The serine 31 residue of the B subunit of Shiga toxin 2 is essential for secretion in enterohemorrhagic Escherichia coli

Abstract

Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin 1 (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.

FIG. 1.

Distribution of Stx1 and Stx2 and effect of overexpressed histidine-tagged StxB in EHEC. EHEC K24 (Stx1), EHEC EDL933 (Stx1 and Stx2), EHEC 86-24 (Stx2), and transformed EHEC 86-24 were fractionated into supernatant and cell-associated fractions. Each 2 μg of protein of the cell-associated fraction (P) and an equal volume of the supernatant fraction (S) were analyzed by SDS-PAGE and immunoblotting using a mixture of anti-Stx1 antiserum, anti-Stx2 antiserum, and anti-His tag antibody. Values represent the ratios of StxA2 protein levels to the cell-associated fraction in the supernatant fraction (upper) and the ratios of StxB-H protein levels to the supernatant fraction in the cell-associated fraction (lower).

FIG. 2.

Effect of chimeric StxB1-H on Stx2 secretion in EHEC 86-24. Each 2 μg of protein of the cell-associated fraction (P) and an equal volume of the supernatant fraction (S) were analyzed by SDS-PAGE and immunoblotting using a mixture of anti-Stx2 antiserum and anti-His tag antibody. Values represent the ratios of StxA2 protein levels to the cell-associated fraction in the supernatant fraction (upper) and the ratios of StxB-H protein levels to the supernatant fraction in the cell-associated fraction (lower).

FIG. 3.

Effect of point-mutated StxB-H on Stx2 secretion in EHEC 86-24. Each 2 μg of protein of the cell-associated fraction (P) and an equal volume of the supernatant fraction (S) were analyzed by SDS-PAGE and immunoblotting using a mixture of anti-Stx2 antiserum and anti-His tag antibody. Values represent the ratios of StxA2 protein levels to the cell-associated fraction in the supernatant fraction (upper) and the ratios of StxB-H protein levels to the supernatant fraction in the cell-associated fraction (lower).

FIG. 4.

Two orthogonal views of the Stx2 B pentamer. (A) View along the five-fold axis. The surface toward the viewer is the sugar-binding surface, which corresponds to the bottom surface in the view shown in B. Each serine 31 residue of the B subunit is located in the shaded circle. Opened arrows indicate the carboxyl-terminal end of each B-subunit monomer. In the Stx2 structure, the A subunit is located on the face opposite the binding sites in A (side of the carboxyl-terminal end in B). Stx1 is also very similar structurally to Stx2. The figures were made with Cn3D software (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml).

FIG. 5.

Effect of StxB2-H (regions A and B) on Stx2 secretion in EHEC 86-24 (A) and size determination of StxB2-H (regions A and B) by gel filtration (B). (A) Two micrograms of protein of the cell-associated fraction (P) and an equal volume of the supernatant fraction (S) were analyzed by SDS-PAGE and immunoblotting using a mixture of anti-Stx2 antiserum and anti-His tag antibody. Values represent the ratios of StxA2 protein levels to the cell-associated fraction in the supernatant fraction (upper) and the ratios of StxB-H protein levels to the supernatant fraction in the cell-associated fraction (lower). (B) Purified StxB2-H (regions A and B) was loaded onto a TSK-G3000SW column and measured by reading the absorbance at 280 nm. Closed arrows indicate the elution of molecular mass standards. The fraction (shaded arrow) of StxB2-H contained the binding activity towards Vero cells, but the fraction (opened arrow) of StxB2-H (regions A and B) showed no binding activity.

FIG. 6.

Growth curve (A) and distribution of Stx1 and Stx2 (B) in EHEC EDLN, isogenic mutant EHEC strain N20-8 (StxB1 subunit, N32S), and EHEC N1-12 (StxB2 subunit, S31N). EHEC strains were fractionated into a supernatant fraction and a cell-associated fraction at mid-log phase (4 h), late log phase (6 h), early stationary phase (8 h), and stationary phase (10 h). Each 2 μg of protein of the cell-associated fraction (P) and an equal volume of the supernatant fraction (S) were analyzed by SDS-PAGE and immunoblotting using a mixture of anti-Stx1 and anti-Stx2 antisera. Closed rectangles, EHEC EDLN; closed circles, EHEC N20-8 (StxB1 subunit, N32S); open circles, EHEC N1-12 (StxB2 subunit, S31N).

FIG. 7.

Distribution of point-mutated holotoxins in EHEC K47. EHEC strain K47 strain retained the stx₂ gene, but no Stx2 protein was detected by immunoblotting. EHEC K47 harboring the stx gene was fractionated into supernatant (S) and cell-associated (P) fractions. The samples were analyzed by SDS-PAGE and immunoblotting using anti-Stx1 or anti-Stx2 antiserum.

FIG. 8.

Comparison of binding between Stx1 and mutant Stx1 (B subunit, N32S) (A) or between Stx2 and mutant Stx2 (B subunit, S31N) (B) to Gb₃ immobilized on ELISA plates coated with Gb₃. Bound holotoxin was detected with anti-Stx1 or anti-Stx2 antiserum, followed by HRP conjugated to anti-rabbit IgG. Closed rectangles, Stx1; open rectangles, mutant Stx1 (B subunit, N32S); closed circles, Stx2; open circles, mutant Stx2 (B subunit, S31N). The data are presented as the percentages of the values for the maximum binding of Stx1 or Stx2 (means ± standard errors; n = 3).