Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

Abstract

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made withgfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33–38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5α and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, 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 reporter protein is a viable approach to investigate in vivo expression ofP. mirabilis virulence genes in experimental urinary tract infection.

Interaction of primary human endometrial cells with Neisseria gonorrhoeae expressing green fluorescent protein

“…To our knowledge, this is the first report of the stable transformation of Neisseria species with GFP, and its subsequent use in infection studies in vitro . GFP has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms (Dhandayuthapani et al ., 1995; Webb et al ., 1997), and more recently has been used in studies of the pathogenicity of various bacterial species, including Salmonella typhimurium , Yersinia pseudotuberculosis and Mycobacterium marinarum (Valdivia et al ., 1996), Bartonella henselae (Dehio et al ., 1998b) and Proteus mirabilis (Zhao et al ., 1998). In the present study, use of the GFP protein offered many advantages, notably obviating exposure of endometrial cells to permeabilizing treatments and subsequent detection of gonococci by indirect labelling with specific antibody and secondary conjugates.…”

Proteus mirabilisand Urinary Tract Infections

“…UreR also positively regulates its own expression when bound to the intergenic region between ureR and ureD, and ureR transcription is repressed by the global regulator H-NS (23). Urease expression is induced in the urinary tract of experimentally-infected mice (24,25).…”

Regulation of capsule gene expression by group A Streptococcus during pharyngeal colonization and invasive infection

“…Transcriptional or translational fusions to gfp have been used to assess the expression of virulence factors both in Gram‐negative and Gram‐positive pathogens (Dhandayuthapani et al ., 1995; Valdivia and Falkow, 1997; Cheung et al ., 1998; Jacobi et al ., 1998; Zhao et al ., 1998; Bubert et al ., 1999). To investigate regulation of GAS capsule gene expression, the GFPuv variant of green fluorescent protein was selected for use as a reporter system because of its low toxicity and high fluorescence emission in bacteria (Crameri et al ., 1996).…”