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Transcytosis of staphylococcal superantigen toxins.

Hamad AR, Marrack P, Kappler JW - J. Exp. Med. (1997)

Bottom Line: We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA.We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA.Our data suggest that these toxins can cross the epithelium in an immunologically intact form.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

ABSTRACT
Staphylococcus aureus produces a set of proteins (e.g., staphylococcal enterotoxin A [SEA], SEB, toxic shock syndrome toxin 1 [TSST-1]) which act both as superantigens (SAgs) and toxins. Although their mode of action as SAgs is well understood, little is known about how they enter the body via the intestine and cause food poisoning. To examine this problem we used an in vitro culture system to study the capacity of class II MHC-negative human intestinal epithelial cells (Caco-2) to transcytose several staphylococcal toxins. We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA. We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA. Our data suggest that these toxins can cross the epithelium in an immunologically intact form. These results may have important implications for the pathogenesis of food poisoning.

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Mutation of residues N23 and F44 affects SEB transcytosis by  Caco-2 cells. (A) The amount of wild type and mutants SEB transcytosed  (apical to basal) per filter in 4 h. The experiment was done as in Fig. 3.  Wild type and mutant SEB were used to construct standard curves. (B)  The amounts of wild type and mutants transcytosed relative to that of  HRP. Each point is the mean of three determinations. SEM was usually  <15%. This experiment is representative of three separate experiments  with similar results.
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Figure 4: Mutation of residues N23 and F44 affects SEB transcytosis by Caco-2 cells. (A) The amount of wild type and mutants SEB transcytosed (apical to basal) per filter in 4 h. The experiment was done as in Fig. 3. Wild type and mutant SEB were used to construct standard curves. (B) The amounts of wild type and mutants transcytosed relative to that of HRP. Each point is the mean of three determinations. SEM was usually <15%. This experiment is representative of three separate experiments with similar results.

Mentions: Residues N23 and F44 of SEB play critical roles in its SAg activity. Previously, we have shown that residue N23 is essential for interaction with the TCR (22), whereas the stretch of six amino acids located in the loop between SEB β strands 1 and 2 (including residue F44) are involved in binding to MHC class II molecules (22, 31, 32). In an attempt to determine if these domains of SEB also may play a role in transcytosis, we compared the rate of transcytosis of SEB through Caco-2 monolayers to that of two SEB mutants: N23 >K and F44 >S. Although, in our experience, these mutations do not affect the sensitivity of SEB detection by the pair of the mAbs used (23), standard curves of mutants were used to calibrate their concentrations in the tested supernatants as an added precaution. These mutant SEBs are reduced in their capacity to stimulate T cells by at least 1,000-fold (22). Either mutation reduced the rate of SEB transcytosis by 50–70% (Fig. 4). These results suggest that these regions of SEB have a function in the transcytosis and provide additional evidence for the specificity of SEB transcytosis by Caco-2 cells.


Transcytosis of staphylococcal superantigen toxins.

Hamad AR, Marrack P, Kappler JW - J. Exp. Med. (1997)

Mutation of residues N23 and F44 affects SEB transcytosis by  Caco-2 cells. (A) The amount of wild type and mutants SEB transcytosed  (apical to basal) per filter in 4 h. The experiment was done as in Fig. 3.  Wild type and mutant SEB were used to construct standard curves. (B)  The amounts of wild type and mutants transcytosed relative to that of  HRP. Each point is the mean of three determinations. SEM was usually  <15%. This experiment is representative of three separate experiments  with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196287&req=5

Figure 4: Mutation of residues N23 and F44 affects SEB transcytosis by Caco-2 cells. (A) The amount of wild type and mutants SEB transcytosed (apical to basal) per filter in 4 h. The experiment was done as in Fig. 3. Wild type and mutant SEB were used to construct standard curves. (B) The amounts of wild type and mutants transcytosed relative to that of HRP. Each point is the mean of three determinations. SEM was usually <15%. This experiment is representative of three separate experiments with similar results.
Mentions: Residues N23 and F44 of SEB play critical roles in its SAg activity. Previously, we have shown that residue N23 is essential for interaction with the TCR (22), whereas the stretch of six amino acids located in the loop between SEB β strands 1 and 2 (including residue F44) are involved in binding to MHC class II molecules (22, 31, 32). In an attempt to determine if these domains of SEB also may play a role in transcytosis, we compared the rate of transcytosis of SEB through Caco-2 monolayers to that of two SEB mutants: N23 >K and F44 >S. Although, in our experience, these mutations do not affect the sensitivity of SEB detection by the pair of the mAbs used (23), standard curves of mutants were used to calibrate their concentrations in the tested supernatants as an added precaution. These mutant SEBs are reduced in their capacity to stimulate T cells by at least 1,000-fold (22). Either mutation reduced the rate of SEB transcytosis by 50–70% (Fig. 4). These results suggest that these regions of SEB have a function in the transcytosis and provide additional evidence for the specificity of SEB transcytosis by Caco-2 cells.

Bottom Line: We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA.We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA.Our data suggest that these toxins can cross the epithelium in an immunologically intact form.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.

ABSTRACT
Staphylococcus aureus produces a set of proteins (e.g., staphylococcal enterotoxin A [SEA], SEB, toxic shock syndrome toxin 1 [TSST-1]) which act both as superantigens (SAgs) and toxins. Although their mode of action as SAgs is well understood, little is known about how they enter the body via the intestine and cause food poisoning. To examine this problem we used an in vitro culture system to study the capacity of class II MHC-negative human intestinal epithelial cells (Caco-2) to transcytose several staphylococcal toxins. We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA. We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA. Our data suggest that these toxins can cross the epithelium in an immunologically intact form. These results may have important implications for the pathogenesis of food poisoning.

Show MeSH
Related in: MedlinePlus