Suppression of Starvation-Induced Autophagy by Recombinant Toxic Shock Syndrome Toxin-1 in Epithelial Cells

<div><p>Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced from <i>Staphylococcus aureus</i>, has been reported to bind directly to unknown receptor(s) and penetrate into non-immune cells but its function is unclear. In this study, we demonstrated that recombinant TSST-1 suppresses autophagosomal accumulation in the autophagic-induced HeLa 229 cells. This suppression is shared by a superantigenic-deficient mutant of TSST-1 but not by staphylococcal enterotoxins, suggesting that autophagic suppression of TSST-1 is superantigenic-independent. Furthermore, we showed that TSST-1-producing <i>S. aureus</i> suppresses autophagy in the response of infected cells. Our data provides a novel function of TSST-1 in autophagic suppression which may contribute in staphylococcal persistence in host cells.</p></div

rTSST-1 suppresses autophagy in nutrient-starved HeLa 229 cells.

<p>HeLa 229 cells were transfected with pEGFP-hLC3 or pEGFP-C2 (Mock). Effect of TSST-1 was observed in nutrient-rich (MEM) and nutrient-starvation (KRB) condition by addition of 10 µg/ml rTSST-1. At 6 h, GFP-LC3 puncta were assessed under confocal microscope (A). GFP-LC3 puncta were counted from 100 cells of 3 independent-experiments (B).</p

Suppression of LC3-II accumulation by rTSST-1 in the cells is superantigenic activity-independent.

<p>Autophagy in HeLa 229 cells was induced by nutrient-starvation (KRB) with or without lysosomal protease inhibitors and 10 µg/ml rTSST-1, mTSST-1, SEA, SEB or SEC. Cells in MEM were used control. At 4 h of induction, LC3-II was detected by Western blotting (A) and the intensity of LC3-II band was quantified (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113018#pone-0113018-g005" target="_blank">Figure 5C</a>. The data is provided as SD of at least 3-independent experiments.</p

rTSST-1 does not enhance autophagosome and lysosome fusion.

<p>HeLa 229 cells were transfected with pEGFP-hLC3. Autophagy was induced under nutrient-starvation condition for 0 and 6 h with or without the addition of 10 µg/ml rTSST-1. Lysosomes were immunostained with LAMP1, lysosomes and GFP-LC3 puncta were observed under confocal microscope (A). GFP-LC3 puncta and overlapping between GFP-LC3 and lysosomes were counted from 100 cells of 2 independent-experiments (B).</p

TSST-1-producing <i>S. aureus</i> suppresses autophagy.

<p>HeLa 229 cells were transfected with pEGFP-hLC3 plasmid and infected with <i>S. aureus</i> 834 or Δ<i>tst</i>. At 6 h of infection, <i>S. aureus</i> cells were immunostained as described in the Experimental procedure. GFP-LC3 and <i>S. aureus</i> cells were observed under confocal microscope (A). LC3-colocalized <i>S. aureus</i> spots are represented and indicated by upper inset and yellow arrowheads, respectively. LC3-free <i>S. aureus</i> spots are represented and indicated by lower inset and white arrowheads, respectively. GFP-LC3 puncta (B), <i>S. aureus</i> cells (C) and colocalization of <i>S. aureus</i> with GFP-LC3 (D) were analyzed from at least 100 cells of 3 independent-experiments.</p

Lysosomal protease inhibitors fail to restore autophagosomes in TSST-1-treated cells.

<p>HeLa 229 cells were transfected with pEGFP/hLC3 and autophagy was induced under nutrient-starvation with or without the addition of 10 µg/ml rTSST-1 and lysosomal protease inhibitors. At the indicating time, the autophagosomal accumulation in the cells was observed by GFP puncta under confocal microscope (A). GFP-LC3 puncta at 4 h were counted from 100 cells of 2 independent-experiments (B).</p

Suppression of autophagy by rTSST-1 is observed by immunostaining and electron microscopy.

<p>Autophagy in HeLa 229 cells was observed in nutrient-rich (MEM) or nutrient-starvation (KRB) condition containing lysosomal protease inhibitors with or without the addition of 10 µg/ml rTSST-1. At 4 h, the cells were fixed and washed. (A) Autophagosomes were stained with anti-LC3 antibody and rhodamine-conjugated anti-rabbit IgG, and then observed under confocal microscope. (B) LC3 puncta were counted from 100 cells of 3 independent-experiments. (C) Autophagosomes were observed under electron microscope. AP indicates autophagosome-like vacuole.</p

Histamine release from intestinal mast cells induced by staphylococcal enterotoxin A (SEA) evokes vomiting reflex in common marmoset.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are known as causative agents of emetic food poisoning. We previously demonstrated that SEA binds with submucosal mast cells and evokes mast cell degranulation in a small emetic house musk shrew model. Notably, primates have been recognized as the standard model for emetic assays and analysis of SE emetic activity. However, the mechanism involved in SEA-induced vomiting in primates has not yet been elucidated. In the present study, we established common marmosets as an emetic animal model. Common marmosets were administered classical SEs, including SEA, SEB and SEC, and exhibited multiple vomiting responses. However, a non-emetic staphylococcal superantigen, toxic shock syndrome toxin-1, did not induce emesis in these monkeys. These results indicated that the common marmoset is a useful animal model for assessing the emesis-inducing activity of SEs. Furthermore, histological analysis uncovered that SEA bound with submucosal mast cells and induced mast cell degranulation. Additionally, ex vivo and in vivo pharmacological results showed that SEA-induced histamine release plays a critical role in the vomiting response in common marmosets. The present results suggested that 5-hydroxytryptamine also plays an important role in the transmission of emetic stimulation on the afferent vagus nerve or central nervous system. We conclude that SEA induces histamine release from submucosal mast cells in the gastrointestinal tract and that histamine contributes to the SEA-induced vomiting reflex via the serotonergic nerve and/or other vagus nerve

Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases