Effects of differential expression of the 49-kilodalton exoenzyme S by Pseudomonas aeruginosa on cultured eukaryotic cells

Abstract

Production of the ADP-ribosylating enzyme exoenzyme S (ExoS) by Pseudomonas aeruginosa has been associated with increased virulence. Previous studies, however, have been unable to confirm an effect of soluble ExoS in cell culture or animal model systems. To determine if bacteria must come in contact with target cells in order for an effect of ExoS to be observed, coculture systems were developed to compare the effects of ExoS- and non-ExoS-producing bacteria on eukaryotic cell function. The two P. aeruginosa strains used in these studies, 388 and 388delta exoS, maintained genetic identity, with the exception that strain 388delta exoS lacked production of the 49-kDa form of ExoS. When bacteria were cocultured with Detroit 532 fibroblastic cells, ExoS-producing 388 bacteria caused a significant decrease in DNA synthesis and viability compared to the decrease caused by non-ExoS-producing 388delta exoS bacteria. Maximal differences between the two strains were observed when 10(4) to 10(7) CFU of bacteria/ml were cocultured with Detroit cells for 4 or 6 h. Both strains were effective in eliminating Detroit cell DNA synthesis after a 20-h coculture period. Secreted ExoS had no effect on Detroit cell growth and viability, indicating that bacteria must have contact with target cells for the effect of ExoS on cellular function to be observed. Similar effects on cell proliferation and viability were observed when the two strains were cocultured with the KB epithelioid cell line. ExoS-associated decreases in eukaryotic cell viability were not found to be mediated by an inhibition of protein synthesis. These studies confirm that the 49-kDa ExoS contributes to the cellular pathogenesis of P. aeruginosa by interfering with eukaryotic cell growth and viability. In addition, the coculture system developed which recognizes this effect should provide a means for defining the function of ExoS in vivo.