Fig 7.

Supplementations that decrease the proportion of anteiso-BCFA reduce the production of LLO and the level of hly transcript. The abundance and activity of LLO production was determined by immunoblotting (A) and hemolytic assay (B), respectively, with overnight cultures of WT L. monocytogenes grown in BHI without supplementation (“−”), with 250 mM sodium butyrate (“B” or “butyrate”), or with 100 mM leucine (“L” or “leucine”). Supernatant fractions from all samples were normalized with BHI based on the culture OD. For immunoblotting, proteins in the normalized supernatant were precipitated with 10% trichloroacetic acid (TCA) and analyzed by SDS-PAGE. “rLLO” denotes recombinant LLO as a positive control. For hemolytic assay, LLO activity in the normalized supernatant was serially diluted and tested against sheep red blood cells. A high absorbance reading indicates greater LLO activity. Averages of triplicates from one experiment were plotted, with error bars denoting standard deviation. The data shown here are representative of at least three independent experiments. (C) For transcriptional analysis of hly, the LLO-encoding gene, WT bacteria from overnight BHI culture were collected, washed, and added to fresh BHI medium supplemented with 250 mM butyrate or 100 mM leucine. The OD was monitored at the indicated sampling time points. Averages of three independent experiments were plotted, with error bars denoting standard deviation. (D) Bacterial samples were harvested at indicated time points postinoculation for RNA extraction, cDNA synthesis, and qPCR analysis. The transcript levels of virulence genes, hly and inlA, were normalized with the transcript level of gyrA, encoding a DNA gyrase subunit A. The data shown here are averages of three independent experiments with error bars denoting the standard deviation. Significant differences are indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.005). The effect of 2MB and isoleucine on LLO production was tested by immunoblotting (E) and hemolytic assay (F) with bacteria grown aerobically overnight in HEPES-buffered BHI (pH 7.0). “−” denotes no supplement, “B” denotes supplementation with butyrate (250 mM) only, “B2” denotes supplementation with butyrate (250 mM) and 2MB (25 mM), and “2” denotes supplementation with 2MB (25 mM) only. Hemolytic units were calculated for leucine- and isoleucine-supplemented samples, denoted by “Leu” and “Ile,” respectively, and are shown below the immunoblot. The data shown here are representative of at least three independent experiments. The hemolysis results shown in panel F are representative of at least three independent experiments. (G) FA analysis was performed by Microbial ID with bacteria harvested and washed from overnight aerobically grown cultures.