USE OF GREEN FLUORESCENT PROTEIN TO ASSESS UREASE GENE-EXPRESSION BY UROPATHOGENIC PROTEUS-MIRABILIS DURING EXPERIMENTAL ASCENDING URINARY-TRACT INFECTION

Proteus mirabilis, a cause of complicated urinary tract infection, exp resses urease when exposed to urea, While it is recognized that the po sitive transcriptional activator UreR induces gene expression, the lev els of expression of the enzyme during experimental infection are not known, To investigate in vivo expression of P. mirabilis urease, the g ene encoding green fluorescent protein (GFP) was used to construct rep orter fusions, Translational fusions of urease accessory gene ureD, wh ich is preceded by a urea-inducible promoter, were made with gfp (modi fied to express S65T/V68L/S72A [B. P, Cormack et al, Gene 173:33-38, 1 996]), Constructs were confirmed by sequencing of the fusion junctions . UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5 alpha and P. mirabilis HI4320, By using Western blotting with ant iserum raised against GFP, expression level was shown to correlate,vit h urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mill urea, Fluorescent E. coli and P. mirabilis bacteri a were observed by fluorescence microscopy following urea induction, a nd the fluorescence intensity of GFP in cell lysates was measured by s pectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusi on plasmid was transurethrally inoculated into the bladders of CBA mic e, One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensit y of bacteria in bladder tissue was higher than that in the kidney, Ki dneys were primarily infected with single-cell-form fluorescent bacter ia, while aggregated bacterial clusters were observed in the bladder, Elongated swarmer cells were only rarely observed, These observations demonstrate that urease is expressed in vivo and that using GFP as a r eporter protein is a viable approach to investigate in vivo expression of P. mirabilis virulence genes in experimental urinary tract infecti on.