05) after the exposure of bacteria on different concentration of pilicides. Only the result for the lowest concentration of pilicide 2 was statistically not significant relatively to the positive control (p = 0.068). The increasing concentration of pilicides also had the influence on adhesion level (p < 0.05). For further
evaluation of the activity of compounds 1 and 2 as inhibitors of Dr fimbriae biogenesis, we used a haemagglutination test (HA) conducted in a manner similar to the case of published data describing the activity of mTOR inhibitor pilicides 1 and 2 as P and type 1 pili biogenesis inhibitors . The assay is based on an analysis of human erythrocyte agglutination mediated by the bacterial cells. The reaction is dependent on the specific interaction between Dr fimbriae and DAF receptor located on the erythrocyte surfaces. The interaction between DAF receptor
and Dr fimbriae is inhibited by the addition of chloramphenicol at a concentration of 2 μM . The specificity of the haemagglutination observed was confirmed by an analysis of its reversibility as a consequence of the addition of chloramphenicol. The observed FHPI manufacturer haemagglutinating ability of the bacteria reflects the amount of Dr fimbriae produced in the presence of the pilicide. The HA-titer, the highest bacterial dilution, in duplicates, which still provides erythrocyte agglutination see more is determined in the experiment (Figure 2). A low HA-titer indicates that a higher concentration of bacteria, with low amount of fimbriae, is required for agglutination to occur. In our assay, the bacteria of E. coli BL21DE3/pBJN406 were grown analogically to the CHO cells adherence experiments, on agar plates containing 3.5 mM pilicide. The fully-fimbriated bacteria grown in the absence of pilicide (positive control) resulted in an HA-titer of 128. The non-fimbriated bacteria E. coli BL21DE3/pACYC184 (negative control) gave an HA-titer of 1. Chorioepithelioma The bacteria cultivated in the presence of pilicides 1 and 2 in media had a reduced HA-titer of 16/32 (Figure 2). The HA-titers were determined as an average from duplicate runs in three independent experiments These results clearly
show that bacteria grown in the presence of these pilicides possess a reduced amount of Dr fimbriae as an effect of blocking the chaperone-usher pathway. Figure 2 Blocking of Dr fimbriae-dependent agglutination of human erythrocytes by pilicides. The following bacterial preparations, normalized to OD600, were used in the hemagglutination assays: negative control – E. coli BL21DE3/pACYC184, grown on TSA plates with 5% DMSO, non-fimbriated strain; positive control – E. coli BL21DE3/pBJN406, grown on TSA plates without pilicide, fully-fimbriated strain; chloramphenicol –E. coli BL21DE3/pBJN406, grown on TSA plates without pilicide, the agglutination experiment was performed in the presence of 2 μM of chloramphenicol; pilicide 1 and pilicide 2 – the E. coli BL21DE3/pBJN406 grown on the TSA plates in the presence of 3.