Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes

Abstract

The primary structure of Escherichia coli hemolysin (HIlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity. Hemolysin also requires an unknown modification by an accessory protein, HlyC, for hemolytic activity. The role of calcium in the interaction of HlyA with erythrocytes was investigated by using recombinant strains which produced inactive hemolysins unmodified by EllyC or deleted of the repeat sequences. 45Ca2' autoradiography of the recombinant hemolysins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium. Inactive hemolysin, unmodified by ElyC, contained the repeated sequences and bound calcium as efficiently as the active, full-length toxin. The binding of the inactive toxins to erythrocytes was investigated by immunoblotting saline-washed, toxin-treated cells with monoclonal antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. The binding of full-length, active hemolysin to erythrocytes was calcium dependent. Inactive hemolysin deleted of the repeat units did not bind to cells. The inactive hemolysin, unmodified by HlyC, bound calcium but did not bind to erythrocytes. These results highlight the importance of calcium in the binding of hemolysin to erythrocytes and suggest that the binding of hemolysin to cells requires an interaction between the calcium-binding repeat domain and the modification produced by the HlyC protein.

The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins

“…As mentioned above, the hemolysin is acylated via an uncharacterized process involving a dimer of HIyC and ACP [18]. In order to stably associate with erythrocytes, calcium must be bound to a region of HIyA which includes the glycine-aspartic acidrich repeats [26,27]. At present it is presumed that the calcium ions bind to HIyA in the cytoplasm prior to its export.…”

“…At present no additional genes have been identified which are required for HlyA export and assembly of the HIyB-HlyD-TolC complex. It should be noted that the HlyA conformation required for secretion competence does not depend on either the acylation or calcification of HlyA because unactivated and calciumfree forms of HIyA are secreted extracellularly to wild type levels [26,27].…”

Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA

“…distal to the bipartite acylation domain (Benz eta/., 1989;Boehm et a/., 1990). However, the presence of excess Ca" ions in the in vitro maturation reaction abolished the acquisition of proA haemolytic activity by preventing acylation at both the KI and the KII site, indicating a major Ca*+-induced change in toxin conformation extending over both FA1 and FAII, comprising more than 200 amino acids.…”

The virulence factors of Bordetella pertussis: a matter of control

“…A repeat domain of adenylate cyclase toxin/haemolysin contains 41 repeats of the glycine-and aspartic acid-rich regions indicative of the RTX family. Homology with other RTX members suggests that this region is involved in host cell recognition and calcium binding [189,190]. In fact, each repeat motif binds a single calcium ion, leading to a major conformational change and possibly the translocation of the catalytic portion of the molecule into host cells [191].…”