Stimulation of Growth and Inhbition of Quorum Sensing by Ginseng in Vibrio harveyi

1. I-027 Stimulation of Growth and Inhbition of Quorum Sensing by Ginseng in Vibrioharveyi J. Makemson, L. Schneper, N. Magicic and K. Mathee Florida International University, Miami, FL Ginseng Treated with DEAE-, Carboxy-methy-, and plain Cellulose Ginseng Stimulates Growth and Inhibits Quorum Sensing Not Affected by Boiling with or without KOH ABSTRACT Experimental Methods: Growth and Luminescence Ginseng root contains (as % dryweight) ~30-45% starch, ~13-14% protein, ~5% crude fiber, 4-5%ginsenosides, and 2.4% minerals (Li et al., 1996. J. Agric. Food Chem. 44:717-720; Woo et al. 2002. Microchem. J. 73:299-306) which accounts for as much as 71% of the dry weight. Ginseng also contains molecules which inhibit quorum sensing in Vibrioharveyi; these molecules extract readily into water or methanol, but not nonpolar solvents. Ginseng extracts possess molecules which enhance the growth of V. harveyi in Autoinducer Bioassay broth (AB broth). Boiling ginseng or treatment with KOH did not affect its inhibitory activity. Treatment of ginseng extract with DEAE-cellulose or Dowex 1-X2 removed part of the inhibitory activity and treatment with carboxymethylcelloulose or cellulose had no effect. The activity adsorbed to DEAE could be eluted with NaCl and no longer stimulated growth, rather simply inhibited quorum sensing. Growth stimulation could be replicated by addition of iron (1 micromolar) but, extraction with chloroform or 10% 8-hydroxyquinoline-chloroform had no effect on growth stimulation suggesting that iron is not responsible for growth stimulation. Thus, it appears that the inhibitory effect is likely due to the combination of a mineral and some unknown negatively charged molecule. • AB Media-Growth and In vivo Luminescence: Inoculate a starter culture, overnight at 25oC with shaking (200 rpm). • Experimental: AB medium 2.5 ml + small volume (µl of extracts or chemicals), inoculate with 25 to 100 μl of overnight culture. • Incubate 25°C with shaking (200 rpm). • Measure OD660 for Growth and Luminescence in a calibrated photometer from the same tubes. • 2. AB Media: Growth and In Vitro + In Vivo Luminescence • Experimental: Overnight culture as above. Inoculate fresh AB (50 ml) with additions or without (control). • Incubate 25oC with shaking (200 rpm) • Measure OD660 for Growth, In Vivo Luminescence (as above) + remove 1 ml, vortex with 10 μl toluene for 20 sec, keep on ice. Assay 5 to 20 μl with the FMNH2 initiated standard luciferase assay with decanal as the fatty aldehyde. Ginseng into Dowex 1-X2 column, Eluted with 0.2M NaCl Ginseng Chemistry Effect of Fe and Mn on Quorum Sensing AB Broth – Effect of Micronutrients: Minerals Chemical Group % Ginseng Dry Weight Starch 30-45% Protein 13-14% Crude Fiber 5% Ginsenosides 4-5% Minerals 2.4% μmoles/gram ginseng K 359 Ca 75 Mg 21.5 Mn 0.8 Zn 0.42 Cu 0.18 Fe 0.10 Ni 0.04 Data above calculated from: Lee, et al., 1996. J. Agric. Food Chem. 44:717-720 Woo, et al., 2002 Microchem J. 73:299-306 VibrioharveyiGROWTH MEDIA Monod Plot Manganese: Luciferase Synthesis and Expression CONCLUSIONS 1. Ginseng contains quorum sensing inhibitory molecules. Ginseng contains iron and manganese that increase growth of V. harveyi in AB broth; iron represses quorum sensing, but manganese stimulates quorum sensing. Other ginseng minerals have no effect on growth or quorum sensing. Negatively charged organic molecule(s) inhibit V. harveyi quorum sensing.