Browsing School, Graduate by Author "Zhao, Hui"
Investigation of the in vivo expression of virulence factors produced by uropathogenic Proteus mirabilisZhao, Hui; Mobley, Harry L. T. (1997)Proteus mirabilis, a motile gram-negative bacterium, is a principal cause of urinary tract infections in patients with functional or anatomic abnormalities of the urinary tract or those with urinary catheters in place. Although a number of putative virulence factors have been determined, when and where these genes are expressed in vivo are not yet known. Therefore two approaches were undertaken: (1) To study the in vivo expression of urease, the gfp gene (which encodes green fluorescent protein) was used to construct reporter fusions with ureD. Wild type P. mirabilis carrying the fusion plasmid was inoculated into experimental mice with ascending UTI. After seven days infection, fluorescent bacteria were detected in the kidney and the bladder by fluorescence microscopy, indicating that the urease genes are actively expressed during infection. Principally, vegetative forms of bacteria colonizing the bladders were observed; (2) MR/P fimbriae produced by P. mirabilis undergo phase variation in vitro. By DNA sequence analysis, an invertible element was found in the intergenic region between mrpI and mrpA. A sigma70 promoter within the invertible element drives the transcription of mrpA when in the ON position. MrpI, a putative recombinase, was able to confer inversion of the mrp switch region from both ON to OFF and OFF to ON. By in vivo PCR, MR/P phase variation could also be detected in vivo. In the absence of urolithiasis, the switch was found >99% in the ON position, suggesting that expression of MR/P fimbriae is selected in P. mirabilis in vivo. In addition, signature-tagged mutagenesis was used to identify more potential virulence genes of P. mirabilis. Random mutations in the chromosome were made by transposon mutagenesis. A 96-mutant pool was experimentally inoculated into the bladders of CBA mice. After two days infection, bacteria from the bladders and the kidneys were collected. By PCR and dot blot, two mutants were reproducibly unrecoverable and found to be attenuated for virulence. The two interrupted genes were cloned and sequenced. By sequence homology search and in vitro analyses, one of them appears to be a gene encoding a protease or collagenase; The other is a gene in the rpoN operon, which also encodes the sigma54 factor of RNA polymerase, and is presumably involved in expression of nitrogen regulated genes.