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Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.

L P Perera1, J E Samuel, R K Holmes

  • 1Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.

Infection and Immunity
|March 1, 1991
PubMed
Summary

The Shiga-like toxin type II (SLT-II) operon requires both its 5' and 3' terminal coding sequences for function. Modifications to the B subunit

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Area of Science:

  • Microbiology and Molecular Biology
  • Bacterial Pathogenesis
  • Toxinology

Background:

  • Enterohemorrhagic Escherichia coli (EHEC) produces Shiga-like toxin type II (SLT-II), a key virulence factor in disease pathogenesis.
  • SLT-II is a bipartite toxin comprising an enzymatically active A subunit and a B subunit responsible for cell binding via globotriaosylceramide.
  • Understanding the functional boundaries of the slt-II operon is crucial for elucidating toxin mechanism and developing countermeasures.

Purpose of the Study:

  • To map the functional boundaries of the slt-II operon.
  • To determine the essential regions of the SLT-II A and B subunits for cytotoxic activity.

Main Methods:

  • Construction and analysis of mutant SLT-II proteins with deletions or extensions at the carboxy terminus of the B subunit.

Related Experiment Videos

  • Construction and analysis of mutant SLT-II proteins with deletions within the A subunit coding region.
  • Assessment of holotoxin cytotoxic activity and A subunit enzymatic activity for all generated mutants.

    • Main Results:

    • Deletion of the final four amino acids of the mature SLT-II B subunit abolished cytotoxic activity.
    • Minor alterations (two amino acid deletion or 21 amino acid extension) at the B subunit's carboxy terminus had minimal impact on cytotoxic activity.
    • Deletion of amino acids 3-18 from the mature SLT-II A subunit completely abolished both holotoxin cytotoxic and A subunit enzymatic activities.

    Conclusions:

    • Both the 5' and 3' terminal coding sequences of the slt-II operon are essential for the functional activity of Shiga-like toxin type II.
    • The integrity of the N-terminal region of the SLT-II A subunit is critical for its enzymatic function and overall toxin activity.
    • The C-terminal region of the SLT-II B subunit plays a role in cytotoxic activity, with specific terminal amino acids being important for function.