Zinc uptake contributes to motility and provides a competitive advantage to Proteus mirabilis during experimental urinary tract infection

Abstract

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 (Zn(2+)) 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 Zn(2+) to the agar prior to inoculation; the addition of Fe(2+) to the agar also partially restored the swarming motility of the znuC::Kan strain, but the addition of Co(2+), Cu(2+), or Ni(2+) 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.

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.

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.

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.

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.

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.

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.

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.