Identification of protease and rpoN-associated genes of uropathogenic Proteus mirabilis by negative selection in a mouse model of ascending urinary tract infection

Proteus mirabilis, a motile Gram-negative bacterium, is a principal cause o f urinary tract infections in patients with functional or anatomical abnorm alities of the urinary tract or those with urinary catheters in place. Thus far, virulence factors including urease, flagella, haemolysin, various fim briae, IgA protease and a deaminase have been characterized based on the ph enotypic traits conferred by these proteins. In this study, an attempt was made to identify new virulence genes of P. mirabilis that may not have iden tifiable phenotypes using the recently described technique of signature-tag ged mutagenesis. A pool of chromosomal transposon mutants was made through conjugation and kanamycin/tetracycline selection; random insertion was conf irmed by Southern blotting of chromosomal DNA isolated from 16 mutants usin g the aphA gene as a probe. From the total pool, 2.3 % (9/397) auxotrophic mutants and 3.5 % (14/397) swarming mutants were identified by screening on minimal salts agar and Luria agar plates, respectively. Thirty per cent of the mutants, found to have either no tag or an unamplifiable tag, were rem oved from the input pool. Then 10(7) c.f.u, from a 96-mutant pool (similar to 10(5) c.f.u. of each mutant) were used as an input pool to transurethral ly inoculate seven CBA mice. After 2 d infection, bacteria were recovered f rom the bladders and kidneys and yielded about 105 c.f.u. as an output pool . Dot blot analysis showed that two of the 96 mutants, designated B2 and B5 , could not be hybridized by signature tags amplified from the bladder outp ut pool. Interrupted genes from these two mutants were cloned and sequenced . The interrupted gene in B2 predicts a polypeptide of 37.3 kDa that shares amino acid similarity with a putative protease or collagenase precursor. T he gene in B5 predicts a polypeptide of 32.6 kDa that is very similar to th at encoded by ORF284 of the rpoN operon controlling expression of nitrogen- regulated genes from several bacterial species. The virulence of the two mu tants was tested further by co-challenging CBA mice with each mutant and th e parental strain. After 1 week of infection, the B2 and B5 mutants were re covered in numbers 100-fold and 1000-fold less than the parental strain, re spectively. Using an in vitro assay, it was shown that the B2 mutant had si gnificantly less (P = 0.0001) extracellular protease activity than the wild -type strain. These findings demonstrate that signature-tagged mutagenesis is a viable approach to identify bacterial genes associated with the abilit y to infect the urinary tract.