title: Dithiothreitol enhances Listeria monocytogenes mediated cell cytotoxicity.
abstract: The hybridoma Ped-2E9 based cytotoxicity assay was developed to distinguish virulent from avirulent Listeria species in 6 hr. The cytotoxicity effect on Ped-2E9 was reported to be primarily due the cytolytic action of listeriolysin O (LLO), produced by L. monocytogenes. In this study, the effect of a reducing agent, dithiothreitol (DTT, 0-2 mM) that is known to activate LLO was investigated to make the Ped-2E9 based cytotoxicity assay an even more sensitive and rapid. Also, we examined the effect of fetal bovine serum (FBS, 0-50%), a common ingredient of tissue culture media on cytotoxicity. A DTT concentration of 0.5 mM gave an optimum cytotoxicity effect, which could be measured by both alkaline phosphatase (AP) and lactate dehydrogenase (LDH) assays in just 1.5-2 hr. FBS, at levels between 10 to 50%, significantly inhibited Listeria-mediated cytotoxicity. Concentrated culture filtrates from L. monocytogenes or LLO producing recombinant L. innocua (prfA+ hlyA+) strain also caused cytotoxicity effects, which were observed by scanning electron microscopy or a cytotoxicity assay in 2-3 hr. Interestingly, addition of DTT to culture filtrates produced 100% cell cytotoxicity in just 15 min. This indicated that LLO activity, which is responsible for Ped-2E9 cytotoxicity, was augmented several folds with the addition of a reducing agent. Examination of Listeria isolates belonging to different serogroups from clinical sources or naturally contaminated meat products with DTT gave cytotoxicity results in 2 hr, which were comparable to the 5-hr assay analyzed concurrently without DTT. These results indicated that DTT, which activated the LLO, could be used in the cytotoxicity assay to enhance Listeria-mediated Ped-2E9 cell cytotoxicity. This knowledge will greatly assist us to develop a user-friendly rapid assay to screen cytopathogenic properties of Listeria species.
authors.affiliation: Department of Life Sciences, Alabama A&M University, Normal 35762, USA.
authors.forename: D G
authors.initials: DG
authors.lastname: Westbrook
authors.forename: A K
authors.initials: AK
authors.lastname: Bhunia
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D003470:Culture Media; D004229:Dithiothreitol; D006801:Humans; D008089:Listeria monocytogenes / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D008460:Meat; D051379:Mice; D008855:Microscopy, Electron, Scanning; D012710:Serum Albumin, Bovine; D014407:Tumor Cells, Cultured
chemical_list: D003470:Culture Media; D012710:Serum Albumin, Bovine; D004229:Dithiothreitol