Cytotoxic and apoptotic effects of recombinant subtilase cytotoxin variants of shiga toxin-producing Escherichia coli

Abstract

In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death.

FIG 1

Cloning strategy for different subAB subunit genes. The genes for the plasmid pO113-encoded subunits SubA₁ and SubB₁ were cloned into the expression vector pET-22b(+) (A and C) using oligonucleotides subAF-His/SubAR-His and SubBF-His/SubBR-His. For the chromosome-located subAB variants, another cloning approach was chosen. (A) In a first PCR, subAB_2-1 and subAB_2-2 were amplified, including their flanking DNA regions. For subAB_2-2, oligonucleotides subA-L/subAB2-3′out were used, and for subAB_2-1, oligonucleotides Linkerfor-subAB/subAB3′tia were used. (B) Amplification of the single-toxin subunits was carried out using the following oligonucleotides: subA_2-2, SubA2-2F-His/SubAR-His; subB_2-2, SubB2F-His/SubB2-2R/His; subA_2-1, SubA2-1F-His/SubAR-His; subB_2-1, SubB2-HisF/SubB2-1R-His. (C) PCR products were cloned into pET-22b(+) as shown and as described in the text. All oligonucleotide sequences are listed in Table 1.

FIG 2

Purification of recombinant SubAB_2-2 subunits. Protein fractions stained with colloidal Coomassie brilliant blue were analyzed by 15% SDS-PAGE. (A) purification of SubA_2-2: lane 1, crude protein extract after lysis of bacteria; lane 2, unbound proteins after Ni-NTA column; lane 3, 5 mM imidazole washing step; lane 4, 20 mM imidazole washing step; lane 5, 40 mM imidazole washing step; lanes 6 to 8, protein elution fractions; lane 9, protein fraction (4 μg) after buffer exchange; M, PageRuler prestained protein ladder (Thermo Scientific, Germany). MW, molecular weight (in thousands). (B) Purification of SubB_2-2: lane 1, crude protein extract after lysis of bacteria; lane 2, unbound proteins after Ni-NTA column; lane 3, 5 mM imidazole washing step; lane 4, 40 mM imidazole washing step; lanes 5 to 7, protein elution fractions; lane 8, protein fraction (3 μg) after buffer exchange; M, PageRuler prestained protein ladder (Thermo Scientific, Germany). Frames in panel A, lane 9, and B, lane 8, label the areas which were used for the determination of protein preparation purity using the software tool ImageJ.

FIG 3

Cytotoxic effects of SubAB_2-2. Vero B4 cells were incubated at 37°C with different concentrations of the single SubA_2-2 (squares) and SubB_2-2 (triangles) toxin subunits and their combination, SubAB_2-2 (diamonds), in the medium. For a control, cells were left untreated. After 72 h, the amount of viable cells was determined. The values of the amount of viable cells were plotted against cell viability of the control cells in percentages. The standard errors are shown in the graph.

FIG 4

Comparison of the cytotoxic effects caused by different SubAB variants on Vero cells. (A) Cytotoxic effects of the different SubAB variants on Vero B4 cells were measured after a 72-h incubation period using the crystal violet staining assay and were compared to each other. Results obtained by the use of different toxin dilutions between 2,500 ng/ml and 20 ng/ml were plotted against the resulting Vero cell viability of the different variants. The amount of viable Vero B4 cells, which were incubated in the absence of SubAB proteins, were calculated as 100%. The standard errors are shown in the graph. (B) Binding of recombinant SubB₁ and SubB_2-1 proteins to Vero and HeLa cells. Confluently grown Vero or HeLa cells in 12-well plates were incubated for 45 min at 4°C in serum-free medium with 5 μg/ml of either biotin-labeled SubB₁ or biotin-labeled SubB_2-1 to enable binding of the SubB proteins to the cells. For a control (con), cells were left untreated. Cells were washed 3 times to remove unbound SubB, lysed in 50 μl of a 2.5-fold SDS sample buffer, and heated for 5 min at 95°C. After SDS-PAGE, the cell-bound biotinylated SubB proteins were detected by Western blotting with streptavidin-peroxidase and the ECL system. The biotin-labeled SubB₁ and SubB_2-1 proteins (0.5 μg each) were included as running controls (right lanes). A cellular protein between a molecular weight (MW) of 70,000 and 100,000 (marked by an asterisk) also was recognized by streptavidin-peroxidase, which indicates comparable protein loading and blotting of all lysate samples.

FIG 5

Cytotoxic effects caused by hybrid SubAB toxins. Recombinant SubA_2-1 and SubB_2-2 (triangles) subunits, as well as SubA_2-2 and SubB_2-1 (squares) subunits, were mixed in vitro and incubated with Vero B4 cells (for details, see the text). Cells also were incubated with the toxins SubAB_2-1 (diamonds) and SubAB_2-2 (circles) using the same approach. The standard errors are shown in the graph.

FIG 6

Cytotoxic effects of SubAB₁ and SubA₁ on HeLa cells. Subconfluently grown HeLa cells were incubated at 37°C with SubA₁ (10 nM) plus SubB₁ (50 nM) or with SubA₁ (10 μg/ml; ∼286 nM) alone. For a control, cells were incubated without any protein. After 24, 46, and 70 h, pictures from the cells were taken to demonstrate the changes in cell morphology.

FIG 7

Binding of SubA₁ to HeLa cells. Cells were incubated for 30 min at 4°C with increasing concentrations of SubA₁ from 1 to 200 μg/ml to enable binding of this protein to the cell surface. Subsequently, the medium was removed and cells were washed to remove any protein, which was not associated with the cells. Cells were lysed and subjected to SDS-PAGE, and the cell-bound His₆-labeled SubA₁ was detected by Western blotting with an antibody against the His₆ tag. For a control, cells were incubated for 30 min at 4°C with His₆-labeled C2I (10 μg/ml), which does not bind to cells, to demonstrate the specificity of the SubA₁ binding. Purified SubA₁ and C2I proteins were used as controls. MW, molecular weight (in thousands).

FIG 8

Continuous presence of SubA₁ in the medium is necessary to induce cytotoxic effects on HeLa cells. Cells were incubated for 1 h at 37°C with SubA₁ (10 μg/ml) to enable internalization of this protein. The medium then was removed and cells were washed to remove the protein. Subsequently, one portion of the cells was incubated further at 37°C in the presence of fresh SubA₁ (10 μg/ml; middle), and another portion was incubated in fresh medium without SubA₁ (lower). For a control, cells were incubated without any protein (upper). Pictures from the cells were taken after 25, 50, and 72 h.

FIG 9

SubA₁ induces apoptosis of HeLa cells. HeLa cells were incubated at 37°C with SubA₁ (10 μg/ml), SubB₁ (50 nM), or the combination of SubA₁ (10 nM) plus SubB₁ (50 nM). For a negative control, cells were left untreated, and for a positive control, cells were incubated with staurosporine, an established inducer of apoptosis. After 72 h, cell viability was determined by FSC/SSC measurements (A) and apoptosis by analysis of DNA fragmentation of PI-stained nuclei (B). Significance was tested by using Student's t test (**, P < 0.005; ***, P < 0.0005); ns, not significant. Cells were incubated for 72 h with or without SubA₁ (10 μg/ml) in the absence (dark gray bars) or presence (light gray bars) of the caspase inhibitor zVAD.fmk (40 μM), and caspase activation was investigated by using the CellEvent caspase-3/7 green detection reagent (C); apoptosis was analyzed by measuring DNA fragmentation of PI-stained nuclei (D).