Rhamnolipid-induced shedding of flagellin from Pseudomonas aeruginosa provokes hBD-2 and IL-8 response in human keratinocytes

Several 'pathogen-associated molecular pattern' (PAMP) of the opportunistic pathogen Pseudomonas aeruginosa activate the innate immune system in epithelial cells. Particularly the production of antimicrobial peptides such as the human beta-defensin-2 (hBD-2) and proinflammatory cytokines as the interleukin (IL)-8 is boosted. In the present study culture supernatants of static grown P. aeruginosa were found to be potent hBD-2 and IL-8 inducers, indicating a soluble or shedded PAMP, comparable to that of heat-killed bacterial supernatants. In subsequent analyses this PAMP was identified as flagellin, the major structural protein of the flagella. Flagellin is known to be an immunostimulatory potent factor, but the mechanisms by which P. aeruginosa is able to remove flagellin from the flagella remain unknown. Here we provide evidence for the presence of a factor responsible for release of flagellin from the flagella. Purification of this factor and subsequent mass spectrometry analyses identified rhamnolipids as responsible agents. Our findings indicate that maybe upon adhesion to surfaces P. aeruginosa alters the outer membrane composition in a rhamnolipid-depending manner, thereby shedding flagellin from the flagella. In turn epithelial cells recognize flagellin and cause the synthesis of antimicrobial peptides as well as recruitment of inflammatory cells by induction of proinflammatory cytokines.