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

Abstract

Background P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues.Methodology/FindingsIntriguingly, 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.ConclusionsIdentification 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.

The attenuated virulence of a Burkholderia cenocepacia paaABCDE mutant is due to inhibition of quorum sensing by release of phenylacetic acid

“…Due to physical proximity and the fact that P. aeruginosa and Bcc species are able to utilize AHL molecules, it is reasonable to assume that there is cross‐talk between bacteria via QS (Lewenza et al ., ; Eberl and Tummler, ). P. aeruginosa is capable of releasing PAA when grown to high‐density levels, which in turn attenuates cytotoxicity (Wang et al ., ). In contrast to P. aeruginosa , where millimolar levels of PAA have an inhibitory effect on growth (Musthafa et al ., ), B. cenocepacia can grow with PAA as a sole carbon source (Law et al ., ) and PAA metabolism is active during Burkholderia growth on NGM (Fig.…”

“…Remarkably, in Pseudomonas aeruginosa , the most common cystic fibrosis pathogen (Kato and Takayama, ), PAA was proposed as an antagonist of pathogenic responses; production of PAA by P. aeruginosa at high cell density levels was demonstrated to reduce cytotoxicity (Wang et al ., ) and exogenous addition of synthetic PAA inhibited virulence traits (Musthafa et al ., ) known to be regulated by quorum sensing (QS) (Smith and Iglewski, ; Schuster and Greenberg, ). Intriguingly, P. aeruginosa genomes do not clearly encode PAA metabolism enzymes (Wang et al ., ) so how PAA is produced or whether exogenous PAA is internalized and metabolized in P. aeruginosa is unknown. Although a direct involvement of QS systems was not demonstrated during these studies, a possible link between PAA metabolism and QS‐regulated virulence emerged.…”

Mimicking the host and its microenvironmentin vitrofor studying mucosal infections byPseudomonas aeruginosa

“…The P. aeruginosa infectious disease process is most frequently studied using a single cell type grown on a nonpermeable surface (plastic or glass), referred to as two‐dimensional (2‐D) monolayers. Two‐dimensional monolayers of different cell lines have been used for study of lung disease (A549, 16HBE, CFBE) and intestinal disease (HeLa, T84, CaCo2, HT‐29) by P. aeruginosa (Saiman et al ., ; Pier et al ., , b; McNamara et al ., ; Moreau‐Marquis et al ., , b; Schaible et al ., ; Wang et al ., ; Weichert et al ., ). In addition, primary cells recovered from nasal brushing of human volunteers, human lung biopsies, human nasal polyps, and mouse lung grown as 2‐D monolayers have been used (Amitani et al ., ; Plotkowski et al ., ; Bajolet‐Laudinat et al ., ; de Courcey et al ., ).…”

Bacterial Interference With Lactate Dehydrogenase Assay Leads to an Underestimation of Cytotoxicity

“…In the present study we investigated whether bacteria that are often recovered from the lower respiratory tract of patients with chronic lung disease (i.e., Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Achromobacter xylosoxidans, Moraxella catarrhalis , and Rothia mucilaginosa ) could influence results obtained with the LDH assay when evaluating bacterial cell-based cytotoxicity in the context of host-pathogen interactions. Based on available literature, high cytotoxicity is generally expected after infection with the pathogens P. aeruginosa, S. aureus, K. pneumoniae , and S. maltophilia (Hawdon et al, 2010 ; Karaba et al, 2013 ; Wang et al, 2013 ; Wen et al, 2018 ). For P. aeruginosa and S. aureus , cytotoxicity toward airway epithelial cells was found to be strain-dependent (Hawdon et al, 2010 ; Strobel et al, 2016 ).…”