Uropathogenic Escherichia coli flagella aid in efficient urinary tract colonization

Abstract

In the murine model of urinary tract infections (UTI), cystitis by uropathogenic Escherichia coli (UPEC) occurs through an intimate relationship with the bladder superficial umbrella cell entailing cycles of adherence, invasion, intracellular bacterial community (IBC) formation, and dispersal (fluxing) from the intracellular environment. IBC dispersal is a key step that results in the spread of bacteria over the epithelial surface to initiate additional rounds of IBC formation. We investigated the role of flagella in mediating adherence and motility during UTI, hypothesizing that the dispersion of the IBC would be incomplete in the absence of motility, thus interrupting the IBC pathway and attenuating the infection. Using gfp reporter fusions, the expression of the flagellar class I flhDC and class III fliC genes was monitored to track key points of regulation throughout the pathogenic cascade. In vitro, growth under conditions promoting motility resulted in the robust expression of both fusions. In contrast, only the class I fusion produced significant expression throughout early stages of IBC development including the dispersion stage. Thus, unlike in vitro modeling of motility, the regulatory cascade appeared incomplete in vivo. Throughout IBC formation, nonmotile DeltafliC mutants achieved the same number of IBCs as the wild-type (wt) strain, demonstrating that flagella are neither essential nor required for first- or second-generation IBC formation. However, in competition experiments between wt and DeltafliC strains, the wt was shown to have a fitness advantage in persisting throughout the urinary tract for 2 weeks, demonstrating a subtle but measurable role for flagella in virulence.

FIG. 1.

In vivo flhDC (class I flagellar gene) transcriptional activity. The bladders of mice infected with 10⁷ bacteria of either the UTI89 class I GFP strain (UTI89 attλ::P_flhDC) (A, B, D, and E) or the UTI89 promoterless GFP reporter strain (UTI89 attλ::PSSH10-1) (C and F) were collected 6 (A to C) or 16 (D to F) hours postinfection, splayed by pinning, fixed with PFA, stained with TO-PRO-3 iodide (nuclear stain; red), and imaged by confocal microscopy at ×63 magnification. Yellow/green staining indicates active transcription. Red staining indicates no transcription. Bar, 10 μm. White arrows indicate autofluorescent vacuoles normally present in superficial facet cells. The UTI89 class I GFP reporter strain resulted in both transcriptionally active and inactive IBCs at 6 h and only active IBCs at 16 h, whereas the control, UTI89 promoterless GFP strain exhibited background GFP levels at both time points.

FIG. 2.

In vivo fliC (class III flagellar gene) transcriptional activity. The bladders of mice infected with 10⁷ UTI89 class III GFP bacteria (UTI89 attλ::P_fliC-FliC_1-15aa-GFP; translational fusion) were collected 6 (A to C), 16 (D to F), or 30 (G to I) hours postinfection, splayed by pinning, fixed with PFA, stained with TO-PRO-3 iodide (nuclear stain; red), and imaged by confocal microscopy at ×63 magnification. Yellow/green staining indicates active transcription. Red staining indicates no transcription. Bar, 10 μm. White arrow indicates autofluorescent vacuoles normally present in superficial facet cells. Open arrowhead indicates GFP-positive bacteria. The UTI89 class III GFP reporter strain resulted primarily in transcriptionally inactive IBCs; however, transcriptionally active bacteria were detected in IBCs from bladders harvested 30 h postinfection.

FIG. 3.

In vitro characterization of the nonmotile UPEC strain UTI89 ΔfliC. (A) Motility analysis of UTI89 ΔfliC, UTI89 ΔfliC/pfliC, and UTI89 ΔfliC/pVC. Strains were grown statically in LB broth and analyzed relative to the similarly grown wild-type UPEC strain UTI89 and UTI89/pVC for motility in 0.25% LB agar alone or supplemented with chloramphenicol (pfliC- and pVC-containing strains). Wells on the right contain 5 mM l-(+)-arabinose, in contrast to the center wells, in which strains are grown without arabinose. (B) Quantified motility. Motility was measured as described in Materials and Methods. (C) Flagellin (H7) immunoblot analysis. Lane 1, UTI89; lane 2, UTI89 ΔfliC; lane 3, UTI89 ΔfliC/pfliC without arabinose; lane 4, UTI89 ΔfliC/pfliC with arabinose; lane 5, UTI89 ΔfliC/pVC without arabinose; lane 6, UTI89 ΔfliC/pVC with arabinose. MW, molecular weight (in thousands). (D and E) Negatively stained electron micrographs of wild-type UTI89 (D) and UTI89 ΔfliC (E). Arrows indicate pili, and the arrowhead indicates the flagellum. (F) Guinea pig erythrocyte hemagglutination (HA). (G) Representative FimCH immunoblot (n = 6). Left lane, UTI89; right lane, UTI89 ΔfliC.

FIG. 4.

IBC formation by the wild-type UPEC strain UTI89 and the nonmotile UPEC strain UTI89 ΔfliC as a function of time. The bladders of mice infected with either wild-type UTI89 (A to C) or the nonmotile UPEC strain (D to F), UTI89 ΔfliC, were collected 6 (A and D), 16 (B and E), and 30 (C and F) hours postinfection, splayed by pinning, stained with Alexa Fluor 633-conjugated WGA, fixed with PFA, and imaged by confocal microscopy at ×63 magnification. Bar, 10 μm. Green staining indicates bacteria. Yellow staining indicates autofluorescent vacuoles. Red staining indicates WGA localization. IBCs arising from the nonmotile UPEC strain UTI89 ΔfliC were indistinguishable from those arising from the wild-type strain UTI89 at all acute cystitis time points examined.

FIG. 5.

Competitive infection of a prototypic cystitis strain, UTI89, and a nonmotile UPEC strain, UTI89 ΔfliC, in the bladders and kidneys of mice. (A) Mice were transurethrally inoculated with either UTI89 or UTI89 ΔfliC, and CFU in the bladders (•) and kidneys (○) was enumerated 2 weeks postinfection. (B) Mice were inoculated with a mixed inoculum (1:1 ratio) of both UTI89 and UTI89 ΔfliC, and CFU were enumerated in bladders and kidneys by replica plating onto LB or LB-kanamycin (50 μg/ml) plates 2 weeks postinfection. The CI of bacteria recovered from the organs were calculated as described in Materials and Methods. Horizontal bars indicate the geometric mean titer or CI of the sample group indicated (n = 5 mice per strain). During the competitive infection, recovery of UTI89 ΔfliC was significantly lower in the bladder (P = 0.008 by the Mann-Whitney U test), indicating a competitive disadvantage during persistent cystitis, while a trend toward significance in the kidney was seen (P = 0.1 by the Mann-Whitney U test).