Abstract
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.
Highlights
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
All toxins bound to erythrocytes and granulocytes in a nonsaturable manner
Toxin mutants in which these lysine residues are replaced with arginine are totally nonhemolytic but retain their capacity to bind to erythrocytes [4, 5] and to liposomes [6]
Summary
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. The lymphocyte function-associated antigen (LFA-1) (CD11a/CD18; ␣L2 integrin), was reported to serve as the receptor for HlyA and Actinobacillus actinomycetemcomitans leukotoxin on polymorphonuclear neutrophils (PMNs) [12]. The authors suggested that nonspecific absorption of HlyA to various cell types might occur that could obscure the receptor-mediate interaction [12] Studies with another RTX toxin, the leukotoxin of Mannheimia hemolytica, indicated that binding to integrin on bovine leukocytes results in activation of the tyrosine kinase signaling cascade [13]. Because HlyA stimulates the respiratory burst in these cells [15,16,17], a unifying concept would be that E. coli hemolysin binds to integrin LFA-1, activating the tyrosine kinase signaling cascade and triggering the respiratory burst. Binding occurs in a nonspecific and nonsaturable manner, and the respiratory burst is triggered directly by pore formation, probably because of flux of extracellular Ca2ϩ into the cells
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