Abstract
Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.
Highlights
We previously described and characterized a monoclonal antibody (mAb) (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB1
Heat-labile enterotoxin (Etx)2 produced by certain strains of Escherichia coli is related to cholera toxin (Ctx) and causes diarrheal disease in humans and animals [2, 3]
The pathogenesis of enterotoxigenic E. coli diarrheal disease requires three processes: firstly, colonization and adherence to the mucosal epithelium by the bacterial fimbriae; secondly, binding of secreted toxin via a specific receptor; and lastly, uptake of the toxin into the cell, causing disruption of the electrolyte balance in gut epithelium [6, 7]
Summary
Heat-labile enterotoxin (Etx)2 produced by certain strains of Escherichia coli is related to cholera toxin (Ctx) and causes diarrheal disease in humans and animals [2, 3]. Using the bind to the receptor GM1, denatured EtxB monomers were in vitro system of toxin reassembly and the highly specific coated directly onto the plastic surface of the microtiter plates. MAbs, we show that the N-terminal region identified by LDS47 Denatured EtxB monomers were prepared by boiling 5 g/ml (APQSITELCS) is crucial for competent pentameric B-subunit purified EtxB in 30 mM dithiothreitol for 5 min.
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