We were further interested in learning if any of the limonoids modulate expression of stx2. Isolimonic acid and ichangin (100 μg/ml) repressed the stx2 by 4.9 and 2.5 fold, respectively (Table 4), while IOAG, isoobacunoic acid and DNAG did not seem to affect the expression of stx2. The culture of EHEC in DMEM was reported to activate LEE expression
[41]. To determine, if isolimonic acid represses LEE under DMEM growth conditions, expression of ler, stx2, escJ and sepZ were measured. Isolimonic acid treatment repressed ler, stx2, escJ and sepZ in DMEM media by >5, 7, 8 and 10 fold whereas, expression of rpoA was unaffected (Figure 4). The escJ and sepZ, which are coded as a polycistronic message, demonstrated differing levels of regulation in presence of isolimonic acid NSC 683864 purchase (Figure 4). However, differential degradation and processing of genes encoded as polycistronic mRNA is well documented [49, 50], and could potentially be the reason of different levels of mRNA transcripts recorded for escJ and sepZ. Figure 4 Expression of LEE encoded genes in DMEM in response to isolimonic acid. Fold change in expression were calculated as isolimonic acid over DMSO. The data represents mean of three biological replicates and SD. The samples were collected at OD600 of 0.5, 1.0 and 2.0 and processed as described in Materials and Methods.
Effect of isolimonic acid on AI-3/epinephrine induced LEE expression AI-3/epinephrine mediated cell-cell signaling regulates biofilm, motility and expression of LEE in EHEC [6, 12, 15]. To ascertain if isolimonic acid interferes with AI-3 signaling, reporter strains TEVS232 and TEVS21 were induced by PM in GSK458 supplier presence of 100 μg/ml isolimonic acid, and β-galactosidase activity was measured. TEVS232 and TEVS21 contain Pazopanib molecular weight single copy operon fusions of LEE1:LacZ and LEE2:LacZ, respectively [41]. Isolimonic acid treatment reduced the expression of LEE1 (TEVS232) and LEE2 (TEVS21) by 46.05 and 34.23%, respectively (Figure 5A and B). Additionally, LEE1 was stimulated by 50 μM epinephrine in presence or absence of 100 μg/ml isolimonic acid and β-galactosidase activity was measured. Isolimonic acid repressed the epinephrine-induced expression
of LEE1 by ≈3.9 fold (74.42 % reduction) (Figure 5C). Figure 5 Effect of isolimonic acid on AI-3/epinephrine mediated signaling. Inhibition of preconditioned media induced β-galactosidase activity in (A) TEVS232 (LEE1) and (B) TEVS21 (LEE2) by 100 μg/ml isolimonic acid or DMSO (control). Preconditioned media was prepared as described in text. (C) Epinephrine induced β-galactosidase activity in TEVS232 in presence of 100 μg/ml isolimonic acid or solvent control (DMSO). The EHEC was grown to OD600 ≈ 0.2, collected by centrifugation and resuspended in preconditioned medium or media supplemented with 50 μM epinephrine. Isolimonic acid or DMSO were added and β-galactosidase activity was measured after 30 min incubation. Asterisk denotes significant (p<0.05) difference from solvent control (DMSO).