Binding of Escherichia coli hemolysin and activation of the target cells is not receptor-dependent.
Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca(2+), which was likely because of passive influx of Ca(2+) through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the beta(2) integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.
Valeva
Angela
A
Institute of Medical Microbiology and Hygiene, University of Mainz, D-55101 Mainz, Germany. avaleva@uni-mainz.de
Walev
Ivan
I
Kemmer
Helene
H
Weis
Silvia
S
Siegel
Isabel
I
Boukhallouk
Fatima
F
Wassenaar
Trudy M
TM
Chavakis
Triantafyllos
T
Bhakdi
Sucharit
S
D000215:Acylation; D019943:Amino Acid Substitution; D001427:Bacterial Toxins; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016897:Respiratory Burst; D017384:Sequence Deletion; D013481:Superoxides / Q000378:metabolism*
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D016169:Lymphocyte Function-Associated Antigen-1; D013481:Superoxides; D002118:Calcium