Article Figures & Data
Figures
- FIG. 1.
(A) Immunoblot of bacterial whole-cell lysates derived from EHEC O113:H21 strain EH41 grown at 25°C or 37°C and detected with anti-H21 FliC antibodies. The asterisk indicates a nonspecific E. coli band that serves as a protein loading control. (B) Adherence (columns a and c) and invasion (columns b and d) of EHEC O113:H21 grown overnight at 25 or 37°C to HCT-8 cells. The results are expressed as the percentage of cell-associated bacteria that resisted killing by gentamicin (invasion) or the percentage of cell-associated bacteria from the original inoculum (adherence) and are the means ± the standard deviation of at least three independent experiments in duplicate wells. The asterisk indicates significantly more than same strain grown overnight at 37°C (P = 0.0072, unpaired, two-tailed t test).
- FIG. 2.
Invasion and adherence of EHEC O113:H21 to HCT-8 cells after preincubation with anti-FliC antibodies. (A) Invasion assay performed with no preincubation (control) (a), preincubation with anti-H21 diluted 1:500 (b), preincubation with anti-H21 diluted 1:1,000 (c), and preincubation with anti-H6 diluted 1:500 (d). The results are expressed as the percentage of cell-associated bacteria that resisted killing by gentamicin (invasion) and are the means ± the standard deviations of at least three independent experiments in duplicate wells. The asterisk indicates significantly lower than control (P < 0.02, unpaired two-tailed t test). (B) Adherence assay performed with no preincubation (control) (a), preincubation with anti-H21 diluted 1:500 (b), preincubation with anti-H21 diluted 1:1,000 (c), and preincubation with anti-H6 diluted 1:500 (d). The results are expressed as the percentage of cell-associated bacteria from the original inoculum (adherence) and are the means ± the standard deviations of at least three independent experiments in duplicate wells.
- FIG. 3.
(A) Immunofluorescence microscopy of wild-type EH41 incubated with anti-H21 antibodies (i), EH41fliC::cat incubated with anti-H21 antibodies (ii), EH41fliC::cat (pTrc:fliCH21) incubated with anti-H21 antibodies (iii), and EH41fliC::cat (pTrc:fliCH6) incubated with anti-H6 antibodies (iv). All fields were also incubated with anti-rabbit fluorescein isothiocyanate-conjugated secondary antibodies, and bacterial nucleic acid was stained with DAPI (4′,6′-diamidino-2-phenylindole). (B) Motility of wild-type EH41, EH41fliC::cat, EH41fliC::cat (pTrc:fliCH21), and EH41fliC::cat (pTrc:fliCH6) in 0.2% semisolid LB agar. Bacteria were incubated overnight at 25°C, after which colony diameter was measured. The results are expressed as the mean of triplicate colonies from three independent experiments.
- FIG. 4.
CLUSTAL W alignment of FliC proteins from E. coli O127:H6 (Scaffold57_cons_ORF_36 from EPEC E2348/69 genome sequence at www.sanger.ac.uk/Projects/Escherichia_Shigella ), E. coli O142:H6 (AAD28526), E. coli O55:H6 (AAD28519), E. coli O113:H21 (DQ862122), E. coli O8:H21 (AAP13312), and E. coli O91:H21 (ABE69176). Shading indicates shared amino acid sequence identity and similarity.
Tables
- TABLE 1.
Relative invasion of and adherence to HCT-8 cells of derivatives of EHEC O113:H21 strains EH41 and 98NK2
Strain % (mean ± SD) of wild-type: Invasiona Adherenceb Derivatives of EH41 EH41 100 ± 82.6 100 ± 60.7 EH41fliC::cat 12.63 ± 5.1* 121.7 ± 29.2 EH41fliC::cat (pTrc:fliCH21) 43.38 ± 7.8 208.5 ± 122.2 EH41fliC::cat (pTrc:fliCH6) 15.42 ± 3.4* 120.6 ± 26.5 Derivatives of 98NK2 98NK2 100 ± 43.4 100 ± 30.9 98NK2ΔfliC 17.4 ± 7.0† 47.9 ± 10.7 ↵ a Results are expressed as a percentage of wild-type EH41 invasion (mean ± standard deviation of at least three independent experiments in duplicate wells). *, Significantly less than EH41fliC::cat (pTrc:fliCH21) (P < 0.005; unpaired, two-tailed t test); †, significantly less than 98NK2 (P < 0.05; unpaired, two-tailed t test).
↵ b Results are expressed as a percentage of wild-type EH41 adherence (mean ± standard deviation of at least three independent experiments in duplicate wells).
