Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract

Abstract

Uropathogenic Escherichia coli (UPEC) causes most uncomplicated urinary tract infections (UTIs) in humans. Flagellum-mediated motility and chemotaxis have been suggested to contribute to virulence by enabling UPEC to escape host immune responses and disperse to new sites within the urinary tract. To evaluate their contribution to virulence, six separate flagellar mutations were constructed in UPEC strain CFT073. The mutants constructed were shown to have four different flagellar phenotypes: fliA and fliC mutants do not produce flagella; the flgM mutant has similar levels of extracellular flagellin as the wild type but exhibits less motility than the wild type; the motAB mutant is nonmotile; and the cheW and cheY mutants are motile but nonchemotactic. Virulence was assessed by transurethral independent challenges and cochallenges of CBA mice with the wild type and each mutant. CFU/ml of urine or CFU/g bladder or kidney was determined 3 days postinoculation for the independent challenges and at 6, 16, 48, 60, and 72 h postinoculation for the cochallenges. While these mutants colonized the urinary tract during independent challenge, each of the mutants was outcompeted by the wild-type strain to various degrees at specific time points during cochallenge. Altogether, these results suggest that flagella and flagellum-mediated motility/chemotaxis may not be absolutely required for virulence but that these traits contribute to the fitness of UPEC and therefore significantly enhance the pathogenesis of UTIs caused by UPEC.

FIG. 1.

Characterization of motility by swimming in soft agar and phase-contrast microscopy. Cultures of the wild type (A), MC4100 (B), and the flagellar mutants and their complements (C to N) were stabbed into 0.25% soft-agar plates and incubated at 30°C. The incubation times for soft-agar plates were 11 h for panel A, 17 h for panels G, H, M, and N, 22 h for panels K and L, 26 h for panels C and D, and 39 h for panels B, E, F, I, and J. The symbols below the images represent motility as assessed by phase-contrast microscopy: nonmotile (−), slightly motile (±), motile (+), and highly motile (+++).

FIG. 2.

Flagellin expression assessed by Western blot analysis. The size of flagellin in UPEC strain CFT073 is predicted to be 60.9 kDa. Flagella from overnight cultures were isolated, denatured, electrophoresed onto SDS-PAGE gels, and subjected to Western blot analysis with antiserum to H1 flagellin. Lane 1, wild type; lane 2, fliC mutant; lane 3, fliC mutant with pfliC; lane 4, motAB mutant; lane 5, motAB mutant with pmotAB; lane 6, cheW mutant; lane 7, cheW mutant with pcheW; lane 8, cheY mutant; lane 9, cheY mutant with pcheY; lane 10, fliA mutant; lane 11, fliA mutant with pfliA; lane 12, flgM mutant; lane 13, flgM mutant with pflgM.

FIG. 3.

Transmission electron micrographs of MC4100 (aflagellated control), wild-type CFT073 (WT) and its ΔfliC mutant, and the ΔfliC mutant complemented with pfliC. Log-phase cultures were added to copper mesh grids coated with poly-l-lysine, fixed with 2.5% glutaraldehyde, and stained with 1% phosphotungstic acid. Bacteria were visualized between ×10,500 and ×19,000 magnification. Bar, 0.5 μm.

FIG. 4.

Independent challenges of mice with E. coli CFT073 and flagellar mutants. The wild type and mutants were individually inoculated transurethrally into the bladders of CBA/J mice. After 3 days, urine, bladder, and kidneys were harvested to determine bacterial concentrations. Each data point represents the log₁₀ CFU per ml of urine or per gram of tissue collected from one mouse. Horizontal bars represent the median values of the populations. The limit of detection of this assay is 10² CFU per ml or per g. ▪, wild type (WT); ▴, mutant.

FIG. 5.

Cochallenges of mice with E. coli CFT073 and flagellar mutants. Three to eight mice were transurethrally inoculated with a 1:1 mixture of the wild type and the mutant. At specific times postinoculation, the mice were sacrificed, and bacterial concentrations were determined. Each data point represents the median log₁₀ CFU per ml of urine or per gram of tissue collected from each group of mice. The limit of detection of this assay is 10² CFU per ml or per g. ▪, wild type; ▴, mutant. §, P < 0.05; *, P < 0.05 and the competitive index in vivo is greater than the competitive index in vitro at corresponding time points (Table 2).

FIG. 6.

Wild-type and fliC mutant coculture and competitive index graphs. (A) The wild type and mutant were mixed 1:1 and passaged every 8 and 16 h for as long as 72 h. The CFU/ml of culture for the wild type and mutant were determined after 24, 48, and 72 h of growth. Replicates of four to five cultures were examined for each test, and each test was repeated. Each point represents the log₁₀ CFU/ml from one coculture. Horizontal bars represent the mean CFU/ml of the populations. ▪, wild type (WT); ▴, mutant. *, P < 0.05 by Student's t test. (B) The competitive index was calculated for the fliC mutant in vitro coculture data (dashed line) and in vivo cochallenge data (solid lines) (▴, urine; •, bladder; ▪, kidneys) as described in the text and in the footnotes to Table 2. The log₁₀ competitive index was plotted against time (hours postinoculation). The dotted line at 10⁰ (or 1) represents a competitive index where there is no competition between the wild type and the mutant. The in vitro competitive indices were used to create a threshold for determining the significance of mutant attenuation in vivo (dashed line). This threshold line was extrapolated to 10⁰ (or 1), where the input CFU ratios are 1:1, to better visualize the cutoff of the threshold.