Pseudomonas aeruginosa Cytotoxicity Is Attenuated at High Cell Density and Associated with the Accumulation of Phenylacetic Acid

BACKGROUND: P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. METHODOLOGY/FINDINGS: Intriguingly, however, in this study we noticed that while a low cell density inoculum of P. aeruginosa caused severe cytotoxicity against human lung tissue cell line A549, increasing the cell density of bacterial inoculum led to decreased cytotoxicity. Addition of the supernatants from high density bacterial culture to low cell density inoculum protected the human cells from bacterial cytotoxic damage, suggesting that P. aeruginosa may produce and accumulate an inhibitory molecule(s) counteracting its pathogenic infection. The inhibitor was purified from the stationary-phase culture supernatants of P. aeruginosa strain PAO1 using bioassay-guided high performance liquid chromatography (HPLC), and characterized to be phenylacetic acid (PAA) by mass spectrometry and nuclear magnetic resonance spectroscopy. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of Type III secretion system (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. CONCLUSIONS: Identification of bacterial metabolite PAA as a T3SS-specific inhibitor explains this intriguing inverse cell-density-dependent-cytotoxicity phenomenon as T3SS is known to be a key virulence factor associated with cytotoxicity and acute infection. The findings may provide useful clues for design and development of new strategies to combat this formidable bacterial pathogen.