Abstract
Introduction. Although there is a significant body of evidence that bacteria activate a variety of pro-inflammatory signals following their interaction with human epithelial cells, there is virtually no information on signaling in the opposite direction. In this study we sought to determine whether host cell factors released by intestinal epithelial cells subjected to stress could signal the opportunistic pathogen, P. aeruginosa, to express a key virulence related protein, the PA-I lectin. Methods. Human intestinal epithelia (Caco-2 bbe) were grown to confluence in HDMEM media. To expose cells to a clinically relevant stress, hyperthermic conditions were created by immersing sealed culture dishes in a 42°C water bath for 23 min, followed by 2 h of recovery at 37°C and 5% CO 2. Control cells were maintained in 37°C at 5% CO 2.. Media were then filtered through a 0.22-μm filter before serial centrifugal membrane filtration using 100, 50, 30, 10, and 3 kDa filters and resuspended in HDMEM. GFP reporter strains of P. aeruginosa carrying a fusion construct for the PA-I lectin were then exposed to the epithelial cell derived filtrates from heat shock stressed Caco-2 cells. Dynamic fluorimetry was used to measure fluorescence (i.e. PA-I activity) over time. To determine if inducing compounds were proteins, fractions capable of activating PA-I were then heat inactivated by heating to 70°C for 30 min and tested against active fractions by the same method. Results. A 30- to 50-kDa media fraction from heat-stressed cells induced a 10-fold increase in PA-I promoter activity over controls, as measured by fluorescence (1082 ± 216%, P < 0.01), while all other fractions showed no effect. Heat inactivation completely eliminated this effect (−156% of control ± 293%, P = NS). Conclusion. Human intestinal epithelial cells release a soluble 30- to 50-kDa compound(s) that can activate a key virulence protein in P. aeruginosa. We speculate that the molecular dialogue between a pathogen and the intestinal epithelium is bidirectional and can be initiated by host stress conditions such as hyperthermia.
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