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J Med Microbiol 57 (2008), 1068-1078; DOI: 10.1099/jmm.0.2008/002071-0
© 2008 Society for General Microbiology
ISSN 1473-5644

Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis

Stephanie D. Himpsl1, C. Virginia Lockatell2, J. Richard Hebel3, David E. Johnson2,4 and Harry L. T. Mobley1

1 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA

2 Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, MD 21201, USA

3 Department of Epidemiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

4 Research Service, Department of Veteran Affairs, Baltimore, MD 21201, USA

Correspondence
Harry L. T. Mobley
hmobley{at}umich.edu

Received 21 March 2008
Accepted 29 April 2008

The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar M-ring protein fliF and hook protein flgE, and multiple metabolic genes.

Abbreviations: MR/P, mannose-resistant/Proteus-like; STM, signature-tagged mutagenesis; UTI, urinary tract infection.




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M. C. Lane, X. Li, M. M. Pearson, A. N. Simms, and H. L. T. Mobley
Oxygen-Limiting Conditions Enrich for Fimbriate Cells of Uropathogenic Proteus mirabilis and Escherichia coli
J. Bacteriol., March 1, 2009; 191(5): 1382 - 1392.
[Abstract] [Full Text] [PDF]


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