Urinary tract infections are the most frequently diagnosed kidney and urological disorders. While E. coli is the most common isolate in normal individuals, those with complicated urinary tracts, i.e., those with functional or anatomic abnormalities or with chronic catheterization are very frequently infected with Proteus mirabilis. In addition to cystitis and acute pyelonephritis, these infections are further complicated by the production of urinary stones, a hallmark of infection with this organism. Urea, the principal nitrogenous waste product of humans (present at nearly 0.5 M in urine) induces urease synthesis in P. mirabilis and is hydrolyzed by this enzyme. NH3, a product of hydrolysis, elevates urine pH causing precipitation of normally soluble ions forming struvite (MgNH4PO4) or carbonate-apatite (Ca10(PO4)6.CO3) stones. Although our understanding of the molecular biology of urease has been significantly advanced over the last five years, there are two areas that remain obscure.
As specific aims, the investigators plan: 1. To determine the mechanism for urea-inducible, UreR-mediated, positive transcriptional activation of the Proteus mirabilis urease gene cluster; and 2. To determine the specific interactions of the urease accessory proteins UreD, UreE, UreF, and UreG with the urease [)UreA-UreB-UreC)3].
In specific aim 1, the regulation of urease gene expression will be examined using DNA-protein interaction methodology including gel shift and footprinting.
In specific aim 2, the role of accessory proteins in assembly of a catalytically active urease will be examined using the yeast two-hybrid system and direct biochemical identification of interacting polypeptides. This project focuses on urease, a documented and potent virulence determinant of P. mirabilis which is central to the pathogenesis caused by this species. By understanding the mechanisms of induction of the urease genes and assembly of active enzyme, the long term objective of effective management of complicated UTI will be realized.
| Shen, Z; Schauer, D B; Mobley, H L et al. (1998) Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB. J Clin Microbiol 36:2447-53 |
