Proteus mirabilis, a common cause of bacteriuria and the most frequently described cause of infection-induced urinary tract and kidney stones, produces a urease that is inducible with urea. This enzyme catalyzes the hydrolysis of urea to CO2 and NH3 initiating formation of struvite (MgNH4PO46H2O) or carbonate-apatite (Ca10(PO4CO3OH)6(OH)) stones. Using a genetically-constructed urease-negative mutant, we have demonstrated the enzyme to be a critical virulence determinant for this uropathogen by evaluation in the CBA mouse model of ascending urinary tract infection. Although we have determined many of the physical properties of the enzyme and the genetic organization and nucleotide sequence of the P. mirabilis urease operon, there are many features of the operon that are not understood including regulation of enzyme synthesis, transcriptional organization, the role of accessory polypeptides and the active site amino acids involved in catalysis. This proposal outlines a series of experiments necessary to gain a better understanding of urease operon of P. mirabilis. Specifically, we propose to: 1) determine whether the P. mirabilis urease operon is regulated by a trans-acting factor which confers inducibility upon the operon in the presence of urea, a nitrogen regulation system (Ntr), catabolite repression, or transcriptional activation by nickel ion; 2) isolate and sequence the regulatory gene(s) for the urease operon; 3) determine the transcriptional organization of the urease operon; 4) measure the inducibility of the urease operon by urease inhibitors; 5) determine the role of accessory polypeptides of the urease operon; and 6) locate the active site of urease by site-directed mutagenesis. These areas represent gaps in our present knowledge of this critical virulence determinant. An understanding of these processes may provide new targets for therapeutic inhibition of urease.
| 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 |
