Effect of GAs on <i>C. albicans</i> infection in <i>Caenorhabditis elegans</i> and mammalian cells.

<p>(A) Rescue of <i>C. albicans</i> infected <i>C. elegans</i> from death by GAs. Larvae of <i>C. elegans</i> fed with yeast cells of <i>C. albicans</i> were incubated in RPMI medium with or without GAs (40 µg/ml) in a 96 well microtiter plate and incubated at 30°C for 2–4 days. Arrow in the top left panel (-GAs) shows the growth of <i>C. albicans</i> hyphae from the dead worms while addition of GAs (+GAs) prevent growth of hyphae from the worms’ body and hence worms survival (top right panel). Small round structures in the background are <i>C. albicans</i> yeast cells. Inset of <i>C. elegans</i> from GAs treated well shows confocal microscopic image of <i>C. elegans</i> containing <i>C. albicans</i> yeast cells in the worm’s gut (arrow). Scale bar (inset) = 10 µm. Bar graph at lower left panel indicates the % worms surviving after 4 days of exposure to GAs or to AMB. Survival of worms was determined by their movements and absence of hyphal growth from worms using microscope. Error bars indicate SD from the averages of 3 independent experiments. (B) GAs are non hemolytic and nontoxic to mammalian cells. Hemolytic assay was performed on tryptic soy agar plate containing human red blood cells (hRBC, 5%) (left side). A diagrammatic representation with sample identity is shown on the right side. Different fractions containing GAs [<i>G. sylvestre</i> extract, GE 1 mg/ml, 4 µl; fraction #194 (4 µl); and purified GAs (40 µg/ml, 4 µl)] were diluted in PBS and spotted on hRBC-agar. Positive controls including actively growing <i>Staphylococcus aureus</i> cells (2 µl) or PBS containing Triton X-100 (1%) (Tri-X) were also spotted on the blood agar medium as controls. Plates were incubated for 24–48 h at 37°C and the results were recorded by image capture. White clear halos around spots indicate hemolytic activity. GAs are not toxic to mammalian kidney epithelial cells (far right sector). Napthaquinone (NAP, 50 µg/ml) killed the kidney epithelial cells whereas solvent control (-GAs), amphotericin B (+AMB) or test compounds (+GAs; 40 µg/ml) did not. Scale bar = 10 µm.