Abstract

Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin alpha/karyopherin alpha 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor alpha, interferon gamma, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor kappa B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses.

Highlights

  • Staphylococcal enterotoxin B (SEB)1 causes a spectrum of human diseases, including food poisoning and non-menstrual toxic shock syndrome (NMTSS) [1, 2]

  • The systemic production of tumor necrosis factor ␣, interferon ␥, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor ␬B in T cells

  • Transgenic Inhibitor of nuclear factor ␬B (NF␬B) Nuclear Import in T Cells Recapitulates the Cell-permeant Effects of Peptides—To explore the relative contribution of NF␬B to the set of SRTFs targeted by a cell-permeant peptide inhibitor of nuclear import, we introduced SEB and D-Gal into C57BL/6 mice expressing an I␬B.DN transgene under the control of a T-cell-specific promoter [29]

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Summary

Staphylococcal Enterotoxin B and a Nuclear Import Inhibitor

Sequences of cell-permeant peptides in single-letter amino acid code comprise membrane-translocating motif (MTM) derived from the hydrophobic region of the fibroblast growth factor 4 signal sequence and cargo of nuclear localization sequence (NLS) derived from nuclear factor ␬B. NLS sequence is cyclized in the cyclized form of SN50 (cSN50) (by the addition of a pair of cysteines) and mutated in SM peptide. SN50 and SM peptides were labeled with FITC to study their delivery and intracellular location in T cells. MTM-deficient N50c peptide was labeled with fluorescein-5-maleimide (FM) via an NH2-terminal cysteine

NLS or its mutant
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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