Year

1999

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Bordetella bronchiseptica is a common pathogen of the mammalian respiratory tract. Infection by this organism results in a number of respiratory conditions which may be fatal to the host. Urease is a potential virulence determinant of B. bronchiseptica that has been implicated in the pathogenesis of several other microorganisms. The urease operon of B. bronchiseptica is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to urease genes from other bacteria. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may be involved in nickel transport across the bacterial membrane. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (bbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. Other members of this family have been shown to regulate the expression of urease in Klebsiella aerogenes (NAC), and to induce the expression of a set of genes in Escherichia coli (OxyR) which protects the bacteria from phagolysosomal attack after intracellular invasion. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was also found 27 bp upstream of the urease promoter in B. bronchiseptica. The results of regulation studies show that urease is repressed by the Bordetella virulence gene locus (bvg). Urease was not inducible by 10 mM urea nor up-regulated in nitrogen limiting conditions. To investigate the role of BbuR in the regulation of urease, BbuR mutants of the BB7865 and the avirulent strain BB7866 were constructed by homologous recombination. The BbuR deficient BB7865 Bl lost the ability to regulate the expression of urease, suggesting that BbuR may be an intermediary in the bvg regulation of urease. As BbuR is homologous to OxyR, and urease has been suggested to protect bacteria intracellularly, an evaluation of the intracellular survival of urease-negative mutants of BB7865 and BB7866 in comparison to their urease-positive parental strains was also undertaken. This experiment demonstrated that increasing the concentration of urea in the assay increased survival of the wild-type but not urease-negative strains after 24 h, suggesting that urease may have a role in intracellular survival. To further address the role of urease in respiratory infection, we compared the ability of BB7865, the bbuR mutant strain BB7865 Bl which constitutively expresses urease, and the urease negative mutant BB7865 U5 to colonise and persist on the murine respiratory tract. The results showed that the constitutive expression, or abolishment of urease activity, had no significant effect on respiratory infection in this model. A DNA probe containing the gene encoding UreA of B. bronchiseptica hybridised to chromosomal DNA from Bordetella pertussis Tohama I indicating the presence of cryptic urease genes in this urease negative species. PCR primers designed to amplify part of ureD and the urease promoters from B. bronchiseptica were also able to amplify identically sized DNA fragments from B. bronchiseptica, B. pertussis and B. parapertussis ATCC15311. Nucleotide sequence analysis of these regions revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself. The biochemical data together with the results from the intracellular invasion experiments suggest that urease is somehow involved in post-infection processes. The possible presence of compensatory mechanisms may explain why this hypothesis is not supported by the murine respiratory colonisation assay data.

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