Association of RTX toxins with erythrocytes.
A critical step in the target cell attack by RTX cytotoxins is their association with target cells. A binding assay was used to study the association of the Escherichia coli hemolysin protein (HlyA) with erythrocytes. Several parameters required for lysis by HlyA were tested for their effects on its initial association with erythrocytes. The results demonstrate that HlyA binding to target cells is independent of several structural components of the active toxin, including the N-terminal hydrophobic region, the glycine-rich repeat region, and the HlyC-dependent acylation of HlyA. Further, the association with erythrocytes was independent of Ca2+ concentration or temperature, while the lytic event is both Ca2+ dependent and temperature dependent. The association of two other RTX toxin proteins, the Pasteurella haemolytica leukotoxin (LktA) and the enterohemorrhagic E. coli toxin (EhxA), were also examined; these toxins bound to erythrocytes much less efficiently than did HlyA. The association of HlyA with erythrocytes occurred rapidly, within 12 s of incubation, and demonstrated no measurable dissociation. HlyA bound to erythrocytes with a maximum of approximately 2,000 molecules per cell. Competition between active HlyA and unacylated HlyA demonstrated no inhibition of binding by unacylated HlyA; rather, active HlyA appeared to displace unacylated HlyA on the cell surface. These data demonstrate that binding and lysis by HlyA are separable events and challenge the concept of nonspecific binding to the cell surface by RTX toxins.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 53706, USA.
M E
ME
Bauer
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Welch
D000215:Acylation; D000217:Acyltransferases; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005098:Exotoxins / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D006461:Hemolysis; D007700:Kinetics; D012756:Sheep; D013696:Temperature
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C097321:EHEC-hlyA protein, E coli; D029968:Escherichia coli Proteins; D005098:Exotoxins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; C018579:leukotoxin; D000217:Acyltransferases; C092237:HlyC protein, E coli; D002118:Calcium