Triggering the ExoS regulon of Pseudomonas aeruginosa: A GFP-reporter analysis of exoenzyme (Exo) S, ExoT and ExoU synthesis

Abstract

The ExoS regulon of Pseudomonas aeruginosa encodes diverse type III secreted effector proteins which have been shown to exert cytotoxic effects in cell culture experiments. However, little information exists about the environmental conditions and stimuli for upregulation of the ExoS regulon. Translational reporter fusion proteins of exoenzyme (Exo) S, ExoT and ExoU, as well as the type II secreted exotoxin A (ETA) to the green fluorescent protein (GFP), were constructed in order to compare exoprotein production under diverse growth conditions. Reporter protein activity was recorded by FACS-analysis and by conventional and confocal laser scanning microscopy. Low ion concentration induced co-ordinated upregulation of ExoS, ExoT and ExoU with a maximum effect at 37 degrees C. A dose-dependent upregulation was seen with human serum or increasing NaCl concentrations. A type III secretion-negative pcrD mutant of P. aeruginosa showed a weak ExoS response to environmental stimuli, compared with the parental strain, suggesting a negative regulatory mechanism. Co-culture with the mammalian cell lines J774A.1 or HeLa led to rapid upregulation of ExoS, ExoT and ExoU synthesis. These data suggest that the ExoS regulon of P. aeruginosa can be triggered by a variety of environmental signals as well as by cell contact with eukaryotic cells.