Contribution of Staphylococcal Enterotoxin B to Staphylococcus aureus Systemic Infection

Abstract

Staphylococcal enterotoxin B (SEB), which is produced by the major human pathogen, Staphylococcus aureus, represents a powerful superantigenic toxin and is considered a bioweapon. However, the contribution of SEB to S. aureus pathogenesis has never been directly demonstrated with genetically defined mutants in clinically relevant strains. Many isolates of the predominant Asian community-associated methicillin-resistant S. aureus lineage sequence type (ST) 59 harbor seb, implying a significant role of SEB in the observed hypervirulence of this lineage. We created an isogenic seb mutant in a representative ST59 isolate and assessed its virulence potential in mouse infection models. We detected a significant contribution of seb to systemic ST59 infection that was associated with a cytokine storm. Our results directly demonstrate that seb contributes to S. aureus pathogenesis, suggesting the value of including SEB as a target in multipronged antistaphylococcal drug development strategies. Furthermore, they indicate that seb contributes to fatal exacerbation of community-associated methicillin-resistant S. aureus infection.

Keywords: Staphylococcus aureus; CA-MRSA; ST59; cytokine storm; sepsis; staphylococcal enterotoxin B; superantigen.

Figure 1.

Presence of the seb gene distinguishes sequence type (ST) 59 community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) from geographically matched predominant hospital-associated (HA) MRSA isolates. The distribution of staphylococcal enterotoxin genes and the tst gene encoding toxic shock syndrome toxin 1 in CA-MRSA ST59, HA-MRSA ST239, and HA-MRSA ST5 clinical isolates from patients obtained at Renji hospital, Shanghai, between 2005 and 2014 was determined by means of analytical polymerase chain reaction. Forty-four isolates were randomly selected for each ST.

Figure 2.

Staphylococcal enterotoxin B (SEB) production from sequence type 59 culture filtrates induces proliferation of human T cells in vitro. A, Culture filtrates were collected from 16-hour cultures grown in tryptic soy broth and probed with anti-SEB antibody. Signal intensities were determined by means of densitometry using ImageJ software. Error bars represents means with standard deviations. Statistical analysis was performed using unpaired Student t tests or 1-way analysis of variance (ANOVA). *P < .05; †P < .01). B, Peripheral blood mononuclear cells from human whole-blood samples were plated at 5 × 10⁴ cells per well and incubated with a 1:1000 dilution of sterile culture filtrates for approximately 48 hours. Adenosine triphosphate (ATP) levels were measured as a function of T-cell proliferation and release of lactate dehydrogenase (LDH) to verify that there were no cytolytic effects. Error bars represent means with standard deviations. Statistical analysis was performed using unpaired Student t tests or 1-way ANOVA, or the respective nonparametric tests (Mann-Whitney and Kruskal-Wallis) if data did not pass normality distribution tests. *P < .05; †P < .01. Abbreviations: NS, not significant; RLUs, relative light units; WT, wild type.

Figure 3.

Staphylococcal enterotoxin B (SEB) contributes to virulence of sequence type (ST) 59 in a mouse systemic infection model. Survival of HLA-DR3 female mice was determined after intravenous challenge with 2 × 10⁸ colony-forming units of Staphylococcus aureus ST59 wild type (WT) (n = 13) or its isogenic seb deletion strain (n = 12). Animals were monitored for up to 8 days (192 hours) after infection. Statistical analysis was performed using the log-rank (Mantel-Cox) test. Data are representative of 2 independent experiments. ‡P < .001.

Figure 4.

Decreased survival in mouse systemic infection is not due to differences in bacterial burden in organs or complete blood cell counts. A, Blood, kidneys, livers, and spleens were collected 12 hours after intravenous challenge with 2 × 10⁸ colony-forming units (CFUs) of Staphylococcus aureus (n = 10/group). Blood and organ homogenates were assessed for CFUs. B, Complete blood cell counts of white blood cells (WBCs), neutrophils, monocytes, and lymphocytes. A, B, Error bars represent means with standard deviations. Statistical analysis was performed using unpaired Student t tests or Mann-Whitney tests if data did not pass normality distribution tests. No significant differences were observed. Abbreviation: WT, wild type.

Figure 5.

Contribution of staphylococcal enterotoxin B (SEB) to pathogenesis in mouse systemic infection is not due to a gene regulatory effect. A, Sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Coomassie stain of filtrates from ST59 wild-type (WT) or Δseb cultures grown for 8 or 16 hours. B, Systemic infection model in WT (C57BL-6NCrl) mice. The infection model was performed as described for Figure 3, except that mice were monitored longer owing to expected lower virulence in WT mice (n = 5 per group).

Figure 6.

Decreased survival in mouse systemic infection is linked to a cytokine storm. Spleen homogenates (A) and serum samples (B) were assessed for concentrations of the cytokines interferon (IFN) γ, interleukin 10 (IL-10), and tumor necrosis factor (TNF) α, 12 hours after infection with 2 × 10⁸ colony-forming units of Staphylococcus aureus wild-type (WT) or Δseb mutant (n = 10 per group). Error bars represent means with standard deviations. Statistical analysis was performed using unpaired Student t tests or Mann-Whitney tests if data did not pass normality distribution tests. †P < .01; ‡P < .001. Abbreviation: NS, not significant.