leukotoxin and Lymphoma

A series of internal deletions in the lktA gene of Pasteurella haemolytica has been constructed. All of the deletions eliminated the lytic activity of the leukotoxin towards the bovine lymphoma cell line, BL-3. Deletions removing segments of the amino-proximal hydrophobic region, which is thought to constitute an essential membrane-spanning domain, were found to agglutinate BL-3 cells. Agglutination was similar to lysis by the wild-type toxin in that it was dependent upon the presence of calcium and required expression of the lktC gene. The agglutinating deletion proteins protected BL-3 cells from lysis by the wild-type toxin in a competitive fashion. This suggests that these mutants bind to a surface feature of the leukocyte which interacts with the native leukotoxin. These findings demonstrate that the cell-binding and lytic domains of the leukotoxin are separable.

Topics: Animals; Bacterial Toxins; Cattle; Cell Line; Cell Survival; Chromosome Deletion; Codon; Exotoxins; Genes, Bacterial; Lymphoma; Mutagenesis, Site-Directed; Pasteurella; Plasmids

Pasteurella haemolytica A1 leukotoxin causes rapid (5 to 15 min) leakage of intracellular K+ and cell swelling and slower (15 to 60 min), Ca2+-dependent formation of large plasma membrane defects (congruent to 100 nm) and leakage of lactate dehydrogenase from bovine lymphoma cells (BL3 cells) (K. D. Clinkenbeard, D. A. Mosier, A. L. Timko, and A. W. Confer, Am. J. Vet. Res., in press). Incubation of BL3 cells in medium made hypertonic by inclusion of 75 mM sucrose blocked leukotoxin-induced cell swelling, formation of large plasma membrane defects, and leakage of lactate dehydrogenase but did not block leukotoxin-induced leakage of intracellular K+. Carbohydrates with molecular weights less than that of sucrose, e.g., mannitol, did not block leukotoxin-induced cell swelling of BL3 cells. Increasing the concentration of mannitol to twice that of sucrose still resulted in no protective effect. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the functional transmembrane pore size formed by leukotoxin in BL3 cells is slightly less than the size of sucrose, i.e., 0.9 nm. Exposure of BL3 cells to leukotoxin for 15 or 45 min followed by the addition of hypertonic sucrose to the incubation medium reversed leukotoxin-induced cell swelling and prevented further leakage of lactate dehydrogenase. Leukotoxin-induced leakage of lactate dehydrogenase required both cell swelling and Ca2+-dependent processes. The Ca2+-dependent steps can occur before or concurrent with cell swelling.

Topics: Animals; Bacterial Toxins; Cattle; Cell Count; Cell Line; Cell Membrane; Cytotoxins; Exotoxins; Ion Channels; L-Lactate Dehydrogenase; Lymphoma; Osmotic Pressure; Pasteurella; Sucrose

Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured bovine lymphoma cells (BL3 cells). Release of 51CrO4(2-) and lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition, leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from leukotoxin-exposed BL3 cells was proportional to the amount of leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of leukotoxin added. Leukotoxin-induced release of LDH required a divalent cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased leukotoxin-induced release of LDH. Divalent cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation. Ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling. Leukotoxin action on BL3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.

Topics: Animals; Bacterial Toxins; Calcium; Cattle; Cell Adhesion; Cell Aggregation; Cell Survival; Exotoxins; L-Lactate Dehydrogenase; Lymphoma; Microscopy, Electron, Scanning; Pasteurella; Potassium; Tumor Cells, Cultured