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

The ExoS regulon of Pseudomonas aeruginosa encodes diverse type III secrete d effector proteins which have been shown to exert cytotoxic effects in cel l culture experiments. However, little information exists about the environ mental conditions and stimuli for upregulation of the ExoS regulon. Transla tional reporter fusion proteins of exoenzyme (Exo) S, ExoT and ExoU, as wel l as the type II secreted exotoxin A (ETA) to the green fluorescent protein (GFP), were constructed in order to compare exoprotein production under di verse growth conditions. Reporter protein activity was recorded by FACS-ana lysis and by conventional and confocal laser scanning microscopy. Low ion c oncentration induced co-ordinated upregulation of ExoS, ExoT and ExoU with a maximum effect at 37 degreesC. A dose-dependent upregulation was seen wit h human serum or increasing NaCl concentrations. A type III secretion-negat ive pcrD mutant of P. aeruginosa showed a weak ExoS response to environment al stimuli, compared with the parental strain, suggesting a negative regula tory mechanism. Co-culture with the mammalian cell lines J774A.1 or HeLa le d to rapid upregulation of ExoS, ExoT and ExoU synthesis. These data sugges t that the ExoS regulon of P. aeruginosa can be triggered by a variety of e nvironmental signals as well as by cell contact with eukaryotic cells. (C) 2000 Academic Press.