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O295 Alveolar macrophage phagocytosis and respiratory burst activity is regulated by Lyn-PI3Kinase-Akt pathway
P. aeruginosa pathogenesis and immunotherapeutics have important implications for the design of strategies addressed at controlling bacterial resistance based on antibiotic restriction policies. Results of studies on antibiotic resistance in settings of minimal antibiotic exposure (including investigations recently carried out in very remote human communities living in the Bolivian Chaco and in the Alto Amazonas jungle of South America, and among wild reptiles (land iguanas) from a remote and protected island of the Gal´apagos archipelago with no documented sources of antibiotic exposure and minimum human contacts) will be critically reviewed in this presentation, and the mechanisms potentially involved in the dissemination of resistant strains and resistance genes unrelated to antimicrobial consumption will be discussed. Results from the characterisation of resistance determinants carried by bacterial isolates from these settings pointed to their likely origin from antibiotic-exposed areas rather than to a local and independent resistance selection process. However, the mechanisms responsible for this flow of resistance genes and for their maintenance and spread in absence of sustained antibiotic use remain elusive.
Reference(s) [1] Gilliver M et al. Antibiotic resistance found in wild rodents. Nature 1999; 401: 233-4. [2] Bartoloni A et al. High prevalence of acquired antimicrobial resistance unrelated to heavy antimicrobial consumption. J Infect Dis 2004; 189: 1291-94. [3] Grenet K et al. Antibacterial resistance, Wayampis Ameridians, French Guyana. Emerg Infect Dis 2004; 10: 1150-3.
P. aeruginosa pathogenesis and immunotherapeutics O295 Alveolar macrophage phagocytosis and respiratory burst activity is regulated by Lyn–PI3Kinase–Akt pathway S. Kannan, A. Audet, H. Huang, M. Wu (Grand Forks, US) Introduction: Alveolar macrophages (AM) form the first line of defence in the lung alveoli. Pseudomonas aeruginosa (PA) is a common hospitalacquired infectious agent that can cause life-threatening infections in susceptible individuals. We hypothesise AM to have important function in early stages of PA infection. Rationale: Recently, we have shown that Lyn tyrosine kinase is critically involved in alveolar epithelial cell invasion with PA through lipid raft mechanism. Lyn is known to be involved in mast cell motility and generation of superoxide free radicals in neutrophils. Thus we propose a role for Lyn in AM function during PA infection. Methods: Akt activity was detected by immunoblotting with phospho Akt (Ser 473) antibody and by in vitro kinase assay. Lyn, PI3Kinase and Akt protein interactions were analysed by co-immunoprecipitation and Lyn-GST pull down assays. Phagosome formation and localisation of signaling proteins were studied by immunostaining with fluorescent antibodies followed by confocal microscopy. Lyn-YFP and PH-AktGFP co-transfected MHS cells were used for studying spatio-temporal association of Lyn and Akt during PA phagocytosis by live cell confocal microscopy. Phagosomes from infected cells were isolated by sucrose density gradient centrifugation for biochemical characterisation. Results: PA-infection induced Akt activity in MHS cells depends on Lyn function. Lyn, PI3Kinase and Akt were actively recruited to phagosome fractions in PA-infected cells. Both Lyn-YFP and PH-AktGFP were found to colocalise in the lamellipodium and phagocytic cup of PA-infected MHS cells from live cell imaging studies. GST pull down assay confirmed Lyn–Akt interaction. Respiratory burst activity depends on Lyn function. Conclusions: Our data indicates that Lyn–PI3Kinase–Akt pathway is crucial for regulating phagocytosis and respiratory burst activity of AM.
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Figure 1. Co-immunoprecipitation analysis shows that Lyn interacts with PI3Kinase (B) and Akt (A) upon PAO1 infection of MHS cells. This interaction is significantly affected by pretreatment with PP2 (Lyn inhibition) or LY2094002 (PI3Kinase inhibitor) (B,C). (D) Lyn dominant negative transfection decreases Lyn and Akt association. GSK3b is a downstream effector of Akt.
O296 EF-Tu, a novel phosphorylcholine-containing protein involved in the interaction of chronic infectious Pseudomonas aeruginosa isolates with the airway epithelial cells M. Barbier, L. Garc´ıa, A. Oliver, J.B. Goldberg, S. Albert´ı (Palma de Mallorca, ES; Charlottesville, US) Pseudomonas aeruginosa PAO1 grown at 22ºC expresses a 43-kDa protein that contains a phosphorylcholine (ChoP) epitope. In other respiratory pathogens, like Streptococcus pneumoniae and Haemophilus influenzae, this motif interacts with the airway epithelial cells via Platelet Activating Factor Receptor (PAFr). Objective: The objective of this study was to identify the 43-kDa ChoP containing protein and characterise its function in the virulence of P. aeruginosa. Methods: To identify the ChoP containing protein, bacterial cell fractions were separated by FPLC. Fractions were analysed by Western blot using specific monoclonal anti-ChoP antibodies and those that contained the ChoP epitope were further purified by SDS-PAGE. A 43-kDa protein containing the ChoP epitope was cut out of the gel, trypsinised, and subjected to capillary LC-MS and MS/MS. ChoP epitope expression of whole cell extracts of 92 genetic unrelated P. aeruginosa isolates (46 from chronic infections and 46 from acute infections) was analysed by Western blot analysis using specific monoclonal anti-ChoP antibody. ChoP epitope expression was also analysed by flow cytometry and immunofluorescent microscopy on intact cells of P. aeruginosa. To determine whether the ChoP epitope was involved in the interaction of the P. aeruginosa isolates with the respiratory epithelial cells, standard invasion assays were performed using 16HBE14- bronchoepithelial cells, either treated or untreated with a PAFr antagonist. Results: The 43-kDa ChoP containing protein was shown to be the elongation factor Tu (EF-Tu). The expression of the ChoP epitope at 37ºC was significantly more frequent in P. aeruginosa strains isolated from chronic infections (70%) than from acute infections (28%). The ChoP epitope was shown to be expressed on the outer surface of the bacterial cell and bacterial invasion was significantly inhibited by treatment with the PAFr antagonist compared to controls. Conclusions: We have demonstrated that in P. aeruginosa, the ChoP epitope is associated with EF-Tu. A high percentage of the isolates from chronic infections, in comparison with those from acute infections, express this epitope at 37ºC. This epitope is present on the cell surface and mediates the invasion of the airway epithelial cells via PAFr.
Reference(s) [1] Gilliver M et al. Antibiotic resistance found in wild rodents. Nature 1999; 401: 233-4. [2] Bartoloni A et al. High prevalence of acquired antimicrobial resistance unrelated to heavy antimicrobial consumption. J Infect Dis 2004; 189: 1291-94. [3] Grenet K et al. Antibacterial resistance, Wayampis Ameridians, French Guyana. Emerg Infect Dis 2004; 10: 1150-3.
P. aeruginosa pathogenesis and immunotherapeutics O295 Alveolar macrophage phagocytosis and respiratory burst activity is regulated by Lyn–PI3Kinase–Akt pathway S. Kannan, A. Audet, H. Huang, M. Wu (Grand Forks, US) Introduction: Alveolar macrophages (AM) form the first line of defence in the lung alveoli. Pseudomonas aeruginosa (PA) is a common hospitalacquired infectious agent that can cause life-threatening infections in susceptible individuals. We hypothesise AM to have important function in early stages of PA infection. Rationale: Recently, we have shown that Lyn tyrosine kinase is critically involved in alveolar epithelial cell invasion with PA through lipid raft mechanism. Lyn is known to be involved in mast cell motility and generation of superoxide free radicals in neutrophils. Thus we propose a role for Lyn in AM function during PA infection. Methods: Akt activity was detected by immunoblotting with phospho Akt (Ser 473) antibody and by in vitro kinase assay. Lyn, PI3Kinase and Akt protein interactions were analysed by co-immunoprecipitation and Lyn-GST pull down assays. Phagosome formation and localisation of signaling proteins were studied by immunostaining with fluorescent antibodies followed by confocal microscopy. Lyn-YFP and PH-AktGFP co-transfected MHS cells were used for studying spatio-temporal association of Lyn and Akt during PA phagocytosis by live cell confocal microscopy. Phagosomes from infected cells were isolated by sucrose density gradient centrifugation for biochemical characterisation. Results: PA-infection induced Akt activity in MHS cells depends on Lyn function. Lyn, PI3Kinase and Akt were actively recruited to phagosome fractions in PA-infected cells. Both Lyn-YFP and PH-AktGFP were found to colocalise in the lamellipodium and phagocytic cup of PA-infected MHS cells from live cell imaging studies. GST pull down assay confirmed Lyn–Akt interaction. Respiratory burst activity depends on Lyn function. Conclusions: Our data indicates that Lyn–PI3Kinase–Akt pathway is crucial for regulating phagocytosis and respiratory burst activity of AM.
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Figure 1. Co-immunoprecipitation analysis shows that Lyn interacts with PI3Kinase (B) and Akt (A) upon PAO1 infection of MHS cells. This interaction is significantly affected by pretreatment with PP2 (Lyn inhibition) or LY2094002 (PI3Kinase inhibitor) (B,C). (D) Lyn dominant negative transfection decreases Lyn and Akt association. GSK3b is a downstream effector of Akt.
O296 EF-Tu, a novel phosphorylcholine-containing protein involved in the interaction of chronic infectious Pseudomonas aeruginosa isolates with the airway epithelial cells M. Barbier, L. Garc´ıa, A. Oliver, J.B. Goldberg, S. Albert´ı (Palma de Mallorca, ES; Charlottesville, US) Pseudomonas aeruginosa PAO1 grown at 22ºC expresses a 43-kDa protein that contains a phosphorylcholine (ChoP) epitope. In other respiratory pathogens, like Streptococcus pneumoniae and Haemophilus influenzae, this motif interacts with the airway epithelial cells via Platelet Activating Factor Receptor (PAFr). Objective: The objective of this study was to identify the 43-kDa ChoP containing protein and characterise its function in the virulence of P. aeruginosa. Methods: To identify the ChoP containing protein, bacterial cell fractions were separated by FPLC. Fractions were analysed by Western blot using specific monoclonal anti-ChoP antibodies and those that contained the ChoP epitope were further purified by SDS-PAGE. A 43-kDa protein containing the ChoP epitope was cut out of the gel, trypsinised, and subjected to capillary LC-MS and MS/MS. ChoP epitope expression of whole cell extracts of 92 genetic unrelated P. aeruginosa isolates (46 from chronic infections and 46 from acute infections) was analysed by Western blot analysis using specific monoclonal anti-ChoP antibody. ChoP epitope expression was also analysed by flow cytometry and immunofluorescent microscopy on intact cells of P. aeruginosa. To determine whether the ChoP epitope was involved in the interaction of the P. aeruginosa isolates with the respiratory epithelial cells, standard invasion assays were performed using 16HBE14- bronchoepithelial cells, either treated or untreated with a PAFr antagonist. Results: The 43-kDa ChoP containing protein was shown to be the elongation factor Tu (EF-Tu). The expression of the ChoP epitope at 37ºC was significantly more frequent in P. aeruginosa strains isolated from chronic infections (70%) than from acute infections (28%). The ChoP epitope was shown to be expressed on the outer surface of the bacterial cell and bacterial invasion was significantly inhibited by treatment with the PAFr antagonist compared to controls. Conclusions: We have demonstrated that in P. aeruginosa, the ChoP epitope is associated with EF-Tu. A high percentage of the isolates from chronic infections, in comparison with those from acute infections, express this epitope at 37ºC. This epitope is present on the cell surface and mediates the invasion of the airway epithelial cells via PAFr.