Abstract
The specificity of the Pasteurella hemolytica leukotoxin suggests a novel toxin receptor on the surface of its target cells. Using a simple competition assay, we have examined the ability of extracts prepared from target and nontarget cells to protect leukocytes from lysis by the leukotoxin. Protective, receptor-like activity was extracted from the toxin sensitive cell line, BL3. Similar activity is not present in extracts prepared from bovine erythrocytes which are resistant to the lytic effects of the leukotoxin. The receptor-like activity is resistant to protease digestion, is chloroform-methanol extractable and is not temperature sensitive. The activity fractionates in the neutral fraction over a DEAE cellulose ion exchange column. These results are consistent with the leukotoxin receptor being a neutral lipid expressed primarily on leukocytes of ruminant origins. The leukotoxin was purified by fusion of the LktA reading frame to sequences encoding a hexahistidine (His6) tag at either its N-or Cterminus. The leukotoxin retained lytic activity and could be efficiently activated by LktC-mediated acylation even when the overexpressed proteins were recovered as inclusion bodies in E. coli. Characterization of the leukotoxin demonstrate that the activated toxin is 20 times more lytic than the protoxin, that both toxin and protoxin are labile at room temperature and that little conformational change could be detected by circular dichroism analysis between the toxin and protoxin in the presence and absence of calcium chloride.
Manley-Barker, Donna Kay (1998). Characterization of the Pasteurella hemolytica leukotoxin and its interaction with target cells. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1998 -THESIS -M365.