5 mM – 92 and 97%; 1.5 mM – 45 and 55%; 2.5 mM – 32 LY3039478 ic50 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 – this website 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. To further evaluate the effect of pilicides on the inhibition of Dr fimbriae production, we quantified the amount of monomeric DraE protein resulting from the denaturation/depolimerization of isolated Dr fimbriae samples using a densitometric analysis of the SDS-PAGE gels stained with Coomassie Blue (Figure 3A-C). The strain E. coli BL21DE3/pBJN406 was grown on agar plates supplemented with
pilicides 1 and 2 at a concentration of 0.5, 1.5, 2.5 Selleck PRN1371 and 3.5 mM. Non-fimbriated E. coli BL21DE3/pACYC184 and fully-fimbriated BL21DE3/pBJN406 strains grown without pilicide were used as the negative and positive controls, respectively. The fimbriae from the bacteria scraped and normalized to OD600 see more were isolated by means of vortexing. Dr fimbriae are very stable structures which require extending heating in Laemmli buffer in order to depolimerize to a monomeric DraE protein. The band of monomeric DraE protein was visible in resolved samples heated for 60 min at 100°C
before electrophoresis. In contrast, there was no band corresponding to monomeric DraE in the samples which had not been denatured thermally before electrophoresis (Figure 3A). This confirms that the isolated fractions only contained Dr fimbriae and were not contaminated by the monomeric, periplasmic form of DraE protein. In order to prove that the heating time for the samples is sufficient for the total denaturation of Dr fimbrial structures to monomeric DraE, we performed a Western blotting analysis with anti-Dr antibodies (Figure 3B). In these experiments, the estimated pilicide effects of compounds 1 and 2 were comparable (Figure 3C). For bacteria cultivated in the presence of 3.5 mM of pilicides 1 and 2, the amount of DraE fimbrial protein was reduced by 75 and 80% in comparison to the fully-fimbriated strain grown without pilicide, respectively. Performing experiments with 0.5, 1.5 and 2.5 mM concentration of pilicides, we analyzed their dose dependent effects on the volume of fimbrial production. At a concentration of 2.