Toxic effects of seu are different from those of other staphylococcal enterotoxins

Abstract

Background/purpose: Staphylococcal enterotoxins (SEs) are soluble extracellular proteins excreted by Staphylococcal bacterial strains, sharing similar structures and virulence. More than 20 genotypes of SEs have been identified, but the toxicity of some new SEs is still unclear.

Methods: In this study, we assessed the toxicity effects of six recombinant SEs (rSEA, rSEO, rSEG, rSEK, rSEU and rSEQ) on Balb/c mice by reverse transcription-polymerase chain reaction (RT-PCR)-based methods and enzyme activity detection.

Results: Except rSEU, the other five SEs resulted in systemic inflammatory responses with a significant increase of spleen and liver index and decrease of thymus index. SEs enhanced the enzyme activities of liver POD, T-SOD, LDH but reduced the activity of liver GSH-PX. The transcription levels of five cytokines were all down-regulated by rSEA, rSEG and rSEQ at a dose of 20 ng/g, which was coincided with the results of Caspase 3 level. The transcription and expression of IFN-γ, IL-4, IL-6, and TNF-α involved in inflammatory response were significantly up-regulated by rSEs at a low dose (20 ng/mL) except rSEU in vitro and in vivo.

Conclusion: Our results reveals that the rSEA, rSEO, rSEG, rSEK, and rSEQ have cytotoxicity and superantigenicity for Balb/c mice except the rSEU enterotoxin.

Keywords: Staphylococcal enterotoxin; cytokine; cytotoxicity; enzyme activity; genotype.

Figure 1

SDS-PAGE and Western-Blot analysis of six rSEs. A. The optimal eluted SEs was analyzed by SDS-PAGE electrophoresis; Lane M represents Protein Marker ranging from 14.4 kDa to 97.4 kDa, Lane 1-6 represent rSEA, rSEO, rSEG, rSEK, rSEU, rSEQ, respectively. B. Western-Blot for the six SEs was analyzed using anti-SE chicken polyclonal antibodies of SEA. Lane M represents Protein Marker ranging from 10 kDa to 170 kDa, Lane 1-6 represent rSEA, rSEO, rSEG, rSEK, rSEU, rSEQ, respectively. rSE, recombinant staphylococcal enterotoxins.

Figure 2

Different doses of rSEs induce distinct T cells proliferation in Balb/c in vitro. MTT assay was carried out in 96 plates after 48 h post treatment at concentrations of 10 ng mL^-1, 20 ng mL^-1, 40 ng mL^-1, 80 ng mL^-1 covering six rSEs, natural SEA, conA (positive control) and PBS (blank control) . Y axis was the stimulation index, which indicates the value in treatment groups compared to PBS control. SI > 1 indicates an increase in lymphocyte proliferation after treatment. All rSE except rSEU exhibited an extremely significant dose-dependent stimulation activity compared to PBS control (P < 0.001), and rSEU has a significant difference (P < 0.05) with conA positive control. Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. *indicates significant difference compared with respective control groups. **, P < 0.01. rSE, recombinant staphylococcal enterotoxins; SI, Stimulation index.

Figure 3

The effect of high dosage enterotoxins to VI. Fold change was calculated of Mouse Viscera (three columns, spleen, liver and thymus) Index after 72 h high-dose (20 ng/g) SEs (rSEA, rSEO, rSEG, rSEK, rSEU, rSEQ and native SEA) stimulation compared with negative treatment (PBS, not shown in the figure). There was no significant difference from rSEU-stimulated mice and control. However, the administration of other five rSEs and SEA leads to significant increase of spleen and liver index and decrease of thymus index (T test, P < 0.05), compared with PBS control. Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. *indicates significant difference compared with respective control groups. *, P < 0.05, **, P < 0.01, respectively for statistically significant, and very significant differences with respect to conA or nSEA positive treatment. VI, Viscera Index; rSE, recombinant staphylococcal enterotoxins; nSE, nature staphylococcal enterotoxins.

Figure 4

The enzymes activities in liver induced by SEs. Relative fold change of EA (four columns, POD, SOD, LDH and GSH-PX) Index inoculated with rSEs (rSEA, rSEO, rSEG, rSEK, rSEU, rSEQ and native SEA) after 72 h (20 ng/g body weight), compared with PBS control. Stimulation of rSEU has no significant influence on the enzyme activity, the other rSEs and SEA enhance the activities of liver POD, T-SOD and LDH, and reduce the liver GSH-PX activity. Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. **, P < 0.01, ***, P < 0.001 respectively for very significant and extremely significant differences with respect to PBS treatment. EA, Enzyme Activity; rSE, recombinant staphylococcal enterotoxins; POD, peroxidase; SOD, superoxide dismutase; LDH, lactate dehydrogenase; GSH-PX, glutathione peroxidase.

Figure 5

The effect of cytokines transcription induced by high dose enterotoxins. Total RNA from SEs (seven groups in X axis) stimulated Balb/c mouse thymus tissue was applied for relative real-time fluorescentive PCR assay of five cytokines, IL-2, IL-4, IL-6, TNF-α, IFN-γ (five columns). Fold change in Y axis represents cytokines change fold after treatment compared to the PBS control. Fold change > 1 indicates an increase in cytokines transcription after treatment, or is on the opposite. The rSEO and rSEK raise the level of TNF-α transcription (T test, P < 0.01). The administration of high dose of rSEA, rSEG, rSEQ and nSEA suppresses the level of cytokines IL-2, IL-4, TNF-α, IFN-γ and IL-6 transcription (T-test, P < 0.05). Except TNF-α, rSEU has no significant effect in other four cytokine transcription (T test, P > 0.05). Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001 respectively for statistically significant, very significant and extremely significant differences with respect to PBS treatment. rSE, recombinant staphylococcal enterotoxins.

Figure 6

Enzyme activity of Caspase 3 in T-lymphocyte Induced by SEs. T-lymphocyte derived from thymuses showed a marked change to SEs induced apoptosis compared with PBS control (P < 0.001), except rSEU (no significant difference). Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. ***, P < 0.001 respectively for extremely significant differences with respect to PBS treatment. rSE, recombinant staphylococcal enterotoxins.

Figure 7

The cytokine content of cell culture supernatant treated with different SEs in vitro. Five cytokines (X axis) and their content (pg/mL, Y axis) in supernatant of thymocytes treated by different SEs and PBS control (eight columns) were determined by ELISA kits. Most cytokines measured were significantly increasing in rSEs-treated groups. In rSEU-treated cells, no significant difference were detected in five cytokines as PBS control. Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001 respectively for statistically significant, very significant and extremely significant differences with respect to PBS treatment. rSE, recombinant staphylococcal enterotoxins.

Figure 8

The cytokine content of sera of mice inoculated by different rSEs in vivo. Five cytokines (X axis) and their content (pg/mL, Y axis) in sera of Balb/c mice inoculated different SEs and PBS contrl (eight columns) were determined by ELISA kits. rSEU treated mice almost have no effect on five cytokine expression as PBS control (T test, P > 0.05). Error bars represent the standard deviations, and statistical significance was determined by using Student’s unpaired t-test. *, P < 0.05, **, P < 0.01, respectively for statistically significant, and extremely significant differences with respect to PBS treatment. rSE, recombinant staphylococcal enterotoxins.