed to P. aeruginosa PA103 or various isogenic PA103 mutants such as exoT, exoU, exoUT, pscJ, which results in a nonfunctional type III secretion apparatus (TTSS), and pilA, a type IV pili mutant, was performed [55]. The authors reported 46 unique genes regulated by PA103. Further analysis defined 28 genes which were TTSS-independently regulated. Among these, several genes, such as VEGF-a, IGFBP3, adrenomedullin, stanniocalcinl, and NF-IL3 are known to be regulated by the transcription factor HIF-1a [62-65], suggesting that PA103 products induce expression of genes regulated by HIFla.

Fourteen genes were found to be regulated by ExoU. This group revealed a striking number of genes involved in transcriptional regulation, such as RhoB, which may inhibit NF-jB activation [45], tristetraprolin, which attenuates the mRNA stability of TNF-a [66], dual specificity phosphatase 1 (DUSP1, MKP1), which inactivates MAP kinases [67], and transcription factor 8 (TCF8, ZEB, NIL2A), which is known to be a transcriptional repressor [68, 69]. In addition, Exo U induced AP-1 activation which coincided with expression of AP-1-regulated genes such as MCP1, and IL-6.

Cystic fibrosis is a monogenic disorder characterized by a dysfunction of the cystic fibrosis transmembrane conductance regulator CFTR [50], resulting in disturbed ion transport across epithelia and a severe form of lung disease that is characterized by chronic infection with P. aeruginosa and S. aureus. The mechanism by which CFTR mutation leads to lung disease is not well understood, although recent studies show that a common mutant form of CFTR (DF408) leads to a cell stress response resulting in increased NF-kB activation [70]. Virella-Lowell et al. [71] addressed the question how CFTR deficiency may affect response of epithelial cells exposed to P. aeruginosa. For this purpose the epithelial cell line IB3-1 showing CFTR genotype DF408 and the isogenic transduced cell line with low-level expression of wildtype CFTR were compared with regard to gene expression. Infection with P. aeruginosa PAO1 showed that CFTR deficiency exaggerated activation of typical NF-jB-activated genes, cytokine receptors such as IL15Ra, IL-18R, and genes involved in activation of IFN signaling such as IRF3 and STAT1. Moreover, enzymes involved in protease inhibition, and enzymes involved in metabolism showed blunted activation. Thus, CFTR mutations change both the constitutive and the P. aeruginosa-induced host gene expression. Further studies will have to reveal which of these changes have an impact on the outcome of the chronic infection.

Taken together, various virulence factors of Pseudomonas account for different activation programs in epithelial cells: (a) flagellin triggers NF-jB activation and proinflammatory gene expression, (b) ExoU accounts for a transcriptional repression program and activation of AP-1, and (c) as yet unrecognized bacterial factors account for a HIF-1a-mediated gene expression program.

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