Adherence of enterohemorrhagic Escherichia coli strains to a human colonic epithelial cell line (T84).

Enterohemorrhagic Escherichia coli (EHEC) produce Shiga-like toxins and attach to certain tissue culture cells. T84 cells are human colonic carcinoma cells. Unlike previously studied cell lines, T84 cells grown on collagen-coated surfaces polarize and produce tight junctions and desmosomes, forming a colonic epithelial cell layer in vitro. The purpose of this study was to examine the attachment of EHEC strains to the T84 cell line as a possibly more relevant in vitro model of EHEC adherence. Twelve EHEC strains were grown overnight in Penassay broth, suspended in minimal essential medium with and without 0.5% mannose, and incubated for 1 to 3 h with 5- to 7-day-old T84 cell monolayers grown on collagen-coated coverslips. The bacteria were removed, and attachment was quantitated microscopically. For both E. coli O157:H7 and other EHEC serotypes, there were marked differences in adherence between strains (range of 152 to 3 bacteria per oil immersion field). Mannose partially inhibited the adherence of some EHEC strains. Adherence to the T84 cells appeared to be related to the amount of pili present and not to the serotype. Electron micrographs showed that a highly adherent strain (strain 43-12) tended to form microcolonies in the area of tight junctions on the T84 cell monolayers. In addition, the attachment of these EHEC strains to T84 cells correlated with their ability to adhere to isolated rabbit colonocytes (r = 0.91, P = 0.00004; without mannose) (r = 0.60, P = 0.04; with mannose). These data show that there are EHEC strain-related differences in adherence which can be demonstrated in a human-derived colonic epithelial cell line (T84) and that these cells can be used to study EHEC adherence.

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells.

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.

Studies of synthetic peptide analogs of the amphipathic helix. Effect of charged amino acid residue topography on lipid affinity.

The amphipathic helix hypothesis for plasma lipoproteins was investigated using synthetic peptides. The lipid-associating properties of two potentially amphipathic model peptides and two analogs were studied by incubating synthetic peptides with small unilamellar vesicles and protein-lipid association examined by equilibrium density centrifugation, leakage of liposome-entrapped fluorescence compounds, intrinsic tryptophan fluorescence, and circular dichroism spectroscopy. The analog peptides were designed to determine the significance of the number and specific location of the charged residues in amphipathic domains of plasma lipoproteins to protein-lipid association. Based on the four procedures used to examine protein-lipid interactions, the two model peptides (18Aa, 18As) were found to associate strongly with liposomes; the two analog peptides (18As1, 18Asr), differing only with respect to the number and/or position of their charged residues, failed to demonstrate similar lipid binding properties. These findings support the earlier suggestions of the importance of the charged residues, but do not define the precise mechanisms involved. Such amino acids may help initiate the lipid-protein association by electrostatic interactions, contribute to the hydrophobicity of the nonpolar face of the helix by the acyl portion of lysine and arginine, and/or complement the charge distribution in the polar head regions of the phospholipid molecules.

Liposome-cell interactions. A rapid assay for cells in suspension culture.

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interation. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.