Search

Background: Participation of renal cells in the pathogenesis of staphylococcal enterotoxin B (SEB) is critical for late cleansing and sequestration of the antigens facilitated by CD1d mediated antigen sensing and recognition. This is a noted deviation from the typical antigen recognition process that recruits the major histocompatibility complex class II (MHC II) molecules. The immunological importance of CD1d is underscored by its influences on the performances of natural killer T-cells and thereby mediates the innate and adaptive immune systems., Results: Using diffraction-based dotReady™ immunoassays, the present study showed that SEB directly and specifically conjugated to CD1d. The specificity of the conjugation between SEB and CD1d expressed on human renal proximal tubule epithelial cells (RPTEC) was further established by selective inhibition of CD1d prior to its exposure to SEB. We found that SEB induced the expression of CD1d on the cell surface prompting a rapid conjugation between them. The mRNA transcripts encoding CD1d remained elevated potentially after completing the antigen cleansing process., Conclusion: Molecular targets associated with the delayed pathogenic response have essential therapeutic values. Particularly in the event of bioterrorism, the caregivers are typically able to intervene much later than the toxic exposures. Given circumstances mandate a paradigm shift from the conventional therapeutic strategy that counts on targeting the host markers responding to the early assault of pathogens. We demonstrated the role of CD1d in the late stage of pathogen recognition and cleansing, and thereby underscored its clinical potential in treating bioweaponizable antigens, such as Staphylococcal enterotoxin B (SEB).

Bacterial infection in burn patients is still a devastating contributor to morbidity and mortality. Little is known regarding the presence of staphylococcal toxins in the burn-injured patient. The aim of this study was to characterize the prevalence of several of these toxins and their relationship to clinical metrics and mortality in burn patients. Levels of exotoxins staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B, toxic shock syndrome toxin 1 (TSST-1), and α-hemolysin were assayed from the serum of 207 adult burn patients aged 16-92 years. Clinical, demographic, and microbiological data from these patients were then compared to toxin levels. Staphylococcal exotoxins α-hemolysin and SEA were present in 45% and 25% of the population, respectively. Bacterial cultures concomitantly showed a high prevalence of Staphylococcus aureus in 48% of patients, of which 59% were methicillin resistant. Several metrics may be predictive of high toxin concentrations of α-hemolysin and TSST-1 and SEA including burn size, length of stay, and bacteremia. Mortality associations indicated that burn size, bacteremia, age, and the presence of α-hemolysin and SEA may be predictors of mortality. A high prevalence of staphylococcal toxin α-hemolysin and superantigens TSST-1 and SEA can be found in the circulation of the adult burn population. The presence of these toxins may contribute to the morbidity and mortality of the burn patient., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Staphylococcal enterotoxin B (SEB) is a biothreat agent, etiologic agent of food poisoning, and potent inducer of toxic shock syndrome. This heat-stable exoprotein is thought to act as a superantigen to induce T cell-specific pathology. Most animal models do not accurately map the clinical syndrome of human SEB exposure. Previously, we have demonstrated the utility of the weanling piglet model of SEB intoxication. Here, we analyze gross and histopathologic specimens from lymphoid tissue of these animals. Hematological testing was completed to observe changes in circulating leukocytes. Further, these leukocytes were differentiated and the subsets were subsequently analyzed using flow cytometry. Cytokine mRNA was quantified in lymphoid tissue and peripheral blood cells and compared to actual protein concentration using ELISA. The mRNA expression levels for several cell markers implicated in T and B cell differentiation were quantified and compared to control animals, as were levels for apoptosis-related genes. Lymphadenopathy was constantly seen post mortem. SEB-exposed animals had a leukocytosis which increased linearly over the time course. Monocyte levels increased over time, while lymphocyte levels peaked at 6h and then returned to baseline. Most cytokines had mRNA levels that were upregulated after exposure. Detection of serum cytokine changes was accomplished; however, these patterns did not always follow those seen in the differentially expressed genes. Both pro- and anti-apoptotic genes were differentially expressed in exposed animals. This paper reports, for the first time, the immunological findings in the weanling piglet model of SEB intoxication. From this work it is clear that there is not one absolute cell-mediated pathway contributing to the pathology these animals exhibit as a result of SEB exposure.

Staphylococcal enterotoxin B (SEB) is a toxic shock-inducing agent produced by Staphylococcus aureus. The hallmark of SEB-induced lethal shock is acute vasodilation leading to severe hypotension. Animal studies reveal that approximately 70% of intravenously administered toxin localizes to renal proximal tubule epithelial cells (RPTEC). This evidence, together with the well-documented role of the kidney in regulation of vascular tone, suggests that molecular events induced in RPTEC by SEB may contribute to the blood pressure dysregulation seen in enterotoxic shock. In an attempt to elucidate these molecular mechanisms, differential display was performed on SEB-treated and untreated RPTEC, and 32 differentially expressed transcripts (DETs) were identified. One of the down-regulated DETs matched the sequence for Rnd3, which normally inhibits Rho protein function. Consistent with Rnd3 down-regulation, message for RhoA was shown to increase upon SEB exposure, and actin stress fiber formation was dramatically increased. Further, SEB-exposed cells showed both increased enzymatic activity of caspase-3 and an increase in the percentage of apoptotic cells. Taken together, these results support the hypothesis that RPTEC undergo apoptosis upon exposure to SEB. Furthermore, these data implicate the involvement of the Rho family proteins in the molecular signaling pathway induced by SEB in RPTEC.

Objectives: The purpose of this study was to identify enterotoxin genes from isolates of coagulase-negative staphylococci and coagulase-positive staphylococci obtained from dairy products, responsible for 16 outbreaks of food poisoning., Methods: From the pool of 152 staphylococcal isolates, 15 coagulase-negative and 15 coagulase-positive representatives were selected for this study. The 15 coagulase-negative isolates were tested for the presence of coa and femA genes, which are known to be characteristic of Staphylococcus aureus. After testing for enterotoxin genes by polymerase chain reaction (PCR), the 30 selected isolates were tested for the presence of toxin by immunoassay., Results: Seven of the coagulase-negative isolates amplified the coa gene and were subsequently reclassified as coagulase-positive. Twenty-one of 30 selected isolates had staphylococcal enterotoxin genes and most of these produced toxin as well. The most frequently encountered enterotoxin genes were sea and seb. Among eight coagulase-negative isolates, five had enterotoxin genes, all of which were found to have detectable toxin by immunoassay., Conclusions: The results from this study demonstrate that coagulase-negative as well as coagulase-positive staphylococci isolated from dairy products are capable of genotypic and phenotypic enterotoxigenicity. Furthermore, these data demonstrate that PCR is a sensitive and specific method for screening outbreak isolates regardless of coagulase expression.

Studies suggest that staphylococcal enterotoxin B (SEB) is initially harbored in the kidney by binding to digalactosylceramide molecules in the proximal tubular cells. However, little is known in regard to the peptide motif within SEB that binds to these cells and imparts toxic effects. Herein, using human kidney proximal tubular cells (PTs) we have performed a systematic study on the binding of various peptides and peptide analogs of SEB and demonstrate a structure-functional relationship. Using [(125)I]labeled SEB peptides, we show a high affinity and displaceable binding of SEB 191-220 to human PT cells. Binding was mitigated by the use of antibody against SEB, by digalactosylceramide (the putative receptor), and by the use of endoglycoceramidase, which selectively removes the oligosaccharide backbones from glycosphingolipids. Our structure/ functional studies revealed that peptide 130-160 induces a concentration-dependent increase in programmed cell death/ apoptosis in human proximal tubular cells. Mechanistic studies further suggest that SEB/SEB peptide (130-160) impart apoptosis via the activation of neutral sphingomyelinase, which hydrolizes sphingomyelin to ceramide and phosphocholine. SEB 130-160 mediated apoptosis was mitigated by preincubation of cells with antibody against SEB and an SEB 130-160 antibody.

Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.

Staphylococcal enterotoxin (SE) B causes serious gastrointestinal illness, and intoxication with this exotoxin can lead to lethal toxic shock syndrome. In order to overcome significant shortcomings of current rodent and nonhuman primate models, we developed a piglet model of lethal SEB intoxication. Fourteen-day-old Yorkshire piglets were given intravenous SEB, observed clinically, and sacrificed at 4, 6, 24, 48, 72, or 96 hrs posttreatment. Clinical signs were biphasic with pyrexia, vomiting, and diarrhea within 4 hrs, followed by terminal hypotension and shock by 96 hrs. Mild lymphoid lesions were identified as early as 24 hrs, with severe lymphadenopathy, splenomegaly, and prominent Peyer's patches found by 72 hrs. Widespread edema-most prominent in the mesentery, between loops of spiral colon, and in retroperitoneal connective tissue-was found in animals at 72 hrs. Additional histologic changes included perivascular aggregates of large lymphocytes variably present in the lung and brain, circulating lymphoblasts, and lymphocytic portal hepatitis. Preliminary molecular investigation using gene array has uncovered several gene profile changes that may have implications in the pathophysiology leading to irreversible shock. Five genes were selected for further study, and all showed increased mRNA levels subsequent to SEB exposure. The use of this piglet model will continue to elucidate the pathogenesis of SEB intoxication and facilitate the testing of new therapeutic regimens that may better correlate with human lesions.

Staphylococcal enterotoxins (SE) are exoproteins produced by Staphylococcus aureus that act as superantigens and have been implicated as a leading cause of food-borne disease and toxic shock. Little is known about how these molecules penetrate the gut lining and gain access to both local and systemic immune tissues. To model movement in vitro of staphylococcal enterotoxins, we have employed a monolayer system composed of crypt-like human colonic T-84 cells. SEB and SEA showed comparable dose-dependent transcytosis in vitro, while toxic shock syndrome toxin (TSST-1) exhibited increased movement at lower doses. Synthetic peptides corresponding to specific regions of the SEB molecule were tested in vitro to identify the domain of the protein involved in the transcytosis of SE. A toxin peptide of particular interest contains the amino acid sequence KKKVTAQELD, which is highly conserved across all SE. At a toxin-to-peptide ratio of 1:10, movement of SEB across the monolayers was reduced by 85%. Antisera made against the SEB peptide recognized native SEB and also inhibited SEB transcytosis. Finally, the conserved 10-amino-acid peptide inhibited transcytosis of multiple staphylococcal enterotoxins, SEA, SEE, and TSST-1. These data demonstrate that this region of the staphylococcal enterotoxins plays a distinct role in toxin movement across epithelial cells. It has implications for the prevention of staphylococcal enterotoxin-mediated disease by design of a peptide vaccine that could reduce systemic exposure to oral or inhaled superantigens. Since the sequence identified is highly conserved, it allows for a single epitope blocking the transcytosis of multiple SE.

Staphylococcus aureus, a gram-positive bacterium, causes toxic shock through the production of superantigenic toxins (sAgs) known as Staphylococcal enterotoxins (SE), serotypes A-J (SEA, SEB, etc.), and toxic shock syndrome toxin-1 (TSST-1). The chronology of host transcriptomic events that characterizes the response to the pathogenesis of superantigenic toxicity remains uncertain. The focus of this study was to elucidate time-resolved host responses to three toxins of the superantigenic family, namely SEA, SEB, and TSST-1. Due to the evolving critical role of melanocytes in the host's immune response against environmental harmful elements, we investigated herein the transcriptomic responses of melanocytes after treatment with 200 ng/mL of SEA, SEB, or TSST-1 for 0.5, 2, 6, 12, 24, or 48 h. Functional analysis indicated that each of these three toxins induced a specific transcriptional pattern. In particular, the time-resolved transcriptional modulations due to SEB exposure were very distinct from those induced by SEA and TSST-1. The three superantigens share some similarities in the mechanisms underlying apoptosis, innate immunity, and other biological processes. Superantigen-specific signatures were determined for the functional dynamics related to necrosis, cytokine production, and acute-phase response. These differentially regulated networks can be targeted for therapeutic intervention and marked as the distinguishing factors for the three sAgs. [ABSTRACT FROM AUTHOR]