A Structure-Function Analysis of Shiga-Like Toxin Type 2 of Enterohemorrhagic Escherichia Coli
Abstract
Shiga toxin of Shigella dysenteriae I and Shiga-like toxins I and II (SLT-I and SLT-II) of enterohemorrhagic Escherichia coli are functionally similar, protein cytotoxins. These toxin molecules have a bipartite molecular structure which consists of an enzymatically active A subunit that inhibits protein synthesis in eukaryotic cells and an oligomeric B subunit that binds to globotriosylceramide glycolipid receptors on eukaryotic cells. The first objective of the project was to generate monoclonal antibodies against SLT-II and to investigate the molecular basis for antigenicity and toxin neutralization. Monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933 strain. Five monoclonal antibodies were isolated that were capable of neutralizing the cytotoxicity of SLT-II, but not SLT-I, Shiga toxin, or a variant of SLT-II produced by E. coli that causes edema disease of swine. All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit. Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass. All five monoclonal antibodies had K light chains. To identify the epitopes of SLT-II reactive with the monoclonal antibodies, a sublibrary of the sIt-II gene was created in lambda ZAP expression vector, and the recombinant phage library was screened with individual SLT-II neutralizing monoclonal antibodies. The sequences of the DNA inserts that expressed the immunoreactive epitopes contained sit-W gene segments larger than 1 kb in size. Therefore, sequence analysis of a larger pool of immunoreactive clones may be needed to identify the minimal coding region of each epitope.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 07, 1990
- Accession Number
- AD1010974
Entities
People
- Liyanage Perera
Organizations
- Uniformed Services University of the Health Sciences