Figure 3

Relative determination of Dr fimbriae amount on bacteria treated with pilicides. (A) SDS-PAGE analysis of the fimbrial fractions isolated from the following bacterial cultures: lanes 1,5 - BL21DE3/pBJN406, grown on TSA plates without the pilicide, fully-fimbriated strain; 2,6 - BL21DE3/pACYC184, non-fimbriated strain; 3,7 and 4,8 - BL21DE3/pBJN406, grown in the presence of 3.5 mM of agents 1 and 2, respectively. Before electrophoresis, the samples from 1 to 4 and from 5 to 8 were incubated for 60 min at 100°C and 25°C, respectively. M – the SDS-PAGE LMW Calibration Kit weight standard. Arrow denoted monomeric DraE protein. (B) Western blotting analysis of the fimbrial fractions, performed to confirm the complete depolymerization of Dr fimbriae during sample denaturation. 1,2,3 – the same samples as in lanes 2,1 and 5 in panel B, respectively. (C) SDS-PAGE analysis of fimbrial fractions isolated from E. coli BL21DE3/pBJN406 grown on TSA plates supplemented with different concentrations of pilicide 1 (Pil1) and pilicide 2 (Pil2). Densitometrically evaluated DraE amounts are expressed as the percentage of mean value present relative to bacterial strain cultivated without pilicide, arbitrary set to 100%: 0.5 mM – 92 and 97%; 1.5 mM – 45 and 55%; 2.5 mM – 32 and 25%; 3.5 mM – 25 and 20% for strains grown in the presence of pilicide 1 and 2, respectively. (D) Evaluation of bacteria fimbriation using an ELISA assay with microtitre plates coated with type IV human collagen. The Dr fimbriae exposed on the bacteria adhered to collagen were visualized using anti-Dr antibodies. The following bacterial preparations were used in the assay: 1 - BL21DE3/pACYC184, non-fimbriated strain; 2-5 - BL21DE3/pBJN406, grown in LB medium supplemented with 3.5, 2.5, 1.5 and 0.5 mM of agent 1, respectively; 6 - BL21DE3/pBJN406, grown in LB medium without the pilicide, fully-fimbriated strain. The bars represent the s.d. of the mean of three independent experiments in duplicate.