As isolimonic acid seems to interfere with AI-3/epinephrine induced pathway, it was possible that this interference is dependent on QseBC. To determine if isolimonic acid inhibits EHEC biofilm formation by affecting QseBC, biofilm formation in EHEC 86–24,

QseC deletion mutant (VS138) and complemented strain VS179 [6] was studied. Since ΔqseBC strain (VS138) did not form appreciable biofilm at 24 h, the biofilms were grown up to 48 h. The biofilm formation in ΔqseBC at 48 h was similar between solvent control (DMSO) and isolimonic acid (p>0.05) (Figure 6A). In contrast, isolimonic acid reduced Epacadostat in vivo the biofilm formation by 61.33% in complemented strain VS179. To further understand the role of QseBC in wild type strain ATCC 43895, plasmid pVS178 (carrying qseBC), was purified from VS179 and introduced into wild type strain. In addition, qseB and qseC were amplified from EHEC genomic DNA, cloned into pBAD33 vector and introduced into EHEC strain ATCC 43895. The expression

of qseBC/qseB/qseC was induced by addition of 0.2% arabinose in the media. Overexpression of qseBC/qseC/qseB formed significantly more biofilm, when compared to EHEC wild type carrying vector alone (Figure 6B). We further measured the effect of isolimonic acid on the biofilm formation in strains overexpressing qseBC/qseC/qseB (Figure 6C). The isolimonic acid treatment did not significantly Dipeptidyl peptidase affect the biofilm formation, measured after 24 h of growth, in EHEC strains overexpressing qseBC/qseC/qseB (Figure 6C). Furthermore, it was possible Selleck MDV3100 that isolimonic acid modulates the expression of qseBC leading to inhibition of biofilm. To determine the effect of isolimonic acid, expression of qseB and qseC was measured by

qRT-PCR. The results indicate that isolimonic acid do not regulate the expression of qseB and qseC (Figure 6C). Altogether, finding of these experiments seem to suggest that isolimonic acid affects the QseBC activity but not the expression to inhibit biofilm formation. Figure 6 Activity of isolimonic acid is dependent on QseBC . Inhibition of biofilm in (A) ΔqseBC mutant and ΔqseBC mutant complemented with qseBC (pVS178). (B) Biofilm formation in EHEC supplemented with qseBC, qseB and qseC. Asterisk denotes significant (p<0.05) difference from vector control. (C) Inhibition of biofilm by 100 μg/ml isolimonic acid in EHEC supplemented with qseBC, qseB and qseC. Asterisk denotes significant (p<0.05) difference from solvent control (DMSO). (D) Expression of qseB and qseC in presence of 100 μg/ml isolimonic acid. The fold changes in expression were calculated as isolimonic acid over DMSO. The experiments were conducted in triplicate and mean ± SD are presented.