Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI). This bacterium adheres to and internalizes within urinary tract cells, where it aggregates and subsequently forms biofilm-like multicellular colonies that protect UPEC from antimicrobial agents and the host's immune system. Here, we show that OmpX, an outer membrane protein, plays a role in the pathogenesis of UPEC in renal cells. Deletion of ompX decreased bacterial internalization and aggregation within kidney epithelial cells and also impaired the colonization of mouse urinary tracts, but the ompX mutant still adhered to the epithelial cells at a level similar to that of the parent strain. FlhD, the master regulator of flagellum-related genes, had a low expression level in the ompX mutant compared to the parent strain, and the ompX mutant exhibited defective motility due to lower flagellar production than the parent strain. The fliC mutant, which lacks flagella, exhibited lower levels of bacterial internalization and aggregation than the parent strain. Additional deletion of ompX in the fliC mutant did not further decrease bacterial internalization. These combined results suggest that OmpX contributes to flagellar production in UPEC and then sustains UPEC virulence associated with bacterial internalization and aggregation within urinary tract cells and colonization in the urinary tract.

Keywords: biofilm; flagella; gene regulation; motility; pathogenesis; pyelonephritis; urinary tract infection; virulence.

FIG 1

Colonization by the parent strain (CFT073) and ompX mutant in the bladders and kidneys of mice with UTIs. The female mice (n = 3 for each group) were infected with the parent strain or ompX mutant. At 48 h postinfection, cell numbers of bacteria isolated from the bladder and kidneys were determined as CFU. We repeated experiments independently three times, but one mouse infected with the parent strain died within 48 h of infection. Each data point represents a sample from an individual mouse (n = 8 for the parent strain and n = 9 for the ompX mutant). Horizontal bars show median values. *, P < 0.05 relative to the value for the parent strain.

FIG 2

Adhesion to and internalization in kidney epithelial cells (HTB-44) of the parent strain (CFT073) and the ompX mutant (A and B) or the parent strain and the ompX mutant carrying pTrc99K (empty vector) or pTrc99KompX (ompX expression plasmid) (C). Values are percent CFU of adhered/internalized (A) and internalized (B and C) bacteria relative to total bacterial cell numbers. Data are means from three independent experiments; error bars indicate standard deviations. *, P < 0.05 relative to values for CFT073 (A and B) or CFT073/pTrc99K (C).

FIG 3

Aggregation within kidney epithelial cells (HTB-44) for the parent strain and the ompX mutant or the parent strain and the ompX mutant carrying pTH18kr (empty vector) or pTH18krompX (ompX expression plasmid). Bacteria carrying a green fluorescence protein (GFP) expression plasmid, pTurboGFP-B, and HTB-44 cells stained with rhodamine-phalloidin were imaged with green and red fluorescence, respectively, using a 100× objective. Images were taken from above (A), and cross-sectional images (B) correspond to the white boxes in panel A. The experiment was repeated twice, and similar results were obtained. (C) Aggregated bacteria within HTB-44 cells were quantified by representing levels of colonized bacteria as areas (in square pixels) of GFP. Microscopy data are means from three fields of view, and error bars indicate standard deviations. **, P < 0.01 relative to the value for the parent strain CFT073.

FIG 4

Motilities and flagellar production for the parent strain (CFT073) and the ompX mutant or the parent strain and the ompX mutant carrying pTrc99K (empty vector) or pTrc99KompX (ompX expression plasmid). (A) Bacterial migration on LB medium containing 0.3% agar. (B) Diameters reflecting bacterial migration on the agar. Data are means from three independent experiments; error bars indicate standard deviations. (C) Flagella and bacterial cells were stained with Victoria blue/tannic acid were pictured using a 100× objective. (D) Ratios of bacteria observed with flagella to ∼120 to 150 randomly selected bacteria on microscopy, presented as percentages. Data are means, and error bars indicate standard deviations. **, P < 0.01 relative to the value for CFT073.

FIG 5

FliC expression in the ompX mutant and contribution of fliC to bacterial adhesion to and internalization within the kidney epithelial cells. (A) Western blots of cell lysates and secreted proteins from the parent strain (CFT073) and the ompX mutant containing a VSVG-tagged FliC expression plasmid (pTH18krfliC-VSVG) and pTrc99A (empty vector) or pTrc99AompX (ompX expression plasmid). Locations of molecular mass standards (in kilodaltons) are shown on the left. VSVG-tagged FliC was visualized by probing with a VSVG antibody. Adhesion to and internalization in kidney epithelial cells (HTB-44) of the parent strain, fliC mutant, and fliC/ompX double mutant (B and C) or the parent strain and the fliC mutant carrying pTrc99A (empty vector) or pTrc99AfliC (fliC expression plasmid) (D). Values are percent CFU of adhered/internalized (B) and internalized (C and D) bacteria relative to total bacterial cell numbers. Data are means from three independent experiments; error bars indicate standard deviations. *, P < 0.05 relative to CFT073 (B and C) or CFT073/pTrc99A (D).

FIG 6

Aggregation within kidney epithelial cells (HTB-44) for the parent strain and the fliC mutant or the parent strain and the fliC mutant carrying pTH18kr (empty vector) or pTH18krfliC-VSVG (fliC expression plasmid). Bacteria carrying a GFP expression plasmid, pTurboGFP-B, and HTB-44 cells stained with rhodamine-phalloidin were imaged with green and red fluorescence, respectively, using a 100× objective. Images were taken from above (A), and cross-sectional images (B) correspond to the white boxes in panel A. The experiment was repeated twice, and similar results were obtained. (C) Aggregated bacteria within HTB-44 cells were quantified by determining levels of colonized bacteria as areas (in square pixels) of GFP. Microscopy data are means from three fields of view, and error bars indicate standard deviations. *, P < 0.05, and **, P < 0.01, relative to CFT073.

FIG 7

Transcript levels and promoter activities of flagellum-related and fimbrial genes in the parent strain (CFT073) and the ompX mutant. (A) Transcript levels were determined relative to that of rpoD. Data are means for two biological replicates; error bars indicate the ranges. (B) β-Galactosidase activities corresponding to flhD, fliA, and fliC promoter activities in the parent strain and the ompX mutant containing pNNflhD-P, pNNfliA-P, or pNNfliC-P, the lacZ reporter plasmid. Data are means from three independent experiments; error bars indicate standard deviations. **, P < 0.01 relative to CFT073.