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.
Full-textDOI: · Available from: Richard B Thompson, Jul 04, 2015
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ABSTRACT: The complexities of bacterial gene expression during mammalian infection cannot be addressed by in vitro experiments. We know that the infected host represents a complex and dynamic environment, which is modified during the infection process, presenting a variety of stimuli to which the pathogen must respond if it is to be successful. This response involves hundreds of ivi (in vivo-induced) genes which have recently been identified in animal and cell culture models using a variety of technologies including in vivo expression technology, differential fluorescence induction, subtractive hybridization and differential display. Proteomic analysis is beginning to be used to identify IVI proteins, and has benefited from the availability of genome sequences for increasing numbers of bacterial pathogens. The patterns of bacterial gene expression during infection remain to be investigated. Are ivi genes expressed in an organ-specific or cell-type-specific fashion? New approaches are required to answer these questions. The uses of the immunologically based in vivo antigen technology system, in situ PCR and DNA microarray analysis are considered. This review considers existing methods for examining bacterial gene expression in vivo, and describes emerging approaches that should further our understanding in the future.Philosophical Transactions of The Royal Society B Biological Sciences 06/2000; 355(1397):601-11. DOI:10.1098/rstb.2000.0601 · 6.31 Impact Factor
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ABSTRACT: Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal-host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro-inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL-8 chemokine and reduced secretion of IL-6 cytokine. Gonococcal challenge also caused increased secretion of TNF-alpha cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non-adherent bacteria may be involved in TNF-alpha induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.Molecular Microbiology 02/2000; 35(1):32-43. DOI:10.1046/j.1365-2958.2000.01694.x · 5.03 Impact Factor
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ABSTRACT: Proteus mirabilis, a gram negative bacterium, represents a common cause of complicated urinary tract infections (UTIs) in catheterized patients or those with functional or anatomical abnormalities of the urinary tract. To systematically identify surface-exposed antigens, proteins in the outer membrane fraction of bacteria were separated by 2D gel electrophoresis and subjected to Western blotting with sera from mice experimentally infected with P. mirabilis. Proteins recognized by sera were identified by mass spectrometry. Thirty-seven antigens (including 24 outer membrane proteins) to which a humoral response had been mounted were identified; these antigens are presumably expressed during infection and therefore represent potential virulence factors. Six representative antigens were selected for further study. Of these antigens, three played no apparent role in pathogenesis, as strains with isogenic mutations were not attenuated in the mouse model of ascending UTI: a putative secreted 5’-nucleotidase (PMI0047), RafY (PMI0288), and FadL (PMI1810). However, two putative iron acquisition proteins, PMI0842 and PMI2596, both contribute to fitness in the urinary tract. The sixth antigen, ZnuB (PMI1150), was annotated as the inner membrane component of the high affinity zinc (Zn2+) transport system ZnuACB. Components of this system have been shown to contribute to virulence in other pathogens; therefore, the role of ZnuACB in P. mirabilis was investigated by constructing a strain with an insertionally interrupted copy of znuC. The znuC::kan mutant was more sensitive to zinc limitation than wild type, was outcompeted by wild type in minimal medium, displayed reduced swimming and swarming motility, and produced less flaA transcript and flagellin protein. Production of flagellin and swarming motility were restored by complementation with znuCB in trans. Swarming motility was also restored by the addition of Zn2+ to agar prior to inoculation. ZnuC offers a competitive advantage during urinary tract infection. Since we demonstrated a role for PMI0842, PMI2596, and ZnuC in UTI, we hypothesize that there is limited iron and zinc present in the urinary tract and that P. mirabilis must scavenge these ions to colonize and persist in the host.