HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Himpsl, S. D. Articles by Mobley, H. L. T. Search for Related Content PubMed PubMed Citation Articles by Himpsl, S. D. Articles by Mobley, H. L. T. Agricola Articles by Himpsl, S. D. Articles by Mobley, H. L. T.

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

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

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

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Proteus mirabilis is a common cause of complicated urinary tract infections (UTIs). These infections are predominant in individuals with urinary tract abnormalities or patients with long-term catheters, such as those in nursing homes or hospitals. Bacteriuria with P. mirabilis may lead to fever, pyelonephritis, bacteraemia and even death (Mobley & Warren, 1987; Setia et al., 1984; Warren et al., 1982, 1987). During P. mirabilis infection, the urinary tract often becomes obstructed by urinary stones resulting from urease production (Griffith et al., 1976). Urease hydrolyses urea into carbon dioxide and ammonia; the liberation of ammonia creates an alkaline environment that causes soluble ions to precipitate from urine producing a urinary stone (Mobley & Hausinger, 1989). Whilst urease is critical for uropathogenesis, other potential virulence determinants have also been identified including fimbriae, flagella, haemolysin, IgA protease and an amino acid deaminase (Allison et al., 1992a, b, 1994; Bahrani et al., 1991; Belas & Suvanasuthi, 2005; Belas et al., 1998; Bondarenko et al., 1993; Chippendale et al., 1994; Jones & Mobley, 1988; Liaw et al., 2004; Loomes et al., 1993; Mobley & Chippendale, 1990; Peerbooms et al., 1983; Senior et al., 1987; Tolson et al., 1997; Walker et al., 1999; Wang et al., 2008; Wassif et al., 1995; Welch, 1987; Wray et al., 1986; Zunino et al., 2003, 2007). To determine the contribution of these proteins to pathogenesis, isogenic mutants have been constructed to determine their effect on virulence (Bahrani et al., 1994; Dattelbaum et al., 2003; Jacobsen et al., 2008; Johnson et al., 1993; Jones et al., 1990; Li et al., 1999, 2002; Massad et al., 1994b; Mobley et al., 1996; Swihart & Welch, 1990; Zunino et al., 2001). To identify additional genes involved in infection or colonization, global techniques such as signature-tagged mutagenesis (STM) have been employed (Burall et al., 2004; Hensel et al., 1995; Zhao et al., 1999).

The application of STM has been successful in identifying a number of virulence genes in many bacterial organisms (Chiang & Mekalanos, 1998; Mei et al., 1997; Sanschagrin et al., 2008; Schwan et al., 1998; Shea et al., 1996). Using this method, mutants in a mixed population can readily be identified by their unique DNA signature tag located at the end of the transposon. This attribute is particularly desirable following infection within a host in which the population of transposon mutants not recovered from an experimental infection (i.e. attenuated mutants) can be identified from among the input pool of mutants. Thus mutants absent from the recovered population, following infection and host colonization, potentially contain transposon insertions within virulence genes. For a recent and thorough review of this technique, see Mazurkiewicz et al. (2006).

Previously, we reported the findings of a non-saturating STM screen in which genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell-surface structures and key metabolic pathways were found to be required for P. mirabilis UTIs (Burall et al., 2004). However, we estimated that the theoretical coverage of the genome in that study was only 46.5 %. To increase coverage to at least 70 %, we carried out a second STM screening of 1880 P. mirabilis strain HI4320 transposon mutants. Performing co-challenge competition experiments using the CBA strain mouse model of ascending UTI, we identified 37 transposon mutants that were significantly outcompeted by wild-type HI4320. The results of this unbiased study when combined with a previous STM study (Burall et al., 2004) and numerous traditional virulence studies (Allison et al., 1992a, b, 1994; Bahrani et al., 1991, 1994; Belas & Suvanasuthi, 2005; Belas et al., 1998; Bondarenko et al., 1993; Chippendale et al., 1994; Dattelbaum et al., 2003; Jacobsen et al., 2008; Johnson et al., 1993; Jones & Mobley, 1988; Jones et al., 1990; Li et al., 1999, 2002; Liaw et al., 2004; Loomes et al., 1993; Massad et al., 1994b; Mobley & Chippendale, 1990; Mobley et al., 1996; Peerbooms et al., 1983; Senior et al., 1987; Swihart & Welch, 1990; Tolson et al., 1997; Walker et al., 1999; Wang et al., 2008; Wassif et al., 1995; Welch, 1987; Wray et al., 1986; Zunino et al., 2001, 2003, 2007) provide a fairly complete picture of urovirulence properties for this agent of complicated UTIs, P. mirabilis. Newly identified genes include fimbrial, flagellar, metabolic and transport genes.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains and plasmids. P. mirabilis strain HI4230 (Tet^R) was originally isolated from the urine of an elderly woman presenting with bacteriuria during long-term catheterization (Mobley & Warren, 1987; Warren et al., 1982). For conjugation, 48 Escherichia coli S17 pir (pUT/mini-Tn5Km2) (Amp^R Kan^R) transformants, each containing a unique DNA signature tag at one end of the mini-Tn5 transposon, were used as donor strains (de Lorenzo et al., 1990; Herrero et al., 1990; Zhao et al., 1999). For identification of P. mirabilis transposon mutants, arbitrary PCR products were cloned into pCR2.1-TOPO (Invitrogen) and maintained in either E. coli DH5 or E. coli TOP10 (Invitrogen).

Culture media and phenotypic testing. P. mirabilis HI4320 was routinely cultured in Luria broth (LB) (l^–1: 0.5 g NaCl, 5.0 g yeast extract, 10.0 g tryptone). Both non-swarming agar (l^–1: 0.4 g NaCl, 5.0 g yeast extract, 10.0 g tryptone, 15.0 g agar, 5.0 ml glycerol) and Luria agar (l^–1: 0.5 g NaCl, 5.0 g yeast extract, 10.0 g tryptone, 15.0 g agar) were used to maintain the transposon mutants and prevent swarming (Belas et al., 1991). Swarming phenotypes of P. mirabilis transposon mutants were compared with the wild-type HI4320 swarming phenotype on swarming agar (Burall et al., 2004). Modified urea segregation agar was used to identify the presence of urease activity in the P. mirabilis transposon mutants as described previously (Burall et al., 2004; Hu et al., 1990). Minimal A medium [l^–1: 10.5 g K₂HPO₄, 4.5 g KH₂PO₄, 0.47 g sodium citrate and 1.0 g (NH₄)₂SO₄] was prepared and autoclaved. After cooling, 1 ml 1 M MgSO₄, 10 ml 20 % glycerol and 1 ml 1 % nicotinic acid were added per litre and used to screen for auxotrophic P. mirabilis transposon mutants (Belas et al., 1991). Minimal A medium agar was made by the addition of 2.5 % Bacto Agar to minimal A medium. Antibiotics were added as necessary at the following concentrations: kanamycin, 25 µg ml^–1; tetracycline, 15 µg ml^–1; and ampicillin, 100 µg ml^–1.

Generation of an STM library using bacterial conjugation. P. mirabilis transposon mutants were constructed and screened using an STM technique adapted by Hensel et al. (1995). Transformation of E. coli S17 pir with each of the 48 pUT/mini-Tn5Km2 plasmids containing a unique DNA signature tag (provided by C. Tang, Imperial College, London, UK) was performed as previously described (Zhao et al., 1999). Unique DNA signature tags were previously screened and selected based on non-cross-reactivity with one another (Burall et al., 2004). Bacterial conjugation reactions between recipient strain P. mirabilis HI4320 and 48 E. coli S17 pir (pUT/mini-Tn5Km2) (Amp^R Kan^R) donor strains were performed as described previously using a modified protocol (Burall et al., 2004). Overnight cultures of P. mirabilis HI4320 (Tet^R) and each of the 48 E. coli donor strains (Kan^R Amp^R) were mixed at a ratio of 2 : 1 or 5 : 1. Following centrifugation and removal of supernatant, LB medium without antibiotic was added to the pellet and held at room temperature for 5 min. After resuspension of the pellet, 50 or 100 µl of the suspension was pipetted onto membranes (0.45 µm pore size) and aseptically positioned on non-swarming agar plates without antibiotic. After drying, plates were incubated at 37 °C for 60–90 min to minimize replication of siblings. Following incubation, membranes were aseptically overlaid with LB medium without antibiotic onto non-swarming agar plates containing kanamycin and tetracycline and incubated at 37 °C overnight. Transconjugants were screened for ampicillin sensitivity to ensure loss of the pUT vector and incorporation of the mini-Tn5 transposon into the genome of P. mirabilis, as described previously (Burall et al., 2004). Simultaneously, screening for resistance to kanamycin and tetracycline was performed to select only P. mirabilis transposon mutants containing the kanamycin transposon. In the library, 59/1920 transconjugants selected (3 % conjugation rate failure) had to be reselected to create pools in which each one of the 48 unique DNA signature tags was represented only once per pool. Within each pool, one of the 48 mutants served as a negative control. The signature tag of the negative control failed to hybridize and did not cross-hybridize with other tags. For this study, 40 pools (1880 tagged transposon mutants not including negative controls) were created, each with 48 P. mirabilis transposon mutants containing a uniquely tagged transposon (Kan^R Tet^R). P. mirabilis mutants (Kan^R Tet^R Amp^S) were stored in 96-well plates at –80 °C in 80 % glycerol.

CBA mouse model of ascending UTI. To identify attenuated signature-tagged mutants, all 1880 P. mirabilis transposon mutants were tested in the CBA/J murine model of ascending UTIs as described previously (Burall et al., 2004; Hagberg et al., 1983; Johnson et al., 1993). For primary screens, each of the 40 pools was transurethrally infected into a total of five mice for a 4-day infection. Subsequent secondary screens of 23 smaller pools containing 20–25 mutants (each DNA signature tag represented only once per pool) were also transurethrally infected into a total of five mice for a 4-day infection. Co-challenge competition experiments between P. mirabilis HI4320 and putatively attenuated P. mirabilis transposon mutants were performed with a modified protocol (Burall et al., 2004). For each of the 93 competition experiments, a total of five mice were infected with either 1x10⁷ or 5x10⁷ c.f.u. of a bacterial suspension containing a 1 : 1 ratio of P. mirabilis HI4320 and a P. mirabilis transposon mutant. It was not uncommon for mice to die midway through the 7-day infection period; this appeared to be caused primarily by obstruction of urine flow by urease-mediated stone formation. In these instances, experiments were repeated with additional mice to achieve data from a minimum of five mice. Viable counts from plates were expressed in c.f.u. (ml urine)^–1 or c.f.u. (g tissue)^–1, as described previously (Burall et al., 2004).

In vitro screening using LB and minimal A media. Prior to co-challenge competition experiments, P. mirabilis transposon mutants putatively attenuated in both in vivo primary and secondary screens were tested in vitro to disqualify mutants that may have been attenuated in the mouse model due only to minor growth defects. In vitro primary and secondary screening using LB and minimal A media was performed as described previously (Burall et al., 2004). Mutants were placed in pools of 15 for primary screening and then regrouped into pools of five to ten for secondary screening.

Labelling and hybridization screening of STM mutants. For all in vivo and in vitro screens, DNA signature tags of bacteria in the input inoculum and from recovered bacteria (output sample) were PCR amplified, labelled with digoxigenin–dUTP (Roche) and hybridized to donor DNA dot-blot membranes containing a representative of each DNA signature tag. For in vivo screens, recovered bacteria were isolated from the bladder and kidneys. Visual comparison of the hybridized blots between input and output samples was performed to identify putatively attenuated transposon mutants. For in vivo primary screens, transposon mutants that lacked a hybridization signal in either or both output samples, had a reduced hybridization signal compared with the input sample or had an intensity equal to or less than that of the negative control were scored anywhere from being slightly attenuated to unrecoverable. Primary P. mirabilis transposon mutants attenuated from the primary screen were regrouped into secondary pools of 20–25 mutants each and subjected to a secondary screen. Mutants of the secondary screen in the output sample lacking a hybridization signal compared with the input sample or having an intensity equal to or less than that of the negative control were considered reproducibly unrecoverable. For in vitro primary and secondary screens, putatively attenuated mutants that were able to survive passage in LB and minimal A media, and thus were recovered as seen by hybridization on donor DNA dot blots, were kept as putatively attenuated mutants to be tested in co-challenge competition experiments. The labelling and hybridization procedure was performed for all input and output samples of primary and secondary in vivo and in vitro screens as described previously with minor modifications (Burall et al., 2004). A two-step PCR was used to amplify the DNA signature tags. A 1 : 5 dilution of sample was used as template for PCR I. Following PCR I, the product was loaded on a 1.6 % (w/v) SeaPlaque agarose gel (Cambrex) and electrophoresed. A 3.5 µl sample of the 80 bp gel slice was used for PCR II. A sample of 10–25 µl of the PCR II digoxigenin-labelled fragments was used for hybridization to donor DNA blots prepared with 10 µl plasmid DNA. The primers used in PCRs I and II are listed in Table 1.

Sequence analysis. Arbitrary PCR and molecular cloning were carried out on P. mirabilis transposon mutants that were statistically outcompeted by wild-type HI4320 to identify the gene in which the transposon was inserted. Arbitrary PCR as described by Bahrani-Mougeot et al. (2002) was carried out as two sequential amplification steps with primers complementary to the transposon end reading outward and arbitrarily designed primers reading inward from the unknown sequence. Arbitrary PCR 1 primer (Arb 1) and primer 1955 were prepared and used under PCR conditions described previously (Burall et al., 2004). Arbitrary PCR 2 was performed with arbitrary PCR 2 primer (Arb 2) and primer 1954 using 1 µl product from arbitrary PCR 1 as described previously (Burall et al., 2004). Primers used in arbitrary PCRs 1 and 2 are listed in Table 1. Gel-isolated arbitrary PCR product was either directly sequenced or cloned into pCR2.1-TOPO (Invitrogen) and sequenced at the Biopolymer Core Facility at the University of Maryland, MD, USA, or at the Sequencing Core Facility at the University of Michigan, MI, USA. The sequences of primers 1955 and 1954 are both located within the transposon. For all sequencing reactions, primer 1954 was used to determine the interrupted gene sequence.

Sequence analysis and translation were performed using online search engines at http://expasy.org. Homology searches (BLAST) of nucleotide and protein sequences (Altschul et al., 1990) were completed using the public database GenBank (http://www.ncbi.nlm.nih.gov). All nucleotide sequences of identified transposon mutants were examined using the Sanger Institute's database of the sequenced and annotated P. mirabilis HI4320 genome at http://www.sanger.ac.uk/Projects/P_mirabilis/ (Pearson et al., 2008). The nucleotide sequence was analysed for presence of the transposon and placed in the Sanger Institute's P. mirabilis BLAST server to query exact sequence matches. By examining matches of unknown nucleotide sequence at the end of the transposon to known P. mirabilis sequence, a transposon insertion could be determined at the exact nucleotide within the genome. The correlating P. mirabilis nucleotide number where the transposon inserted and that of the Sanger Institute's annotation of P. mirabilis was used to identify the gene.

SDS-PAGE and Western blot analysis. Attenuated transposon mutants of P. mirabilis confirmed by co-challenge competition experiments were analysed by Western blotting for the presence of flagellin (Mobley et al., 1996) and the MrpA fimbrial subunit (Burall et al., 2004; Li et al., 1999). Bacterial suspensions were standardized at OD₆₀₀=0.8 and protein was precipitated using TCA. Samples were denatured for 5 min by boiling in sample buffer, separated by 15 % SDS-PAGE gel and transferred to a PVDF membrane (Millipore). In Fig. 1(a), samples were standardized using a 2-D Quant kit (Amersham) and 11 µg protein was loaded onto the gel. After Western blotting, membranes were incubated with rabbit polyclonal antiserum against either flagellin or MrpA followed by a secondary goat anti-rabbit IgG conjugated to horseradish peroxidase. Membranes were developed using ECL Plus Western blotting detection reagents (GE Healthcare).

View larger version (72K): [in this window] [in a new window]   Fig. 1. (a) Flagella and mannose-resistant/Proteus-like fimbriae production by wild-type P. mirabilis HI4320 and selected STM transposon mutants shown by Western blotting of standardized whole-cell preparations reacted with rabbit anti-FlaA and anti-MrpA, respectively. The molecular masses of the FlaA and MrpA subunit proteins are indicated in kDa. Mutant 2E2, in which metabolic gene cyaA is disrupted, appeared to be deficient in both FlaA and MrpA expression. As expected, mutant 8A6, which disrupted fliF, lacked FlaA but appeared to have increased MrpA production compared with wild-type HI4320. Likewise, mutant 21B2, which disrupted flgE, lacked FlaA but did not appear to have an increase in MrpA. Interestingly, the putative adhesin disrupted in mutant 36D2 did not produce MrpA. Metabolic mutant 10E4 and unidentified mutants 15B4 and 17A4 did not produce MrpA. Mutant 38E2, which disrupted a multicopy oxidase, did not produce FlaA. (b–g) Swarming phenotypes of P. mirabilis (b) and selected transposon mutants (c–g) after spotting overnight cultures onto swarming agar and incubating for 18 h. Mutants showed deficient to aberrant swarming patterns in comparison with wild-type P. mirabilis HI4320 (WT) with vertical lines depicting the edge of the outer swarming raft.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

Elimination of auxotrophic mutants or mutants with growth defects

The 217 transposon mutants identified as attenuated in both primary and secondary in vivo screens were tested in vitro to identify those with minor growth defects that may decrease generation time in rich LB medium and to identify mutants with auxotrophic mutations by lack of growth in minimal A medium. Following passage of the 217 transposon mutants in vitro, 141 mutants were not outcompeted in either LB or minimal A medium. The remaining 76 transposon mutants were placed in secondary pools and each was retested in both LB and minimal A media. Of these transposon mutants, 21 were outcompeted after performing morning and afternoon passages for 4 days in LB and minimal A media. Thus a total of 196 transposon mutants were not outcompeted in LB or minimal A medium and displayed no growth defects after primary and secondary in vitro screens.

Co-challenge and identification of attenuated mutants

CBA mice were co-challenged with wild-type P. mirabilis HI4320 and each of the 93 transposon mutants that were considered most attenuated out of the 196 transposon mutants based on hybridization intensity results from the dot blots. Of the 93 transposon mutants tested, 37 were statistically outcompeted by wild-type HI4320. Attenuation by at least 100-fold in urine, bladder or kidney samples was observed in 15 of the 37 statistically significant transposon mutants outcompeted in the co-challenge experiment (Table 2).

Transposon mutants 11B2 and 20C4, also containing unique tags, had identical disruptions within cpsF, which encodes a glycosyltransferase required for lipopolysaccharide core modification (Klena et al., 1992; MacLachlan et al., 1991; Parker et al., 1992). Previously, this mutant has also been found to compromise urinary tract virulence (Allison et al., 1994). The aberrant swarming phenotype of mutant 20C4 was similar to that of a P. mirabilis U6450 TnphoA insertion mutant of cpsF unable to assemble capsular polysaccharide (Gygi et al., 1995). Upon further analysis, mutant 11B2 produced a swarming phenotype like that of HI4320 and not of 20C4. It is possible that there was a compensatory mutation in transposon mutant 11B2 restoring the swarming phenotype to that of HI4320 or that the difference in the tag sequence itself somehow governed this phenotype.

Transport

The disrupted gene of transposon mutant 7A1, exbD, encodes a biopolymer transport protein. ExbD works in conjunction with TonB and ExbB to form an energy transducing system that uses the proton motive force of the cytoplasmic membrane to transport micronutrients across the outer membrane into the cell. The TonB–ExbB–ExbD system is essential for internalization of iron–siderophore complexes across outer-membrane receptors (Braun et al., 1996; Fischer et al., 1989). Thus these mutants would be outcompeted in the iron-limited urinary tract (Alteri & Mobley, 2007).

Gene regulation

Transposon mutant 4B6 was identified as disrupting the transcriptional activator protein NhaR, a DNA-binding protein of the LysR family of transcriptional regulators, and was attenuated in the urine (P=0.001) and kidneys (P=0.004) and overall (P<0.0005) in vivo. NhaR expression has been shown to be induced in response to alkaline conditions and activates transcription of the pgaABCD operon required for poly-β-1,6-N-acetyl-D-glucosamine production (Goller et al., 2006); the latter promotes E. coli fitness by aiding cell-to-cell adhesion, attachment to surfaces and stabilization of biofilm structures (Itoh et al., 2005). Unlike uropathogenic E. coli, P. mirabilis produces urease, which creates an alkaline microenvironment (Jones et al., 1990). When P. mirabilis infects the urinary tract, urease-mediated urea hydrolysis increases environmental pH, which may cause activation of NhaR; this could lead to increased expression of genes that may ultimately contribute to the persistence of infection. Presently, gene targets of the P. mirabilis NhaR regulator have yet to be identified. The finding of nhaR in our STM screening provides evidence that the urinary tract is alkaline in the presence of P. mirabilis and supports the premise that P. mirabilis HI4320 responds to alkaline conditions by producing virulence factors to aid in UTIs.

Cell-surface structures and motility

In our previous STM study (Burall et al., 2004), only one motility-related gene, cheW, was identified. Here, we report findings of three additional motility genes important in P. mirabilis HI4320 colonization. Two genes of the flagellum regulon, fliF and flgE, were identified in our STM screen. fliF encodes protein subunits that make up the MS ring of the flagellum, which is the first synthesized substructure and is crucial for flagellar assembly (Grunenfelder et al., 2003; Kubori et al., 1992; Wu & Newton, 1997). The other flagellar transposon mutant had a disruption in the hook protein encoded by flgE. This mutant can form the PL ring complex and distal rod, but cannot assemble a complete flagellum (Komoriya et al., 1999). Both Western blot and swarming results of the flgE and fliF transposon mutants were identical; no flagellin protein was detected and a swarming-deficient phenotype was observed, probably due simply to lack of flagella synthesis. In addition, mutant 8A6, in which the transposon disrupts fliF, appeared to be overproducing mrpA (Fig. 1a).

The five known P. mirabilis fimbriae are mannose-resistant/Proteus-like fimbriae (MR/P) (Sareneva et al., 1990), P. mirabilis fimbriae (Massad & Mobley, 1994), ambient-temperature fimbriae (Massad et al., 1994a), non-agglutinating fimbriae (Tolson et al., 1995; Wray et al., 1986) and P. mirabilis P-like pili (Bijlsma et al., 1995). The completed annotation of the P. mirabilis HI4320 genome has revealed an additional 12 putative fimbrial operons (Pearson et al., 2008). Mutant 4A3 contained a disruption of the gene that encodes PMI3001, a putative fimbrial protein, which is located within one of these newly identified fimbrial operons. PMI3001 is located in fimbrial operon 14 (Pearson et al., 2008). Mutant 4A3 was attenuated and outcompeted by wild-type HI4320 in the murine model of ascending UTI, suggesting that this newly identified fimbrial operon may play a role in the pathogenesis of UTIs, as has been shown for the mrp operon (Bahrani et al., 1994).

Transposon mutant 36D2 was outcompeted in vivo and contained a disruption in PMI2575, which encodes a putative adhesin-type autotransporter. Autotransporters have been associated with virulence functions such as adhesion, aggregation, invasion, biofilm formation and toxicity (Alamuri & Mobley, 2008; Wells et al., 2007). By Western blot analysis, it appeared that mutant 36D2 was negative for synthesis of the MrpA fimbrial subunit (Fig. 1). There is no clear explanation, however, as to why a mutation in this adhesin would result in lack of MrpA expression.

Metabolism

A total of seven metabolic genes were identified in our STM screen including those involved in nucleotide, carbohydrate and amino acid metabolism. Mutation of these genes did not interfere with growth of P. mirabilis in vitro in rich medium, although disruption of guaB and aceE resulted in an increased lag phase compared with wild-type HI4320 in minimal A medium, probably due to defects in nucleotide metabolism and central metabolism, respectively. Transposon mutants 5F1 and 2E2 had disruptions in genes involved in purine metabolism. Mutant 5F1 disrupted guaB encoding inosine 5'-monophosphate dehydrogenase, which catalyses the first step of GTP synthesis. The cyaA gene disrupted in transposon mutant 2E2 encodes adenylate cyclase, which catalyses the breakdown of ATP to 3',5'-cyclic adenosine monophosphate (cAMP). Interestingly, this mutant was unable to produce either FlaA or MrpA. Four genes were involved in carbohydrate metabolism. Mutant 11G3 was disrupted in cbbC, a probable molybdopterin-containing oxidoreductase; this is similar to formate dehydrogenase, which is involved in glyoxylate, dicarboxylate and methane metabolism. Transposon mutant 11D4 disrupted edd, encoding 6-phosphogluconate dehydratase, which is involved in the Entner–Doudoroff pathway. Mutant 10E4 disrupted sdhC, which encodes the succinate dehydrogenase cytochrome b₅₅₆ subunit, which is part of the citric acid or TCA cycle. This mutant was also found by Western blot analysis not to produce MrpA, the major fimbrial subunit of MR/P fimbria. Mutant 8B5 had a transposon disruption in aceE encoding pyruvate dehydrogenase, which is involved in glycolysis. Mutant 15C3 had a disruption in sdaA encoding L-serine deaminase 1, which catalyses L-serine to pyruvate and ammonia and is involved in amino acid metabolism of glycine, serine, threonine and cysteine.

Three additional mutants identified as PMI1184, PMI1193 and PMI1000 encoded putative exported proteins and a putative phage-related protein, respectively (Table 3). A total of seven mutants (11A4, 15A6, 15B4, 17F5, 22F6, 33B5 and 37D5) were unable to be identified due to limited or poor DNA sequencing results despite persistent attempts. An additional mutant, 17A4, had a transposon insertion in the intragenic region between PMI1420 and PMI1421.

Phenotypic characterization

All 37 attenuated transposon mutants were examined for synthesis of known P. mirabilis virulence factors including flagella and fimbriae. Production of flagella and MR/P fimbriae was analysed using rabbit serum raised against flagellin and the MrpA subunit, respectively, and analysed by Western blotting. Of the 37 attenuated mutants, a total of eight had a defect in producing either of these proteins: four were unable to produce FlaA and five were unable to produce MrpA (Table 3; Fig. 1). This suggests the possibility that pleiotropic effects resulted from certain sites of transposon insertion. Modified urea segregation agar was used to identify urease-negative mutants in the library of P. mirabilis transposon mutants. Of 1880 transposon mutants tested, however, none of the STM mutants were deficient for urease enzymic activity. Screening on swarming agar was performed to identify mutants with aberrant or deficient swarming phenotypes compared with the swarming phenotype of wild-type HI4320. Visual comparison of the swarming differentiation patterns such as the number and length of swarming rafts was made between HI4320 and each transposon mutant. Of 1880 transposon mutants tested, eight had swarming patterns distinct from that of the wild-type HI4320. Swarming patterns varied from no swarming rafts, multiple short swarming rafts and a small star-shaped raft to elongated swarming rafts (Table 3; Fig. 1). The growth phenotypes of the 37 attenuated transposon mutants were verified on minimal A medium agar and also by independent growth curves in LB and minimal A media. After 24 h, all mutants grew as well as wild-type HI4320 on minimal A medium and in independent LB growth curves (data not shown). Two transposon mutants with disruptions in aceE and guaB demonstrated an increased lag phase (12 and 23 h, respectively) relative to wild-type HI4320 (2.5 h) but displayed identical exponential growth rates to the wild-type in independent liquid cultures of minimal A medium.

Conclusions

For this STM study, a total of 1880 P. mirabilis HI4320 transposon mutants were screened for virulence following transurethral challenge of approximately 500 CBA mice. A total of 40 pools, each containing 48 mutants, were tested in a primary in vivo challenge. A total of 23 groups of approximately 20–25 mutants were tested in a secondary murine screen. All attenuated mutants were tested for growth defects in LB and minimal A media. Mutants outcompeted by the wild-type strain in vitro were eliminated from further consideration. In total, 37/1880 STM transposon mutants were identified as being attenuated in vivo.

In comparison with our previous STM study (Burall et al., 2004), many mutants had transposon disruptions in genes belonging to similar classifications, although no single gene was identified in both studies. Further analysis revealed that genes identified in both studies did not belong to similar putative operons. The total number of transposon mutants created in both STM studies (3968) predicts a theoretical coverage of 69.6 % of the P. mirabilis HI4320 genome. The incomplete saturation of the genome may explain why the same gene was not identified in both STM studies. Together with the numerous virulence studies in which targeted mutants have been assessed for virulence (see Introduction), we are approaching a complete cataloguing of urovirulence genes for P. mirabilis, a prominent cause of urolithiasis, catheter-associated UTIs and other complicated UTIs.

P. mirabilis HI4320 expresses virulence factors that facilitate infection of the bladder and ascension of the ureters to the kidneys to cause pyelonephritis. Using STM, we identified 37 P. mirabilis HI4320 mutants that were significantly attenuated or exhibited reduced fitness in the murine model of ascending UTIs. Virulence factors identified by STM included genes associated with cellular processes, transport, regulation, cell-surface structures and motility, and metabolism, which contribute to the uropathogenesis of P. mirabilis. To satisfy molecular Koch's (Falkow's) postulates for these putative virulence genes, it will be necessary to construct specific mutants in the discovered genes, assess their attenuation and complement the mutants with the cloned determinants.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Alamuri, P. & Mobley, H. L. T. (2008). A novel autotransporter of uropathogenic Proteus mirabilis is both a cytotoxin and an agglutinin. Mol Microbiol 68, 997–1017.[CrossRef][Medline]

Allison, C., Coleman, N., Jones, P. L. & Hughes, C. (1992a). Ability of Proteus mirabilis to invade human urothelial cells is coupled to motility and swarming differentiation. Infect Immun 60, 4740–4746.[Abstract/Free Full Text]

Allison, C., Lai, H. C. & Hughes, C. (1992b). Co-ordinate expression of virulence genes during swarm-cell differentiation and population migration of Proteus mirabilis. Mol Microbiol 6, 1583–1591.[Medline]

Allison, C., Emody, L., Coleman, N. & Hughes, C. (1994). The role of swarm cell differentiation and multicellular migration in the uropathogenicity of Proteus mirabilis. J Infect Dis 169, 1155–1158.[Medline]

Alteri, C. J. & Mobley, H. L. (2007). Quantitative profile of the uropathogenic Escherichia coli outer membrane proteome during growth in human urine. Infect Immun 75, 2679–2688.[Abstract/Free Full Text]

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]

Bahrani, F. K., Johnson, D. E., Robbins, D. & Mobley, H. L. (1991). Proteus mirabilis flagella and MR/P fimbriae: isolation, purification, N-terminal analysis, and serum antibody response following experimental urinary tract infection. Infect Immun 59, 3574–3580.[Abstract/Free Full Text]

Bahrani, F. K., Massad, G., Lockatell, C. V., Johnson, D. E., Russell, R. G., Warren, J. W. & Mobley, H. L. (1994). Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection. Infect Immun 62, 3363–3371.[Abstract/Free Full Text]

Bahrani-Mougeot, F. K., Buckles, E. L., Lockatell, C. V., Hebel, J. R., Johnson, D. E., Tang, C. M. & Donnenberg, M. S. (2002). Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol Microbiol 45, 1079–1093.[CrossRef][Medline]

Belas, R. & Suvanasuthi, R. (2005). The ability of Proteus mirabilis to sense surfaces and regulate virulence gene expression involves FliL, a flagellar basal body protein. J Bacteriol 187, 6789–6803.[Abstract/Free Full Text]

Belas, R., Erskine, D. & Flaherty, D. (1991). Transposon mutagenesis in Proteus mirabilis. J Bacteriol 173, 6289–6293.[Abstract/Free Full Text]

Belas, R., Schneider, R. & Melch, M. (1998). Characterization of Proteus mirabilis precocious swarming mutants: identification of rsbA, encoding a regulator of swarming behavior. J Bacteriol 180, 6126–6139.[Abstract/Free Full Text]

Bijlsma, I. G., van Dijk, L., Kusters, J. G. & Gaastra, W. (1995). Nucleotide sequences of two fimbrial major subunit genes, pmpA and ucaA, from canine-uropathogenic Proteus mirabilis strains. Microbiology 141, 1349–1357.[Abstract/Free Full Text]

Bondarenko, V. M., Parkhomenko, L. V. & Koval'chuk, V. K. (1993). The secreted proteolytic enzymes of Proteus mirabilis. Zh Mikrobiol Epidemiol Immunobiol 2, 41–47.[Medline]

Braun, V., Gaisser, S., Herrmann, C., Kampfenkel, K., Killmann, H. & Traub, I. (1996). Energy-coupled transport across the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro, and leucine 132 in the periplasmic region and aspartate 25 in the transmembrane region are important for ExbD activity. J Bacteriol 178, 2836–2845.[Abstract/Free Full Text]

Burall, L. S., Harro, J. M., Li, X., Lockatell, C. V., Himpsl, S. D., Hebel, J. R., Johnson, D. E. & Mobley, H. L. (2004). Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold. Infect Immun 72, 2922–2938.[Abstract/Free Full Text]

Chiang, S. L. & Mekalanos, J. J. (1998). Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization. Mol Microbiol 27, 797–805.[CrossRef][Medline]

Chippendale, G. R., Warren, J. W., Trifillis, A. L. & Mobley, H. L. (1994). Internalization of Proteus mirabilis by human renal epithelial cells. Infect Immun 62, 3115–3121.[Abstract/Free Full Text]

Dattelbaum, J. D., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. (2003). UreR, the transcriptional activator of the Proteus mirabilis urease gene cluster, is required for urease activity and virulence in experimental urinary tract infections. Infect Immun 71, 1026–1030.[Abstract/Free Full Text]

de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172, 6568–6572.[Abstract/Free Full Text]

Fischer, E., Gunter, K. & Braun, V. (1989). Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB. J Bacteriol 171, 5127–5134.[Abstract/Free Full Text]

Goller, C., Wang, X., Itoh, Y. & Romeo, T. (2006). The cation-responsive protein NhaR of Escherichia coli activates pgaABCD transcription, required for production of the biofilm adhesin poly-beta-1,6-N-acetyl-D-glucosamine. J Bacteriol 188, 8022–8032.[Abstract/Free Full Text]

Griffith, D. P., Musher, D. M. & Itin, C. (1976). Urease. The primary cause of infection-induced urinary stones. Invest Urol 13, 346–350.[Medline]

Grunenfelder, B., Gehrig, S. & Jenal, U. (2003). Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation. J Bacteriol 185, 1624–1633.[Abstract/Free Full Text]

Gygi, D., Rahman, M. M., Lai, H. C., Carlson, R., Guard-Petter, J. & Hughes, C. (1995). A cell-surface polysaccharide that facilitates rapid population migration by differentiated swarm cells of Proteus mirabilis. Mol Microbiol 17, 1167–1175.[CrossRef][Medline]

Hagberg, L., Engberg, I., Freter, R., Lam, J., Olling, S. & Svanborg Eden, C. (1983). Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin. Infect Immun 40, 273–283.[Abstract/Free Full Text]

Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E. & Holden, D. W. (1995). Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403.[Abstract/Free Full Text]

Herrero, M., de Lorenzo, V. & Timmis, K. N. (1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172, 6557–6567.[Abstract/Free Full Text]

Hu, L. T., Nicholson, E. B., Jones, B. D., Lynch, M. J. & Mobley, H. L. (1990). Morganella morganii urease: purification, characterization, and isolation of gene sequences. J Bacteriol 172, 3073–3080.[Abstract/Free Full Text]

Hunstad, D. A., Justice, S. S., Hung, C. S., Lauer, S. R. & Hultgren, S. J. (2005). Suppression of bladder epithelial cytokine responses by uropathogenic Escherichia coli. Infect Immun 73, 3999–4006.[Abstract/Free Full Text]

Itoh, Y., Wang, X., Hinnebusch, B. J., Preston, J. F., III & Romeo, T. (2005). Depolymerization of beta-1,6-N-acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacteriol 187, 382–387.[Abstract/Free Full Text]

Jacobsen, S. M., Lane, M. C., Harro, J. M., Shirtliff, M. E. & Mobley, H. L. (2008). The high-affinity phosphate transporter Pst is a virulence factor for Proteus mirabilis during complicated urinary tract infection. FEMS Immunol Med Microbiol 52, 180–193.[CrossRef][Medline]

Johnson, D. E., Russell, R. G., Lockatell, C. V., Zulty, J. C., Warren, J. W. & Mobley, H. L. (1993). Contribution of Proteus mirabilis urease to persistence, urolithiasis, and acute pyelonephritis in a mouse model of ascending urinary tract infection. Infect Immun 61, 2748–2754.[Abstract/Free Full Text]

Jones, B. D. & Mobley, H. L. (1988). Proteus mirabilis urease: genetic organization, regulation, and expression of structural genes. J Bacteriol 170, 3342–3349.[Abstract/Free Full Text]

Jones, B. D., Lockatell, C. V., Johnson, D. E., Warren, J. W. & Mobley, H. L. (1990). Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection. Infect Immun 58, 1120–1123.[Abstract/Free Full Text]

Justice, S. S., Hunstad, D. A., Harper, J. R., Duguay, A. R., Pinkner, J. S., Bann, J., Frieden, C., Silhavy, T. J. & Hultgren, S. J. (2005). Periplasmic peptidyl prolyl cis-trans isomerases are not essential for viability, but SurA is required for pilus biogenesis in Escherichia coli. J Bacteriol 187, 7680–7686.[Abstract/Free Full Text]

Justice, S. S., Lauer, S. R., Hultgren, S. J. & Hunstad, D. A. (2006). Maturation of intracellular Escherichia coli communities requires SurA. Infect Immun 74, 4793–4800.[Abstract/Free Full Text]

Klena, J. D., Ashford, R. S., II & Schnaitman, C. A. (1992). Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen. J Bacteriol 174, 7297–7307.[Abstract/Free Full Text]

Komoriya, K., Shibano, N., Higano, T., Azuma, N., Yamaguchi, S. & Aizawa, S. I. (1999). Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium. Mol Microbiol 34, 767–779.[CrossRef][Medline]

Kubori, T., Shimamoto, N., Yamaguchi, S., Namba, K. & Aizawa, S. (1992). Morphological pathway of flagellar assembly in Salmonella typhimurium. J Mol Biol 226, 433–446.[CrossRef][Medline]

Li, X., Johnson, D. E. & Mobley, H. L. (1999). Requirement of MrpH for mannose-resistant Proteus-like fimbria-mediated hemagglutination by Proteus mirabilis. Infect Immun 67, 2822–2833.[Abstract/Free Full Text]

Li, X., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. (2002). Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria, a bladder colonization factor of uropathogenic Proteus mirabilis. Mol Microbiol 45, 865–874.[CrossRef][Medline]

Liaw, S. J., Lai, H. C. & Wang, W. B. (2004). Modulation of swarming and virulence by fatty acids through the RsbA protein in Proteus mirabilis. Infect Immun 72, 6836–6845.[Abstract/Free Full Text]

Loomes, L. M., Kerr, M. A. & Senior, B. W. (1993). The cleavage of immunoglobulin G in vitro and in vivo by a proteinase secreted by the urinary tract pathogen Proteus mirabilis. J Med Microbiol 39, 225–232.[Abstract/Free Full Text]

MacLachlan, P. R., Kadam, S. K. & Sanderson, K. E. (1991). Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2. J Bacteriol 173, 7151–7163.[Abstract/Free Full Text]

Massad, G. & Mobley, H. L. (1994). Genetic organization and complete sequence of the Proteus mirabilis pmf fimbrial operon. Gene 150, 101–104.[CrossRef][Medline]

Massad, G., Bahrani, F. K. & Mobley, H. L. (1994a). Proteus mirabilis fimbriae: identification, isolation, and characterization of a new ambient-temperature fimbria. Infect Immun 62, 1989–1994.[Abstract/Free Full Text]

Massad, G., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. (1994b). Proteus mirabilis fimbriae: construction of an isogenic pmfA mutant and analysis of virulence in a CBA mouse model of ascending urinary tract infection. Infect Immun 62, 536–542.[Abstract/Free Full Text]

Mazurkiewicz, P., Tang, C. M., Boone, C. & Holden, D. W. (2006). Signature-tagged mutagenesis: barcoding mutants for genome-wide screens. Nat Rev Genet 7, 929–939.[CrossRef][Medline]

Mei, J. M., Nourbakhsh, F., Ford, C. W. & Holden, D. W. (1997). Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol 26, 399–407.[CrossRef][Medline]

Mobley, H. L. & Chippendale, G. R. (1990). Hemagglutinin, urease, and hemolysin production by Proteus mirabilis from clinical sources. J Infect Dis 161, 525–530.[Medline]

Mobley, H. L. & Hausinger, R. P. (1989). Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev 53, 85–108.[Abstract/Free Full Text]

Mobley, H. L. & Warren, J. W. (1987). Urease-positive bacteriuria and obstruction of long-term urinary catheters. J Clin Microbiol 25, 2216–2217.[Abstract/Free Full Text]

Mobley, H. L., Belas, R., Lockatell, V., Chippendale, G., Trifillis, A. L., Johnson, D. E. & Warren, J. W. (1996). Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun 64, 5332–5340.[Abstract/Free Full Text]

Parker, C. T., Pradel, E. & Schnaitman, C. A. (1992). Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12. J Bacteriol 174, 930–934.[Abstract/Free Full Text]

Pearson, M. M., Sebaihia, M., Churcher, C., Quail, M. A., Seshasayee, A. S., Abdellah, Z., Arrosmith, C., Atkin, B., Chillingworth, T. & other authors (2008). The complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility. J Bacteriol 190, 4027–4037.[Abstract/Free Full Text]

Peerbooms, P. G., Verweij, A. M. & MacLaren, D. M. (1983). Investigation of the haemolytic activity of Proteus mirabilis strains. Antonie Van Leeuwenhoek 49, 1–11.[CrossRef][Medline]

Sanschagrin, F., Kukavica-Ibrulj, I. & Levesque, R. C. (2008). Essential genes in the infection model of Pseudomonas aeruginosa PCR-based signature-tagged mutagenesis. Methods Mol Biol 416, 61–82.[CrossRef][Medline]

Sareneva, T., Holthofer, H. & Korhonen, T. K. (1990). Tissue-binding affinity of Proteus mirabilis fimbriae in the human urinary tract. Infect Immun 58, 3330–3336.[Abstract/Free Full Text]

Schwan, W. R., Coulter, S. N., Ng, E. Y., Langhorne, M. H., Ritchie, H. D., Brody, L. L., Westbrock-Wadman, S., Bayer, A. S., Folger, K. R. & Stover, C. K. (1998). Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models. Infect Immun 66, 567–572.[Abstract/Free Full Text]

Senior, B. W., Albrechtsen, M. & Kerr, M. A. (1987). Proteus mirabilis strains of diverse type have IgA protease activity. J Med Microbiol 24, 175–180.[Abstract/Free Full Text]

Setia, U., Serventi, I. & Lorenz, P. (1984). Bacteremia in a long-term care facility. Spectrum and mortality. Arch Intern Med 144, 1633–1635.[Abstract/Free Full Text]

Shea, J. E., Hensel, M., Gleeson, C. & Holden, D. W. (1996). Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci U S A 93, 2593–2597.[Abstract/Free Full Text]

Swihart, K. G. & Welch, R. A. (1990). Cytotoxic activity of the Proteus hemolysin HpmA. Infect Immun 58, 1861–1869.[Abstract/Free Full Text]

Tolson, D. L., Barrigar, D. L., McLean, R. J. & Altman, E. (1995). Expression of a nonagglutinating fimbria by Proteus mirabilis. Infect Immun 63, 1127–1129.[Abstract/Free Full Text]

Tolson, D. L., Harrison, B. A., Latta, R. K., Lee, K. K. & Altman, E. (1997). The expression of nonagglutinating fimbriae and its role in Proteus mirabilis adherence to epithelial cells. Can J Microbiol 43, 709–717.[Medline]

Walker, K. E., Moghaddame-Jafari, S., Lockatell, C. V., Johnson, D. & Belas, R. (1999). ZapA, the IgA-degrading metalloprotease of Proteus mirabilis, is a virulence factor expressed specifically in swarmer cells. Mol Microbiol 32, 825–836.[CrossRef][Medline]

Wang, W. B., Chen, I. C., Jiang, S. S., Chen, H. R., Hsu, C. Y., Hsueh, P. R., Hsu, W. B. & Liaw, S. J. (2008). Role of RppA in the regulation of polymyxin B susceptibility, swarming, and virulence factor expression in Proteus mirabilis. Infect Immun 76, 2051–2062.[Abstract/Free Full Text]

Warren, J. W., Tenney, J. H., Hoopes, J. M., Muncie, H. L. & Anthony, W. C. (1982). A prospective microbiologic study of bacteriuria in patients with chronic indwelling urethral catheters. J Infect Dis 146, 719–723.[Medline]

Warren, J. W., Damron, D., Tenney, J. H., Hoopes, J. M., Deforge, B. & Muncie, H. L., Jr (1987). Fever, bacteremia, and death as complications of bacteriuria in women with long-term urethral catheters. J Infect Dis 155, 1151–1158.[Medline]

Wassif, C., Cheek, D. & Belas, R. (1995). Molecular analysis of a metalloprotease from Proteus mirabilis. J Bacteriol 177, 5790–5798.[Abstract/Free Full Text]

Welch, R. A. (1987). Identification of two different hemolysin determinants in uropathogenic Proteus isolates. Infect Immun 55, 2183–2190.[Abstract/Free Full Text]

Wells, T. J., Tree, J. J., Ulett, G. C. & Schembri, M. A. (2007). Autotransporter proteins: novel targets at the bacterial cell surface. FEMS Microbiol Lett 274, 163–172.[CrossRef][Medline]

Wray, S. K., Hull, S. I., Cook, R. G., Barrish, J. & Hull, R. A. (1986). Identification and characterization of a uroepithelial cell adhesin from a uropathogenic isolate of Proteus mirabilis. Infect Immun 54, 43–49.[Abstract/Free Full Text]

Wu, J. & Newton, A. (1997). Regulation of the Caulobacter flagellar gene hierarchy; not just for motility. Mol Microbiol 24, 233–239.[CrossRef][Medline]

Zhao, H., Li, X., Johnson, D. E. & Mobley, H. L. (1999). Identification of protease and rpoN-associated genes of uropathogenic Proteus mirabilis by negative selection in a mouse model of ascending urinary tract infection. Microbiology 145, 185–195.[Abstract/Free Full Text]

Zilsel, J., Ma, P. H. & Beatty, J. T. (1992). Derivation of a mathematical expression useful for the construction of complete genomic libraries. Gene 120, 89–92.[CrossRef][Medline]

Zunino, P., Geymonat, L., Allen, A. G., Preston, A., Sosa, V. & Maskell, D. J. (2001). New aspects of the role of MR/P fimbriae in Proteus mirabilis urinary tract infection. FEMS Immunol Med Microbiol 31, 113–120.[CrossRef][Medline]

Zunino, P., Sosa, V., Allen, A. G., Preston, A., Schlapp, G. & Maskell, D. J. (2003). Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice. Microbiology 149, 3231–3237.[Abstract/Free Full Text]

Zunino, P., Sosa, V., Schlapp, G., Allen, A. G., Preston, A. & Maskell, D. J. (2007). Mannose-resistant Proteus-like and P. mirabilis fimbriae have specific and additive roles in P. mirabilis urinary tract infections. FEMS Immunol Med Microbiol 51, 125–133.[CrossRef][Medline]

This article has been cited by other articles:

				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]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Himpsl, S. D. Articles by Mobley, H. L. T. Search for Related Content PubMed PubMed Citation Articles by Himpsl, S. D. Articles by Mobley, H. L. T. Agricola Articles by Himpsl, S. D. Articles by Mobley, H. L. T.