PMC

Photomicrographs were taken 6 hr later from either untreated control (HK-2 cells) (A), or treated with supernatants from CFT073 WT (B), csrA (C), barA (D), uvrY (E), hlyD (F), barA/p-barA (G), uvrY/p-uvrY (H) and hlyD/p-hlyD (I). The rounding and detachment of cells in the treated monolayer are marked with arrow or arrowhead. The pictures were representative of three individual experiments.

A set of twelve 12-day-old SPF embryonated eggs were inoculated through allantoic cavity with 5×10³ CFU of bacteria CFT073 WT (▪), DH5αΚ12 (□), SM3009 (barA::cm) (▴), SM3012 (barA/p-barA) carrying pSM1 (Δ), SM3010 (uvrY::cm) (•), SM3013 (uvrY/p-uvrY) carrying pSM2 (○), SM3011 (csrA::cm) (♦) and SM3014 (csrA/p-csrA) carrying pSM7 (X). The results were scored as live, morbid or dead. The results were representative of two independent experiments.

CBA/J mice were infected transurethrally with mixture of 5×10⁷ CFU of WT bacteria and 5×10⁷ CFU of mutant bacteria. A. Bacteria were recovered 72 hr later from mice infected with CFT073 WT urine (○), SM3009 (barA::cm) urine (•), CFT073 WT bladder (Δ), SM3009 (barA::cm) bladder (▴), CFT073 WT kidneys (□), SM3009 (barA::cm) kidneys (▪) and were expressed in Log₁₀ CFU. B. Bacteria were recovered 72 hr later from mice infected with CFT073 WT urine (○), SM3010 (uvrY::cm) urine (•), CFT073 WT bladder (Δ), SM3010 (uvrY::cm) bladder (▴), CFT073 WT kidneys (□), SM3010 (uvrY::cm) kidneys (▪) and were expressed in Log₁₀ CFU. The results were representative of two independent experiments. Star denotes P<0.01.

LPS was extracted from equal number of cells and separated on 12% SDS-PAGE gel and visualized with silver stain. The upper bands represent the O antigen and the lower bands represent the core LPS antigen. The results were representative of three individual experiments. Lanes: 1. CFT073 wt, 2. barA, 3. barA/p-barA, 4. uvrY, 5. uvrY/p-uvrY, 6. csrA, 7. csrA/p-csrA, 8. protein marker.

Secreted supernatants were filter purified and separated on 4–20% SDS-PAGE gel and stained with Coomassie blue. Bands of the interest were incised and subjected to mass spectrometry analysis. Star indicates the location of the hemolysin protein band. The results were representative of three individual experiments. Lanes: 1. protein marker, 2. LB media, 3. CFT073 wt, 4. barA, 5. barA/p-barA, 6. uvrY, 7. uvrY/p-uvrY, 8. csrA, 9. csrA/p-csrA, 10. protein marker.