Proteus mirabilis represents only a small percentage of the bacteria for causing uncomplicated cystitis or acute pyelonephritis in the normal host, but its importance in UT1 is underscored when the incidences of infection in patients with complicated urinary tracts, i.e., those with functional or anatomic abnormalities or with chronic instrumentation, are examined. In these cases, P. mirabilis is a more frequent uropathogen and its infection may be more severe due to the formation of urinary stones caused by the breakdown of urea by urease. Urease and a set of other virulence factors, including flagella and a IgA-degrading metalloprotease (ZapA), are coordinately regulated as part of a cycle of cellular differentiation and behavior known as swarming. For this reason, it has been postulated that a swarmer cell differentiation and swarming behavior are important in P. mirabilis virulence and UTI. We have identified two metalloproteases: ZapA and ZapE. ZapA is an important virulence factor of P. mirabilis, as we have shown by comparing the survival of ZapA-mutants with the isogenic wild type strain during UTI. The function of os not known, however its amino acid sequence suggests that ZapE may function either as a cytolysin or hemolysin. While it is not known that ZapA has the ability to degrade host immunoglobulins, the dramatic attenuation of virulence in strains lacking ZapA suggests that ZapA and ZapE may have a broader spectrum of activity associated with virulence. This project proposes to test the hypothesis that the roles of ZapA and ZapE in UTI extend beyond cleavage of IgA and IgG, and that the principal substrates of these metalloproteases are other proteins found in the urinary tract, specifically epithelial cell membrane proteins. To test this hypothesis, a panel of proteins consisting of cytoskeletal and extracellular cell matrix proteins of urinary epithelial cells, as well as proteins involved in the host defense mechanism, will be tested as potential substrates for metalloproteases. The activity of each protease will be assessed using (1) purified protein substrates, (1) bladder and kidney epithelial cell cultures, and (3) in our mouse model of UTI, in which the in vivo effects of proteolysis on bladder and kidneys will be measured. For these experiments, we will isolate and purify each protease and construct isogenic mutants lacking either or both protease-encoding genes. As a Specific Aim, we propose: 1. To characterize the role of the ZapA and putative ZapE metalloproteinases in UTI.
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