Determining the Presence of Superantigens in Coagulase Negative Staphylococci from Humans

<div><p>Superantigens (SAgs) are important virulence factors in <i>S</i>. <i>aureus</i>. Recent studies identified their presence in animal coagulase-negative staphylococci (CNS). The emergence of human-associated SAg⁺ CNS would mark a prodigious shift in virulence capabilities. We examined CNS isolates from healthy human nares and diseased individuals, and determined that no known SAgs were present.</p></div

Species and sources of the staphylococcal isolates.

<p>^<u>a</u> Samples obtained from nasal swabs of healthy volunteers.</p><p>Species and sources of the staphylococcal isolates.</p

Prevalence of SAg genes in staphylococcal isolates.

<p>Prevalence of SAg genes in staphylococcal isolates.</p

SAg gene profile of nasal <i>S</i>. <i>aureus</i> isolates.

<p>Conventional PCR and specific primers for known SAg genes were utilized [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143341#pone.0143341.ref017" target="_blank">17</a>].</p

Local Epidermal Growth Factor Receptor Signaling Mediates the Systemic Pathogenic Effects of <i>Staphylococcus aureus</i> Toxic Shock Syndrome

<div><p>Secreted factors of <i>Staphylococcus aureus</i> can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an <i>ex vivo</i> porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live <i>S</i>. <i>aureus</i>. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an <i>S</i>. <i>aureus</i> exotoxin-mediated disease and may represent an attractive host target for therapeutics.</p></div

EGFR signaling mediates the PVM IL-8 response to <i>S</i>. <i>aureus</i>.

<p>PVM explants were inoculated with ~ 10⁷ CFU/explant and incubated for 6 h ± 4 μg/explant AG1478 (AG) or 4 μl/explant 10% DMSO vehicle prior to processing for IL-8 production (via ELISA) and CFU determination. (A-E) White bar, uninfected control (CNTL), black bars indicate presence of bacteria. Incubation of PVM with AG prior to infection with (<b>A</b>) MNPE, (<b>B</b>) MNPE <i>-tstH</i>, (<b>C</b>) MN8, or (<b>D</b>) MN8 <i>-tstH</i> significantly reduced IL-8 production (asterisks, <i>p</i> < 0.0002). DMSO alone had no effect.</p

α-toxin induces AREG shedding and IL-8 production from HVECs.

<p>HVECs were exposed to α-toxin for 6 h and then processed for toxicity via MTT assay or IL-8 secretion and AREG shedding via ELISA. Where inhibitors were used, they were applied to HVECs 30 min prior to addition of α-toxin. (<b>A</b>) IL-8 secretion from and (<b>B</b>) viability of HVECs exposed to various doses of α-toxin. For both curves, the asterisks indicate doses showing significant differences from 0 (<i>p</i> < 0.0001). (<b>C</b>) AREG shedding and (<b>D</b>) IL-8 secretion in response to α-toxin is dampened in the presence of TAPI-1 and AG1478. White bars indicate media alone, black bars represent α-toxin treatment at 1 μg/ml. Asterisks indicate significant differences from α-toxin (AT) alone (<i>p</i> < 0.0001). (<b>E</b>) AREG shedding and (<b>F</b>) IL-8 secretion in response to α-toxin are dampened in ADAM10 (A10) and ADAM17 (A17) KD cell lines. White bars indicate media alone on negative control (NC) siRNA cells, black bars represent α-toxin treatment at 1 μg/ml. Asterisks indicate significant differences from α-toxin treated NC cells (<i>p</i> < 0.0001).</p

EGFR signaling is required for mTSS progression <i>in vivo</i>.

<p>(<b>A, B</b>) WT MN8 or MN8 <i>-tstH</i> were administered at 5 x 10⁸ twice daily for 3 days or until death, <i>N</i> = 1, n = 4. (<b>A</b>) Survival is significantly increased (<i>p</i> < 0.0069) and (<b>B</b>) fever is generally reduced in animals challenged with MN8 <i>-tstH</i> versus WT MN8. (<b>C, D</b>) In separate experiments, rabbits were intravaginally challenged with ~ 10¹⁰ MN8 + 8 mg/ml AG1478 or 30% beta cyclodextrin vehicle twice daily for 4 days or until death, <i>N</i> = 1, n = 5. (<b>C</b>) Survival is significantly increased in animals treated with AG1478 (<i>p</i> < 0.03). (<b>D</b>) Fever is generally decreased in animals treated with AG1478 reaching significance at 24 and 36 h, just prior to the death of the majority of infected, untreated animals (<i>p</i> < 0.005).</p

IL-8 production from PVM in response to TSST-1 and α-toxin is dependent on EGFR signaling.

<p>PVM explants were exposed to TSST-1 and/or α-toxin for 6 h and then processed for IL-8 production via ELISA. Where inhibitors were used, they were applied to explants 30 minutes prior to addition of toxin(s). IL-8 is produced in response to both (<b>A</b>) TSST-1 and (<b>B</b>) α-toxin in a dose-dependent manner. For both curves, the asterisks indicate doses showing significant differences from 0 (<i>p</i> < 0.0004). (<b>C</b>) IL-8 production in response to high doses of both TSST-1 (20 μg/explant) and α-toxin (AT) (2 μg/explant) is completely abrogated in the presence of AG1478 (AG– 40 μg/explant), but the dextrin vehicle (Dex– 10 μl of 15%) alone has no affect. Checkered bars represent TSST-1 treatment and striped bars represent AT treatment. Asterisks indicate significant differences from media alone, while crosses indicate significant differences from toxin alone (<i>p</i> < 0.0003). (<b>D</b>) Low doses of TSST-1 (5 μg/explant) and AT (25 ng/explant) have an additive effect on IL-8 production that is reduced to basal levels in the presence of AG1478 (4 μg/explant) with no dextrin vehicle effect (10 μl of 15%). White bars indicate media alone, checkered bars represent TSST-1 treatment, striped bars represent AT treatment, black bars represent TSST-1 + AT treatment. Asterisk indicates significant difference from media, TSST-1 and AT alone (<i>p</i> < 0.03), while cross indicates significant difference from TSST-1 + AT (<i>p</i> < 0.0009).</p

Fusions of 5′ <i>selu</i> and <i>5</i>′ <i>seg</i> regions of the <i>egc</i> induce GFP expression in RN4220.

<p>GFP expression as measured by relative fluorescence units (RFU) indicates 5′ <i>selu</i> and 5′ <i>seg</i> induce expression of GFP in RN4220 indicating potential promoter elements within the <i>egc</i>. Data displayed are means ± SEM, **<i>P</i> ≤ 0.01, Mann-Whitney test. <i>P</i> ≤ 0.05 is considered statistically significant.</p