Zinc Uptake Contributes to Motility and Provides a Competitive Advantage to Proteus mirabilis during Experimental Urinary Tract Infection

Strains and culture conditions. P. mirabilis HI4320 was cultured from the urine of a catheterized nursing home patient with bacteriuria (46). Luria broth (LB) (per liter, 10 g tryptone, 5 g yeast extract, and 0.5 g NaCl) and nonswarming agar (per liter, 10 g tryptone, 5 g yeast extract, 0.5 g NaCl, and 15 g agar) were used to culture bacteria. Minimal A medium was prepared as previously described (10). All cultures were incubated at 37°C with aeration unless otherwise noted. When appropriate, kanamycin or ampicillin was added to the medium at a final concentration of 25 μg/ml or 100 μg/ml, respectively. Metal chelation was achieved by the addition of N,N,N′,N′-Tetrakis (2-pyridylmethyl)ethylenediamine (TPEN; Sigma-Aldrich), dissolved in ethanol prior to use. Metals used to supplement a medium were obtained from the following sources: FeCl₂ (Fisher Scientific), Cu(II)SO₄·5H₂O (Sigma), Co(II)Cl₂·6H₂O (Sigma), NiSO₄·6H₂O (Sigma), and ZnSO₄·7H₂O (J.T.Baker). A Bioscreen C growth curve analyzer (Growth Curves, United States) was used for independent growth curves.

Construction of mutants.Insertional mutants were constructed using the TargeTron system (Sigma) as described for P. mirabilis by Pearson and Mobley (53). Briefly, genes were disrupted by the insertion of an intron, targeted specifically to the gene of interest by using a set of three primers (IBS, EBS1d, and EBS2; listed in Table 1) in a mutagenic PCR. This mutated region of the intron was ligated into the vector pACD4K-C. The resultant plasmids were sequenced to confirm proper retargeting of the intron. Plasmids containing a correctly retargeted intron were electroporated into electrocompetent P. mirabilis HI4320 containing the helper plasmid pAR1219 (17). Since the intron contains a kanamycin resistance gene, transformants were selected on agar containing kanamycin and screened by PCR for an insertion in the appropriate gene, using the screening primers listed in Table 1.

Complementation.The results of quantitative reverse transcriptase PCR (qRT-PCR) indicated that the znuC::Kan mutant contained a polar mutation that resulted in reduced expression of znuB (data not shown); therefore, the znuCB genes were employed for complementation studies. The znuCB genes were amplified from wild-type P. mirabilis HI4320 genomic DNA using primers listed in Table 1 (znuCBcompFor and znuCBcompRev) and cloned into pCR2.1-TOPO (Invitrogen). The resultant plasmid, pTOPO-znuCB, was transformed into electrocompetent E. coli TOP10 (Invitrogen); transformants were selected on agar containing kanamycin. Restriction enzymes HindIII and XhoI (New England Biolabs) were used to digest pTOPO-znuCB and the vector pACYC177 (New England Biolabs); the insert was ligated into the digested vector using T4 DNA ligase (Promega). The resultant plasmid, pZnuCB, was transformed into P. mirabilis HI4320 by electroporation to yield the complemented [znuC::Kan(pZnuCB)] strain, which was selected for on agar containing ampicillin. pACYC177 was also transformed into the znuC::Kan strain for use as an empty vector control [znuC::Kan(pEV) strain].

qRT-PCR.Log-phase culture (0.5 ml) was added to 1 ml RNAprotect solution (Qiagen). RNA was isolated using the RNeasy mini prep protocol (Qiagen) according to the manufacturer's directions. Samples were treated with DNase (Turbo DNA-free DNase; Ambion), and cDNA was synthesized using the Superscript first-strand synthesis system (Invitrogen). Samples were analyzed by RT-PCR with primers specific to rpoA to confirm lack of product in negative controls with no reverse transcriptase added. qRT-PCRs were performed in duplicate and contained 30 ng cDNA and 12.5 μl 2× SYBR green PCR master mix (Stratagene) per reaction mixture. Primers were used at 100 nM (for rpoA and znuA) or 200 nM (all others). qRT-PCR was performed with an Mx3000P thermal cycler (Stratagene). Data were normalized to the expression of rpoA. For experiments examining expression under conditions of zinc limitation, 35 μM TPEN (or ethanol, as a control) was added to cultures 30 min prior to RNA isolation.

Swimming and swarming motility.Swimming motility was measured by stabbing the inoculum into soft agar swim plates (per liter, 10 g tryptone, 0.5 g NaCl, 2.5 g agar). Swarming motility was measured by spotting 5 μl of the inoculum onto the surface of swarming agar plates (per liter, 10 g tryptone, 10 g NaCl, 5 g yeast extract, 15 g agar). The inocula were late-logarithmic-phase bacterial cultures adjusted to an optical density at 600 nm (OD₆₀₀) of 1.0. Both swim and swarm plates were incubated at 30°C for the times indicated in the legend for Fig. 5. Statistical analyses were performed using the two-tailed Student t test with a 95% confidence interval.

Western blot assay.Overnight cultures of bacteria were adjusted to an OD₆₀₀ of 1.0. Cells were collected from 1 ml of the adjusted culture, resuspended in 100 μl of 2× Laemmli buffer (35), and boiled for 10 min. The proteins present in 10 μl of each sample were loaded onto 12.5% acrylamide gels and separated by polyacrylamide gel electrophoresis. Duplicate gels were run; one gel was stained with Coomassie to confirm that protein levels were similar in each sample, and the other gel was used for Western blotting. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore) at 100 V for 1 h at 4°C. The membrane was blocked overnight at 4°C in 5% milk dissolved in TBS-T (0.05% Tween 20, 100 mM Tris [pH 7.5], 9% NaCl). The membrane was incubated for 45 min at room temperature with anti-FlaA antibody (9), as recently described (52). Following three quick rinses with TBS-T, the membrane was subjected to one 15-min and three 5-min washes in TBS-T. The secondary antibody, peroxidase-conjugated goat anti-rabbit IgG (Sigma), was diluted 1:10,000 in TBS-T and applied at room temperature with shaking for 45 min. After the wash steps were repeated, detection was performed using an Amersham ECL Plus Western blotting detection system (GE Healthcare) following the protocol recommended by the manufacturer.

CBA/J mouse model of urinary tract infection.Cochallenge and independent challenges of CBA/J mice were carried out as previously described using a modification (31) of the Hagberg protocol (25). Briefly, single colonies were picked from a fresh plate and used to start overnight cultures in LB. On the day of inoculation, cultures were diluted with fresh LB to an OD₆₀₀ of ≈0.2. For independent challenges, mice were inoculated transurethrally with 50 μl of culture containing approximately 10⁷ CFU. (Actual inputs were 1.41 × 10⁷ CFU per mouse for mice infected with the wild type and 2.36 × 10⁷ CFU per mouse for mice infected with the znuC::Kan strain.) For cochallenge experiments, the wild type and mutant cultures were mixed in a 1:1 ratio and mice were transurethrally inoculated with 50 μl of the mixture containing approximately 10⁷ CFU. (Actual input for the cochallenge experiment contained, per mouse, 6.15 × 10⁶ CFU of the wild type and 1.08 × 10⁷ CFU of the znuC::Kan strain.) After seven days, urine was collected, mice were euthanized, and bladders and kidneys were harvested and transferred to sterile tubes containing phosphate-buffered saline. Tissues were homogenized using an Omni TH homogenizer (Omni International) and plated using a spiral plater (Autoplate 4000; Spiral Biotech). For independent challenge experiments, all samples were plated on plain agar. For cochallenge experiments, all samples were plated on both plain agar and agar containing kanamycin.

Statistical analysis.Statistical significance was assessed by using the Mann-Whitney test and Wilcoxin matched pairs test for independent and cochallenge experiments, respectively. All statistical analyses were performed using GraphPad Prism (version 3.00 for Windows; GraphPad Software, San Diego, CA).

ZnuC plays a role in zinc acquisition in P. mirabilis.In our previous study, ZnuB was identified on an immunoblot of membrane proteins using sera from mice experimentally infected with P. mirabilis (47). The antisera recognized ZnuB, indicating that this protein is expressed in vivo. As homologous ZnuACB uptake systems have been shown to contribute to virulence in other bacterial pathogens, its role in P. mirabilis was investigated.

We hypothesized that the expression of a divalent cation transporter would be regulated and could be induced under zinc restriction. To test this hypothesis, bacteria were cultured in the presence and absence of the zinc chelator TPEN and the expression levels of znuACB were measured by qRT-PCR (Fig. 1). All three genes were upregulated >20-fold under zinc restriction. Ethanol, used to solubilize TPEN, had no effect on induction.

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FIG. 1.

Expression of znuACB is induced by zinc limitation. Wild-type P. mirabilis was cultured in the absence or presence of TPEN. Gene expression was analyzed by qRT-PCR. Data were normalized to the expression of rpoA and are presented as the fold change in expression compared to the expression in culture in LB without TPEN. Black bars, LB supplemented with 35 μM TPEN; gray bars, LB supplemented with solvent (ethanol). Error bars show standard errors of the means.

To demonstrate a growth requirement for zinc, a mutation was constructed in the putative transport system using a method that has been successful in generating P. mirabilis mutants (53). znuC was inactivated by the insertion of an intron containing a kanamycin resistance gene, resulting in the znuC::Kan strain. Insertion of the intron within znuC was confirmed by PCR (data not shown).

There was no difference in the growth rates of the znuC::Kan strain and the wild type when cultured independently in LB, minimal medium, or pooled human urine (data not shown). However, when the two strains were cultured together, and therefore put in direct competition for resources, the znuC::Kan strain was outcompeted by the wild-type strain in minimal medium (Fig. 2A). The difference in growth was overcome by the addition of 1 mM ZnSO₄ to the culture; under those conditions, the znuC::Kan strain was maintained in the culture, along with the wild-type strain, for the duration of the experiment (Fig. 2B). The maintenance of the znuC::Kan strain for the duration of the experiment could be achieved by the addition of as little as 5 μM ZnSO₄ (the lowest concentration tested) to the culture medium (data not shown). The outcompetition phenotype was observed during coculture in minimal medium but not in LB (data not shown), suggesting that the higher concentration of zinc in LB is sufficient to sustain the growth of P. mirabilis without a functioning high-affinity uptake system, even when it is put in direct competition with the wild-type strain for this necessary nutrient. To confirm results from coculture experiments, the growth of the znuC::Kan strain was analyzed under zinc limitation achieved by the addition of TPEN to the culture medium. Whereas little difference was observed in the growth rates of the wild type and the znuC::Kan strain in LB, the znuC::Kan strain was more sensitive than the wild type to the addition of 30 μM TPEN (Fig. 3). The growth defect observed in response to the addition of TPEN to the medium was zinc specific; when 30 μM ZnSO₄ was added to the TPEN-containing medium, the growth of both strains was restored to levels indistinguishable from the growth observed in plain LB (data not shown). Taken together, these data suggest that the ZnuACB system functions in zinc acquisition in P. mirabilis, consistent with its function in other bacterial species.

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FIG. 2.

The znuC::Kan strain is outcompeted by the wild type during coculture in minimal medium. Cultures were inoculated with approximately equal amounts of the wild type and the znuC::Kan strain. At indicated time points, samples were taken for plating and the culture was repassaged (1:100) into fresh medium. Filled symbols, wild type; open symbols, znuC::Kan strain. (A) Growth in minimal A medium. Dashed gray line designates the limit of detection (100 CFU/ml). (B) Growth in minimal A medium supplemented with 1 mM ZnSO₄.

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FIG. 3.

The znuC::Kan strain is more sensitive to TPEN than the wild type. The wild type and the znuC::Kan strain were cultured in either plain LB or LB supplemented with 30 μM TPEN. Growth was monitored by recording OD₆₀₀ at 15-min intervals over a 24-h time period; for clarity, only 30-min time points are shown. Filled symbols, wild type; open symbols, znuC::Kan strain; circles, growth in LB; diamonds, growth in LB supplemented with 30 μM TPEN.

Zur represses the expression of znuACB.Analogous to the E. coli system (50, 51), we hypothesized that znuACB would be repressed by the Zn-binding repressor Zur, which is distally encoded on the P. mirabilis chromosome (zur, PMI2743, and znuACB, PMI1150-1152). PMI2743 was annotated as Zur in the original P. mirabilis HI4320 genome annotation (55); it is 49.7% identical and 54.7% similar to the Zur amino acid sequence encoded by the UPEC strain CFT073. To investigate the role of Zur in P. mirabilis, zur was insertionally inactivated by the method described above, resulting in a zur::Kan strain. Again, this mutation was confirmed by PCR (data not shown). As assessed by qRT-PCR, the levels of expression of znuACB increased >20-fold in the zur::Kan strain (Fig. 4A). These results support the hypothesis that Zur acts as a repressor of znuACB in P. mirabilis. Interestingly, the expression of the znuACB genes was further increased in the zur::Kan strain by culturing this strain in the presence of TPEN (Fig. 4A), suggesting that there is an additional (Zur independent and metal dependent) mechanism that contributes to the regulation of znuACB in P. mirabilis.

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FIG. 4.

The zur::Kan strain is more sensitive to zinc than the wild-type strain. (A) The wild type and the zur::Kan strain were cultured in LB. Gene expression was analyzed by qRT-PCR. Data were normalized to the expression of rpoA and are presented as the fold change in expression in the zur::Kan strain compared to expression in the wild type. White bars, expression in zur::Kan strain cultured in LB; black bars, expression in zur::Kan strain cultured in LB supplemented with 35 μM TPEN. Error bars show standard errors of the means. (B) The wild type and the zur::Kan strain were cultured in plain LB, as well as LB supplemented with 500 μM, 750 μM, or 1 mM ZnSO₄. Filled symbols, wild type; open symbols, zur::Kan strain; circles, LB; triangles, LB plus 500 μM ZnSO₄; diamonds, LB plus 750 μM ZnSO₄; inverted triangles, LB plus 1 mM ZnSO₄. (C) The zur::Kan strain was cultured in LB supplemented with zinc either at the start of the experiment or after the OD₆₀₀ reached >0.3. Filled symbols, cultures with zinc added at beginning; open symbols, cultures spiked with zinc during growth; triangles, LB plus 500 μM ZnSO₄; diamonds, LB plus 750 μM ZnSO₄; inverted triangles, LB plus 1 mM ZnSO₄. Arrow depicts when zinc was added to (open symbol) cultures.

We hypothesized that since the zur::Kan strain may overproduce the ZnuACB high-affinity uptake system, this strain may be hypersensitive to zinc toxicity. To test this hypothesis, the zur::Kan strain and the wild-type strain were cultured independently in LB supplemented with 500 μM, 750 μM, and 1 mM ZnSO₄. As predicted, the zur::Kan strain was more sensitive to excess zinc than the wild-type strain (Fig. 4B). Interestingly, when cultured with zinc at concentrations of 500 μM ZnSO₄ or above, the zur::Kan strain displayed an extended lag phase until the culture reached a specific density (OD₆₀₀ ≈ 0.3). Once the culture reached this density, log-phase growth commenced. To rule out the possibility of a secondary or suppressor mutation leading to the ability of the zur::Kan strain to grow after an extended lag phase, cultures were passaged again into fresh medium supplemented with the same concentration of ZnSO₄. If a suppressor mutation were responsible for the outgrowth, we would expect no extended lag phase to occur. However, an identical pattern of growth was observed (that is, an extended lag phase until the OD₆₀₀ exceeded 0.3) (data not shown); therefore, we concluded that zinc toxicity, rather than a suppressor mutation, was the cause of this extended lag period during growth. In addition, culturing the zur::Kan strain in preconditioned medium did not affect the occurrence of this extended lag phase (data not shown).

We further hypothesized that if zur::Kan cells were able to divide (albeit slowly) in the presence of zinc, the culture would ultimately reach a density such that the amount of zinc present in the culture could be evenly shared by a greater number of cells and therefore become less of a burden on individual cells, at which point they would be able to overcome the concentration-dependent growth restriction. We reasoned that if the zur::Kan culture was allowed to reach this critical density prior to the addition of zinc, zinc toxicity should not cause the extended lag phase observed in cultures with zinc present from the point of inoculation. To test this hypothesis, the zur::Kan strain was used to inoculate two types of cultures, either LB supplemented with ZnSO₄ at the inception of the experiment or, alternately, LB supplemented with the same concentration of ZnSO₄ after the culture reached an OD₆₀₀ of >0.3. Indeed, when ZnSO₄ was added to cultures of the zur::Kan strain that had already passed an OD₆₀₀ of 0.3, these cultures were able to grow at a much higher rate than their counterparts with ZnSO₄ added at the beginning, even though the paired cultures ultimately contained the same concentration of zinc (Fig. 4C).

Zinc uptake contributes to swarming and swimming motility.It has been previously reported that PpaA, a P-type ATPase homologous to ZntA that functions in zinc efflux (58), plays a role in swarming in P. mirabilis. A strain with a transposon insertion in the gene ppaA showed a delayed swarming response compared to that of the wild type (36). Since disrupted zinc efflux affects swarming motility, we hypothesized that zinc uptake would also affect motility. To test this assertion, the motility of the znuC::Kan and zur::Kan strains was investigated. There was no difference in the swimming or swarming motility of the zur::Kan strain compared to those of the wild type (data not shown). However, the znuC::Kan strain displayed a modest but statistically significant reduction in swimming motility compared to that of the wild-type strain (P = 0.025) (Fig. 5L). Swarming motility was also affected; the znuC::Kan strain displayed significantly reduced swarming motility compared to that of the wild-type strain (P = 0.0082) (compare Fig. 5A and B and see I). In addition, the appearance of the swarming colony of the znuC::Kan strain was grossly different than that of the wild-type swarming colony. Wild-type swarming colonies displayed a smooth appearance; in contrast, znuC::Kan swarming colonies had a fractured appearance, with jagged projections advancing at the swarm front rather than the smooth colony edge observed with the wild type. Upon close inspection, gaps were present in the advancing colony edge containing these projections; the swarming colony did not cover the entire agar surface in the same way that the wild-type swarming colony did. Interestingly, the wild-type strain displayed a similar fractured pattern and reduced swarming radius when inoculated onto swarming agar supplemented with TPEN (Fig. 5E). This result suggests that the defect observed in the znuC::Kan strain is a result of zinc limitation.

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FIG. 5.

The znuC::Kan strain swarms significantly less than the wild type. (A to F) Swarming agar plates were spotted with 5 μl of log-phase culture. After the inoculation spot dried, plates were incubated at 30°C. Plates shown are representative examples measured 24 h postinoculation. (A) Wild type. (B) znuC::Kan strain. (C) znuC::Kan(pEV) strain. (D) znuC::Kan(pZnuCB) strain. (E) Wild type inoculated on agar containing 20 μM TPEN. (F) znuC::Kan strain inoculated on agar supplemented with 250 μM ZnSO₄. (G and H) Gram-stained wild-type and znuC::Kan cells, respectively, taken from the leading edge of a colony on swarm agar. Images shown were taken at the same magnification. (I) Swarm radii of wild-type, znuC::Kan, znuC::Kan(pEV), and znuC::Kan(pZnuCB) strains measured 16 h postinoculation. Data are from three independent experiments and were analyzed by paired t test. Asterisks above bars denote significance compared to results for the wild type. *, P < 0.05; **, P < 0.01. (J) Cultures of the znuC::Kan strain were spotted on swarming agar supplemented with ZnSO₄. Swarm radii were measured 20 h postinoculation. Data are from a representative experiment and were analyzed by unpaired t test. Asterisks denote significance compared to results for plates with no zinc added. *, P < 0.05; **, P < 0.001. (K) Cultures of the znuC::Kan strain were spotted on plain swarming agar or swarming agar supplemented with 50 μM CuSO₄, CoCl₂, NiSO₄, or FeCl₂. Swarm radii were measured 20 h postinoculation. Dotted line represents mean swarm radius of the znuC::Kan strain spotted on agar containing 50 μM ZnSO₄ for comparison (refer to data shown in J). Data are from a representative experiment and were analyzed by unpaired t test. Asterisks denote significance compared to results for plain swarming agar plates. *, P < 0.005. (L) Swimming radii of the wild-type, znuC::Kan, znuC::Kan(pEV), and znuC::Kan(pZnuCB) strains measured 16 h postinoculation. Data are from three independent experiments and were analyzed by paired t test. *, P < 0.05. Radii of the wild type and the znuC::Kan(pZnuCB) strain were not significantly different (P = 0.0508). Error bars show standard errors of the means.

The swarming defects of the znuC::Kan strain were complemented by adding znuCB back in trans on a plasmid (P = 0.0448 compared to an empty vector control) (compare Fig. 5C and D and see I). The complemented strain also regained swimming motility (P = 0.0382 compared to an empty vector control) (Fig. 5L). It should be noted that only partial complementation was achieved in swarming motility; although the complemented strain swarmed less than the wild type, this difference was not significant (P = 0.0508) (Fig. 5I). The swarming defect observed with the znuC::Kan strain could also be complemented by the addition of ZnSO₄ to the swarming agar prior to inoculation (compare Fig. 5B and F and see J); the swarming motility of the wild-type strain was not significantly affected by the addition of 250 μM ZnSO₄ to the agar (data not shown). Under both complementation conditions, the znuC::Kan swarming colony morphology returned to a noticeably smoother appearance. Swarming by the znuC::Kan strain could not be restored by the addition of Co²⁺, Cu²⁺, or Ni²⁺ to the swarming agar (Fig. 5K). When Fe²⁺ was added to the medium, the znuC::Kan strain swarmed more than on plain swarming agar; however, the radius of swarming motility did not reach the level achieved with the addition of ZnSO₄ and the colony retained the fractured morphology (Fig. 5K and data not shown).

For swarming motility to occur, P. mirabilis must first differentiate into the elongated swarmer cell morphology (57). Gram staining of bacteria taken from the edge of the swarming front of both the znuC::Kan strain and the wild type revealed that cells from both plates were elongated (Fig. 5G and H). Swarmer cells are typically defined as those with a length of more than 10 μm (16, 29, 44); an average of 89.4% of cells sampled from the edge of a swarming wild-type colony and an average of 75.2% of cells sampled from a “swarming” znuC::Kan colony were longer than 10 μm. Although this difference is statistically significant (P < 0.05), it is difficult to interpret these data since znuC::Kan swarming colonies do not display typical swarming colony behavior, as described above; these colonies do not appear to undergo the standard 2-h cycling between swarmer cells and consolidate cells that is well-documented for the wild-type strain (54). Therefore, it is difficult to say that the “swarming” samples we collected from znuC::Kan colonies are, indeed, at the “swarming” stage of their life cycle or even if that normal swarming stage occurs as it does in the wild-type strain. However, since we observed cells longer than 10 μm, the swarming defect of the znuC::Kan strain does not appear to be the result of a block in the elongation process.

One of the hallmark characteristics of a swarm cell is the large number of flagella synthesized by this morphotype. To determine if znuC::Kan cells are capable of producing flagella at levels similar to the levels in wild-type cells, flagellin transcripts and protein in the znuC::Kan strain were assessed by qRT-PCR and Western blotting, respectively. The transcription of flaA, which encodes flagellin, was reduced approximately 18-fold in the znuC::Kan strain compared to the transcription level in the wild type (Fig. 6A), which resulted in the production of lower levels of FlaA protein (Fig. 6B). The transcription of other motility genes was also affected in the znuC::Kan strain; the levels of flhD, which encodes the so-called master regulator of flagellar motility FlhD, and of flgA and flgC, which both encode proteins in the basal body of the flagellar structure, were also reduced in the znuC::Kan strain compared to their levels in the wild type (Fig. 6A). The FlaA protein levels were restored in the complemented strain. The transcription of flaA was also slightly reduced in the wild type cultured in LB containing TPEN (approximately 1.8-fold; data not shown).

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FIG. 6.

Flagellin transcript and protein levels are decreased in the znuC::Kan strain. (A) Results of analysis of motility gene transcripts by qRT-PCR. Data were normalized to the expression of rpoA and are presented as the fold change compared to expression in the wild type. Error bars show standard errors of the means. (B) Results of Western blotting with anti-FlaA antibody.

ZnuC contributes to but is not required for virulence of P. mirabilis in the urinary tract.Because the ZnuACB system has been shown to contribute to the virulence of several pathogens, its role in pathogenesis was investigated in the murine model of ascending urinary tract infection that has been well established for virulence factor assessment in P. mirabilis (2, 13, 27, 32, 76). CBA/J mice were transurethrally inoculated with approximately 1 × 10⁷ CFU of either the wild-type or znuC::Kan strain. After seven days, mice were sacrificed and bacteria present in urine, bladders, and kidneys were quantified. We found that the znuC::Kan strain colonized mice in numbers similar to the wild-type strain (Fig. 7A).

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FIG. 7.

ZnuC contributes to fitness of P. mirabilis in the urinary tract but is not required for infection. Mice were infected transurethrally, and after seven days, urine, bladders, and kidneys were quantitatively cultured. Each symbol represents a datum from an individual mouse. Solid symbols, mice infected with wild type; open symbols, mice infected with znuC::Kan strain. Bars represent the medians. Limit of detection is 100 CFU/ml of urine or gram of tissue. (A) Independent challenge. Mice were infected with approximately 10⁷ CFU of either the wild type or the znuC::Kan strain. (B) Cochallenge. Mice were infected transurethrally with approximately 10⁷ CFU of a 1:1 mix of the wild type and the znuC::Kan strain.

However, to assess the contribution of the ZnuACB transport system to the fitness of P. mirabilis in the murine urinary tract, we conducted a cochallenge experiment in which the znuC::Kan strain was put in direct competition with the wild type during infection. Ten CBA/J mice were infected transurethrally with a 1:1 mixture of the wild type and the znuC::Kan strain. Following a seven-day infection, the znuC::Kan strain was outcompeted more than 10,000-fold by the wild type in the urine (P < 0.005) and was unrecoverable from the bladder and kidneys (P < 0.01 and P < 0.005, respectively) (Fig. 7B). Taken together, these data reveal that while ZnuC is not required for P. mirabilis to colonize the host, it offers a competitive advantage during urinary tract infection.

Similar to the phenotypes observed with the znuC::Kan strain, the zur::Kan strain was also statistically significantly outcompeted by the wild-type strain during cochallenge (wild type, median 9.29 × 10³ CFU/g bladder tissue and median 8.52 × 10² CFU/g kidney tissue, and zur::Kan strain, median 1.00 × 10² CFU/g bladder tissue and median 1.00 × 10² CFU/g kidney tissue; for bladder, P = 0.0105, and for kidney, P = 0.0043) but colonized mice in numbers similar to the wild type during independent challenge (data not shown). However, these data are difficult to interpret because the growth defect observed during the cochallenge experiment is not limited to in vivo conditions; the zur::Kan strain was also outcompeted by the wild type during a week-long coculture in vitro, indicating that this strain has a subtle growth defect (data not shown). We hypothesize that this growth defect may be caused by the energy burden due to the production of the ZnuACB high-affinity zinc uptake system when it is not necessary or appropriate.