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Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin.

Bunger JC, Melton-Celsa AR, Maynard EL, O'Brien AD - Toxins (Basel) (2015)

Bottom Line: Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease.We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability.We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA. jbunger@gmail.com.

ABSTRACT
Shiga toxin (Stx) is an AB5 ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

No MeSH data available.


Related in: MedlinePlus

Crystal structure of A2 peptide and B subunits from Stx2a. A ribbon representation of the crystal structure of the A2 peptide shows the relative locations of the amino acids at positions 291 and 297 in comparison to positions 16 and 24 from the B subunits. The A2 peptide is colored red with a space-filled model of phenylalanine at position 291 depicted in teal and lysine at position 297 highlighted in green. The B subunits are colored blue with a space-filled model of aspartic acid at positions 16 and 24 highlighted in grey. Part (A) shows the binding face of Stx2a; Part (B) shows a horizontal view of the toxin with the binding face at the bottom. The PDB accession number for Stx2a is 1R4P. In this representation, the A1 portion of the structure is not shown.
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toxins-07-02306-f005: Crystal structure of A2 peptide and B subunits from Stx2a. A ribbon representation of the crystal structure of the A2 peptide shows the relative locations of the amino acids at positions 291 and 297 in comparison to positions 16 and 24 from the B subunits. The A2 peptide is colored red with a space-filled model of phenylalanine at position 291 depicted in teal and lysine at position 297 highlighted in green. The B subunits are colored blue with a space-filled model of aspartic acid at positions 16 and 24 highlighted in grey. Part (A) shows the binding face of Stx2a; Part (B) shows a horizontal view of the toxin with the binding face at the bottom. The PDB accession number for Stx2a is 1R4P. In this representation, the A1 portion of the structure is not shown.

Mentions: Crystal structure analysis of Stx2a predicts that the C-terminus of the A2 peptide may partially block one of the binding sites of a B subunit for Gb3 (Figure 5A,B) [13]. In addition, we have shown that for Stx2d/d removal of the two C-terminal amino acids from A2 increases its toxicity and capacity to bind Gb3 [19,20]. After making intermediate toxins between Stx2c/c and Stx2d/d, we found that phenylalanine at 291 when present in conjunction with glutamic acid at position 297 (Stx2c/d) was the least toxic protein in vitro and in vivo. We speculate that this combination of amino acids may represent a relatively unstable molecule with a reduced capacity to form holotoxin. However, serine at position 291 in wild-type Stx2d/d or the chimeric protein Stx2d/c was positively correlated with toxicity in vivo regardless of the amino acid at position 297, a finding that highlights the importance of the amino acid at position 291.


Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin.

Bunger JC, Melton-Celsa AR, Maynard EL, O'Brien AD - Toxins (Basel) (2015)

Crystal structure of A2 peptide and B subunits from Stx2a. A ribbon representation of the crystal structure of the A2 peptide shows the relative locations of the amino acids at positions 291 and 297 in comparison to positions 16 and 24 from the B subunits. The A2 peptide is colored red with a space-filled model of phenylalanine at position 291 depicted in teal and lysine at position 297 highlighted in green. The B subunits are colored blue with a space-filled model of aspartic acid at positions 16 and 24 highlighted in grey. Part (A) shows the binding face of Stx2a; Part (B) shows a horizontal view of the toxin with the binding face at the bottom. The PDB accession number for Stx2a is 1R4P. In this representation, the A1 portion of the structure is not shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488704&req=5

toxins-07-02306-f005: Crystal structure of A2 peptide and B subunits from Stx2a. A ribbon representation of the crystal structure of the A2 peptide shows the relative locations of the amino acids at positions 291 and 297 in comparison to positions 16 and 24 from the B subunits. The A2 peptide is colored red with a space-filled model of phenylalanine at position 291 depicted in teal and lysine at position 297 highlighted in green. The B subunits are colored blue with a space-filled model of aspartic acid at positions 16 and 24 highlighted in grey. Part (A) shows the binding face of Stx2a; Part (B) shows a horizontal view of the toxin with the binding face at the bottom. The PDB accession number for Stx2a is 1R4P. In this representation, the A1 portion of the structure is not shown.
Mentions: Crystal structure analysis of Stx2a predicts that the C-terminus of the A2 peptide may partially block one of the binding sites of a B subunit for Gb3 (Figure 5A,B) [13]. In addition, we have shown that for Stx2d/d removal of the two C-terminal amino acids from A2 increases its toxicity and capacity to bind Gb3 [19,20]. After making intermediate toxins between Stx2c/c and Stx2d/d, we found that phenylalanine at 291 when present in conjunction with glutamic acid at position 297 (Stx2c/d) was the least toxic protein in vitro and in vivo. We speculate that this combination of amino acids may represent a relatively unstable molecule with a reduced capacity to form holotoxin. However, serine at position 291 in wild-type Stx2d/d or the chimeric protein Stx2d/c was positively correlated with toxicity in vivo regardless of the amino acid at position 297, a finding that highlights the importance of the amino acid at position 291.

Bottom Line: Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease.We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability.We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA. jbunger@gmail.com.

ABSTRACT
Shiga toxin (Stx) is an AB5 ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

No MeSH data available.


Related in: MedlinePlus