EF-Tu, a novel phosphorylcholine-containing protein involved in the interaction of chronic infectious Pseudomonas aeruginosa isolates with the airway epithelial cells
Abstract number: 1732_190
Barbier M., García L., Oliver A., Goldberg J.B., Albertí S.
Pseudomonas aeruginosa PAO1 grown at 22°C 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 37°C 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 37°C. This epitope is present on the cell surface and mediates the invasion of the airway epithelial cells via PAFr.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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