Characterization of monoclonal antibodies against the Escherichia coli hemolysin

Abstract

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.

Characterization of a neutralizing monoclonal antibody to Pasteurella haemolytica leukotoxin

“…Monoclonal antibodies (MAbs) prepared against the alpha-hemolysin have helped to further define important domains within the toxin (22,30), including a potentially important domain between the membrane-spanning region and the repeating glycine-rich domain that contains an epitope which, when bound by antibody, results in potent neutralization (31,37). Similar investigations have not been performed with MAbs directed against P. haemolytica Al leukotoxin.…”

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

“…Furthermore, deletion of amino acids 10 to 19 and 9 to 37 from the N-terminal end of plasmid-encoded HIyA results in an increase of the haemolytic activity by factors of 1.5 and 2.5, respectively [8]. The influence of the whole N-terminal region on the haemolytic activity and channel-formation seems to be rather small since a mutant missing 66 amino acids from the N-terminus is still haemolytically active [18]. It is interesting to note that the channels formed by the mutant with the deletion of amino acids 9 to 37 have a lifetime in lipid bilayer membranes which considerably exceeds 1 min [8].…”

Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3‐dependent vs . ‐independent macrophage cell death and host colonization

“…Western blotting was performed as previously described in Hohenhaus et al (29). Membranes were incubated with a primary antibody against hemolysin (H10, mouse mAb, isotype G1) (31) or outer membrane protein A (OmpA) (rabbit pAb; Antibody Research Corporation, St. Peters, MO, USA) at a dilution of 1:20,000 and 1:10,000, respectively. Horseradish peroxidase-conjugated antimouse or -rabbit IgG antibodies (Cell Signaling Technology, Danvers, MA, USA), which were used as secondary antibodies (1:2500 dilution), were detected using chemiluminescence and Super RX films (Fujifilm, Tokyo, Japan) or Amersham Imager 600 (GE Healthcare, Waukesha, WI, USA).…”