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Proteus mirabilis, a Gram-negative bacterium, represents a common cause of complicated urinary tract infections in catheterized patients or those with functional or anatomical abnormalities of the urinary tract. ZnuB, the membrane component of the high-affinity zinc (Zn2+) transport system ZnuACB, was previously shown to be recognized by sera from infected mice. Since this system has been shown to contribute to virulence in other pathogens, its role in Proteus mirabilis was investigated by constructing a strain with an insertionally interrupted copy of znuC. The znuC::Kan mutant was more sensitive to zinc limitation than the wild type, was outcompeted by the wild type in minimal medium, displayed reduced swimming and swarming motility, and produced less flaA transcript and flagellin protein. The production of flagellin and swarming motility were restored by complementation with znuCB in trans. Swarming motility was also restored by the addition of Zn2+ to the agar prior to inoculation; the addition of Fe2+ to the agar also partially restored the swarming motility of the znuC::Kan strain, but the addition of Co2+, Cu2+, or Ni2+ did not. ZnuC contributes to but is not required for virulence in the urinary tract; the znuC::Kan strain was outcompeted by the wild type during a cochallenge experiment but was able to colonize mice to levels similar to the wild-type level during independent challenge. Since we demonstrated a role for ZnuC in zinc transport, we hypothesize that there is limited zinc present in the urinary tract and P. mirabilis must scavenge this ion to colonize and persist in the host.
Proteus mirabilis, a motile Gram-negative bacterium, represents a common cause of complicated urinary tract infections (71). These infections typically occur in patients with functional or anatomical abnormalities of the urinary tract or patients subjected to long-term catheterization (such as those with spinal cord injuries or elderly patients residing in nursing homes). The serious sequelae (including catheter encrustation, stone formation, renal scarring, and potential for progression to bacteremia) that can result from these P. mirabilis infections (46, 70, 71), as well as the difficulty in treating them, have sparked active research into the mechanisms of pathogenesis (2, 13, 19, 27, 28, 65, 78) and the identification of potential vaccine candidates (1, 40-42, 47, 56, 61). One of the most notable characteristics of P. mirabilis is swarming motility, a specialized form of flagellum-mediated motility during which bacteria differentiate into elongated hyperflagellated cells; these differentiated cells migrate together en masse (57). Swarming motility is clinically relevant, as P. mirabilis is capable of swarming across the surface of urinary catheters (67).
Our previous study, aimed at identifying antigens using sera from mice with experimental urinary tract infections (30), revealed that a protein annotated as ZnuB is an antigen expressed in vivo (47). ZnuB is homologous to the inner membrane component of the ZnuACB high-affinity zinc (Zn2+) transport system (51). The additional components of this ABC transporter are ZnuA, a periplasmic binding protein, and ZnuC, a cytoplasmic ATPase; P. mirabilis also encodes proteins homologous to ZnuA and ZnuC.
Zinc is essential for life but also toxic at high concentrations (48); thus, its intracellular concentration must be carefully regulated (12). In bacteria, this regulation is achieved primarily by coordinated efforts to import and export zinc in environments where the ion is limited or present in excess, respectively (26). In conditions where zinc is low, high-affinity uptake systems are employed to import zinc into the cell. In Escherichia coli, this process is achieved via the ZnuACB system (51). This system is synthesized from two divergent transcripts, znuA and znuCB. Under zinc-replete conditions, the regulator Zur (zinc uptake repressor) binds as a dimer in the intergenic region between znuA and znuC and represses transcription; when zinc becomes limited, the genes are derepressed (50). Zur is exquisitely sensitive to changes in the zinc concentration in the cell; differences can be sensed in the femtomolar range (49). Under moderate conditions in which zinc is neither limited nor toxic, zinc is brought into the cell through a lower-affinity transporter, namely, ZupT (23), which has broad metal specificity and is expressed constitutively at low levels (22). In addition, PitA, an inorganic phosphate transporter in E. coli, and CitM, a citrate transporter in Bacillus subtilis, are capable of transporting zinc (7, 34).
Much like iron, the level of zinc available in the host is presumed to be limited (43, 75) and may actually decrease in response to bacterial infection (20). Therefore, it is not surprising that the ZnuACB system has been found to contribute to virulence in a number of pathogens, including Campylobacter jejuni (18), Salmonella enterica serovar Typhimurium (6, 14), Haemophilus ducreyi (39), Brucella abortus (33, 74), and Pasteurella multocida (21). Recently, ZnuACB was shown to contribute to the ability of uropathogenic E. coli (UPEC) to colonize the urinary tract (60), suggesting that the urinary tract may be limited in zinc, as previously demonstrated for iron (5, 59, 62, 64).
Zinc uptake is uncharacterized in P. mirabilis. We hypothesized that the ZnuACB system functions as a zinc transport system in P. mirabilis and contributes to virulence, especially considering it is expressed in vivo (47). In this study, we show that the presence of ZnuC allows P. mirabilis to grow to a higher density under zinc limitation and yields a competitive advantage during growth in minimal medium. ZnuC is required for motility; a strain with an interrupted copy of the gene swims and swarms significantly less than the wild type and produces less flagellin, the major subunit of flagella. In addition, znuA and znuCB appear to be regulated by Zur. We show, for the first time, that the ability to import zinc contributes to the fitness of P. mirabilis during experimental urinary tract infection in the mouse model of this disease.
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.
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 Table1)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 Table11.
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 Table11 (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].
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 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. Fig.5.5. Statistical analyses were performed using the two-tailed Student t test with a 95% confidence interval.
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.
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 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).
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. (Fig.1).1). All three genes were upregulated >20-fold under zinc restriction. Ethanol, used to solubilize TPEN, had no effect on induction.
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. (Fig.2A).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. (Fig.2B).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. (Fig.3).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.
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. (Fig.4A).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. (Fig.4A),4A), suggesting that there is an additional (Zur independent and metal dependent) mechanism that contributes to the regulation of znuACB in P. mirabilis.
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. (Fig.4B).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. (Fig.4C4C).
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. (Fig.5L).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. (Fig.5E).5E). This result suggests that the defect observed in the znuC::Kan strain is a result of zinc limitation.
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. (Fig.5L).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. (Fig.5I).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. (Fig.5K).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. (Fig.5K5K 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. (Fig.6A),6A), which resulted in the production of lower levels of FlaA protein (Fig. (Fig.6B).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. (Fig.6A).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).
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. (Fig.7A7A).
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. (Fig.7B).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.
This is the first study to demonstrate the importance of zinc acquisition by P. mirabilis in the urinary tract during infection. We have shown that ZnuC, a component of a putative zinc ABC transporter that imports this critical ion, contributes to the fitness of P. mirabilis in the mouse model of ascending urinary tract infection. Based on growth differences in independent cultures supplemented with TPEN and coculture in minimal medium, ZnuC (presumably as a component of ZnuACB) appears to function as a zinc transport system in P. mirabilis. In addition, znuA and znuCB appear to be repressed by Zur since the expression levels of all three genes in a zur mutant were increased compared to their expression in the wild type. The overexpression of this high-affinity zinc uptake system rendered P. mirabilis hypersensitive to ZnSO4, demonstrating the importance of the ability to regulate zinc homeostasis. We also discovered that zinc acquisition is required for normal swimming and swarming motility. The statistically significant reduction in swarming of the znuC::Kan strain was complemented by expressing ZnuCB in trans or by adding ZnSO4 to the swarming agar prior to inoculation, suggesting that the defect resulted specifically from low levels of intracellular zinc in the mutant.
The results from the cochallenge infection with the wild type and the znuC::Kan strain in vivo suggest that there is a limited supply of zinc in the urinary tract. When the znuC::Kan strain was put in direct competition with the wild type for this nutrient, the mutant failed to thrive and was unrecoverable from infected animal tissue. However, during independent challenge, the znuC::Kan strain was able to utilize the zinc present and colonized the urinary tract to levels similar to the wild type. This result suggests that in the absence of competition, the znuC::Kan strain has sufficient mechanisms for acquiring this critical ion, perhaps through the use of lower-affinity transport systems. A similar phenotype was observed during experimental urinary tract infection with a derivative of the UPEC strain CFT073 lacking a functional ZnuACB transport system; the mutant was outcompeted in bladders and kidneys during cochallege (60). However, in contrast to P. mirabilis, UPEC CFT073 required functional ZnuACB to reach optimal infection levels in the kidney during independent challenge; although there was no difference in bladder colonization, CFT073 ΔznuA was recovered from infected kidneys in significantly fewer numbers than controls (60). The reason for this disparity is not immediately clear, but one possible explanation could be a difference in low-affinity zinc uptake between the two species. In any case, taken together, the data from two different uropathogens align well and support the hypothesis that zinc is a critical nutrient during infection of the urinary tract.
The ability of the znuC::Kan strain to colonize the urinary tract during independent challenge suggests that this mutant is capable of using other means to bring zinc into the cell. ZupT, previously thought to be a zinc-specific low-affinity transporter (23), is in fact a transporter with broad metal specificity (22). However, based on the genome sequence and annotation of P. mirabilis HI4320 (55), this strain appears to lack a ZupT homolog. Other transporters known to import zinc include the inorganic phosphate transporter PitA (7) and the citrate transporter CitM (34). P. mirabilis appears to encode proteins homologous to both PitA and CitM, but the contribution of either of these proteins to zinc acquisition in this species is currently unknown.
It should be noted that we cannot rule out the possibility that the colonization defect of the znuC::Kan strain observed during cochallenge could be due, at least in part, to the reduction in motility observed with this strain. We observed only a modest defect in swimming motility in the znuC::Kan strain, while swarming motility was greatly affected; the znuC::Kan strain produced less flagellin than the wild-type strain. Flagellum-mediated motility has been implicated as a virulence factor in P. mirabilis (45). A FlaA− strain of P. mirabilis did not differentiate into swarmer cells (8), but we observed elongated forms of znuC::Kan cells and know this strain is capable of at least this step in the differentiation process. The contribution of swarmer cells and swarming motility (as opposed to the production of flagella and swimming motility) to virulence is unclear. The expression of virulence genes has been linked to swarmer cell differentiation (4). However, the presence of swarmer cells in vivo is debated (3, 29). In addition, some studies conclude that motile, nonswarming strains have a reduced capacity to cause infection (3), while others found that nonflagellated strains (which are therefore incapable of swarming) are still able to cause infection (38, 77).
It is also possible that the colonization defect of the znuC::Kan strain observed during cochallenge could be due to changes in the expression of virulence factors other than flagella. For example, we determined that the expression of zapA, which encodes the secreted zinc metalloprotease ZapA (72), was reduced approximately 9-fold in the znuC::Kan strain compared with its expression in the wild-type strain (data not shown). ZapA can cleave a broad range of host proteins, including antimicrobial peptides and immunoglobulins, and has been shown to be a virulence factor for P. mirabilis during infection (11, 68). It is possible that this decreased expression of zapA could contribute to the phenotype observed during the znuC::Kan and wild-type strain cochallenge experiment; it is currently unknown if the expression or production of any other virulence factors are affected in the znuC::Kan strain.
In P. mirabilis, it was previously demonstrated that a strain with an interrupted copy of the ZntA zinc efflux protein homolog PpaA also displayed reduced swarming (36). This phenotype is interesting because we note that proteins with seemingly opposing functions (zinc uptake and efflux for ZnuC and PpaA, respectively), when interrupted, both affect swarming motility. Perhaps zinc homeostasis (as opposed to exclusively uptake or efflux) plays a role in motility. Indeed, in E. coli, the transcription of genes involved in flagellar biosynthesis is downregulated in response to treatment with TPEN (63), while the transcription of some motility-related genes is increased in E. coli treated with zinc (37).
As described above, swarming motility requires a large number of flagella per bacterium. The production of flagella occurs via a regulatory cascade mediated by the “master regulators” FlhDC (reviewed in references 15 and 66). The flhDC genes comprise class I of the flagellar regulon and activate the expression of the class II genes, whose products include proteins necessary to form the basal body and hook of the flagellum, as well as FliA. FliA is the transcription factor required to synthesize proteins present in class III, including FlaA. FlhC, part of the FlhDC complex that induces the flagellar cascade, was recently determined to have a zinc-binding site (69). It is unknown what, if any, role zinc binding has on FlhDC function. However, if FlhDC requires zinc for its function, the levels of intracellular zinc in the znuC::Kan strain may not be sufficient to fulfill the normal requirement for optimal FlhDC activity. We hypothesize that this potential reduction in FlhDC activity could represent one explanation for the reduced motility of the znuC::Kan strain; reduced FlhDC activity would result in reduced transcription of flagellar genes and, therefore, reduced motility. Indeed, we observed a stepwise reduction in the transcription of genes in the flagellar cascade in the znuC::Kan strain. The flhD (class I), flgA and flgC (class II), and flaA (class III) transcripts were reduced in the znuC::Kan strain relative to their levels in the wild-type strain. Class III gene expression was affected more than class II gene expression, which in turn was more affected than class I gene expression; these data make sense given what is known about the hierarchical regulation of flagellar genes. We were slightly surprised to observe a decrease, albeit a modest one, in the expression of flhD; we would predict that the protein functions of FlhDC, rather than their expression levels, would be affected by low levels of zinc in the cell, but it is possible that there is a feedback mechanism that affects gene expression. This hypothesis could help explain the differences in the effects mutation of ZnuC had on swimming and swarming motility; swarming motility, which has a greater requirement for flagellin, was affected to a greater degree. Furthermore, this phenomenon may not be specific to P. mirabilis, since motility was also reduced in UPEC mutants lacking functional zinc uptake systems (24, 60). To elucidate the mechanism of interplay between zinc homeostasis and motility, further studies are required.
The ability of P. mirabilis to employ swarming motility, although described for more than a century (73), is still not well understood and remains a target of active research. Deciphering this behavior would not only benefit our understanding of a facet of the life cycle of this pathogenic bacterium but could potentially lead to important therapeutic targets, since P. mirabilis may gain access to the body by swarming over the surface of catheters. We have shown, for the first time, that the ability to acquire zinc via a high-affinity transport system is another piece of this proverbial puzzle.
This work was supported in part by Public Health Service Grants AI43363 and AI059722 from the National Institutes of Health.
Editor: S. R. Blanke
Published ahead of print on 12 April 2010.