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 with gfp (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 DH5alpha 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 of P. mirabilis virulence genes in experimental urinary tract infection.

FIG. 1

Construction of a ureD-gfp fusion. Intact ureR and part of ureD were cloned as an EcoRI/BamHI fragment from pMIR10DZ (18), which is a subclone of pMID1010, into pBluescript to form pURE-RD. A fragment carrying the gfp open reading frame (see text) amplified by PCR from pGFPmut2 (9) was cloned into pCRScript; the BamHI fragment from this plasmid was isolated and ligated into BamHI-digested pURE-RD. The final construct was designated pURE-RD-GFP.

FIG. 2

Western blot analysis of UreD-GFP fusion protein induction by urea. E. coli (A) and P. mirabilis (B) carrying pURE-RD-GFP were uninduced or induced by urea (10, 50, 100, 200, and 500 mM). Soluble protein from these strains was electrophoresed on an SDS–12% polyacrylamide gel. A polyclonal antiserum raised in rabbits against recombinant GFP was used for Western blotting.

FIG. 3

Fluorescence emission spectra of soluble extracts from E. coli DH5α(pURE-RD-GFP) (A) and P. mirabilis HI4320(pURE-RD-GFP) (B). Bacteria were induced with 0 (——), 50 (———), and 250 (–––) mM urea.

FIG. 4

In vivo expression of UreD-GFP by P. mirabilis infecting the bladders and kidneys of CBA mice. Thin sections of bladders (A to H) and kidneys (I to K) obtained from CBA mice infected with P. mirabilis HI4320(pURE-RD-GFP) were observed by fluorescence microscopy. Fluorescent bacteria are identified by arrows. (F and H) Phase-contrast images of panels E and G, respectively.