Apoptosis observed in murine peritoneal macrophages treated with interferon gamma through staphylococcal enterotoxin-dependent cell-mediated cytotoxicity

The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon gamma. Interferon gamma-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.     

Rabbit enterotoxaemia: Purification and preliminary characterisation of a toxin produced by Clostridium spiroforme

Abstract A culture filtrate of Clostridium spiroforme grown in a brain-heart-infusion peptone medium at 37°C for 24 h was concentrated by ultrafiltration, and the toxic fraction separated by ion exchange chromatography. This material was dermonecrotic in depilated guinea pigs, lethal in mice, enterotoxic in rabbit ileal loops and infant mice and cytophathic for HeLa cells. All activity was neutralised by Clostridium perfringens Type E antitoxin. Analysis by size exclusion high-performance liquid chromatography produced a single symmetrical peak corresponding to an M _r of 41 000 to 42 000.     

A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE‐like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)‐PCR analysis method established. LA‐PCR products of 13–17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb‐harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq‐harboring SaPIj11 and non‐superantigen‐harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA‐PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.     

Cholera toxin: A paradigm for multi-functional engagement of cellular mechanisms (Review)

Cholera toxin (Ctx) from Vibrio cholerae and its closely related homologue, heat-labile enterotoxin (Etx) from Escherichia coli have become superb tools for illuminating pathways of cellular trafficking and immune cell function. These bacterial protein toxins should be viewed as conglomerates of highly evolved, multi-functional elements equipped to engage the trafficking and signalling machineries of cells. Ctx and Etx are members of a larger family of A-B toxins of bacterial (and plant) origin that are comprised of structurally and functionally distinct enzymatically active A and receptor-binding B sub-units or domains. Intoxication of mammalian cells by Ctx and Etx involves B pentamer-mediated receptor binding and entry into a vesicular pathway, followed by translocation of the enzymatic A1 domain of the A sub-unit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase and a sequence of events culminating in life-threatening diarrhoeal disease. Importantly, Ctx and Etx also have the capacity to induce a wide spectrum of remarkable immunological processes. With respect to the latter, it has been found that these toxins activate signalling pathways that modulate the immune system. This review explores the complexities of the cellular interactions that are engaged by these bacterial protein toxins, and highlights some of the new insights to have recently emerged.     

Abl-SH3 binding protein 2, 3BP2, interacts with CIN85 and HIP-55

The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.     

Mesenchymal stem cells preconditioned by staphylococcal enterotoxin B enhance survival and bacterial clearance in murine sepsis model

Sepsis, a health-threatening progressive infectious disease, is the major cause of morbidity and mortality worldwide. Cell therapy using mesenchymal stromal cells (MSCs) is an innovative strategy with excessive therapeutic potential in the treatment of sepsis. Staphylococcal enterotoxin B (SEB) preconditioning aims to prolong the interval of survival of transplanted MSCs which induces the production of cytoprotective agents, anti-apoptotic and anti-inflammatory factors. The MSCs were preconditioned with an optimum dose of SEB (470 μmol/L). The expression levels of apoptosis genes and antibacterial activity of MSC and SEB-MSC and their conditioned medium (CM), as well as cell survival, were studied in vitro in an oxidative stress and serum deprivation condition. Following treatment of the septic mice with MSCs and SEB-MSCs, pro/anti-inflammatory cytokines, hematological factors, bacterial clearance and animal survival were assessed. The apoptotic and pro-inflammatory cytokine's genes expression was down-regulated while antibacterial peptides and anti-inflammatory cytokines were up-regulated in SEB-MSC–treated mice. The animal survival rates were improved; bacterial clearance was enhanced in the peritoneal fluids, blood and organs; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were reduced in blood, compared with saline and MSCs alone. This research concludes that transplantation of SEB-MSCs presents improved therapeutic effects on a live bacterial model of sepsis.     

Inhibition of Escherichia coli heat-labile enterotoxin by neoglycoprotein and anti-lectin antibodies which mimic GM1 receptor

Escherichia coli producing heat-labile enterotoxin is responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of this toxin are responsible for the binding to the receptor, the complex ganglioside GM1 which has galactose as its terminal sugar. In this study we showed that analogs of galactose (gal) and N-acetylgalactosamine (GalNAc) interfere with the binding of heat-labile toxin to GM1. Antibodies to lectins which mimic sugar structures and neoglycoprotein were employed. These compounds were able to inhibit heat-labile toxin activity efficiently in Vero cells: 37 microg of IgG-enriched fraction from an antiserum inhibited up to 70% of this activity, and 50% of the binding of heat-labile toxin to GM1. Neoglycoprotein was more efficient than antibodies, since 2.5 microg of this ligand completely abolished the activity of heat-labile toxin on Vero cells. These data suggest that these molecules could be developed for prophylaxis and diagnosis of diarrhea caused by heat-labile toxin.     

Actin cross-linking domain of Aeromonas hydrophila repeat in toxin A (RtxA) induces host cell rounding and apoptosis

The repeat in toxin (Rtx) of an environmental isolate ATCC 7966 of Aeromonas hydrophila consists of six genes (rtxACHBDE) organized in an operon similar to the gene organization found for the Rtx of the Vibrio species. The first gene in this operon (rtxA) encodes an exotoxin in vibrios, while other genes code for proteins needed for proper activation of RtxA and in secretion of this toxin from Vibrio cholerae. However, the RtxA of ATCC 7966, as well as from the clinical isolate SSU of A. hydrophila, was exclusively expressed and produced during co-infection of this pathogen with the host, e.g., HeLa cells, indicating that rtxA gene expression required host cell contact. Within the RtxA, an actin cross-linking domain (ACD) exists and to investigate the functionality of this domain, several truncated versions of ACD were generated to discern its minimal biological active region. Such genetically modified genes encoding ACD, which were truncated on either the NH(2) or the COOH terminal, as well as on both ends, were expressed from a bidirectional promoter of the pBI-enhanced green fluorescent protein (EGFP) vector in a HeLa-Tet-Off cell system. We demonstrated that only the full-length ACD of RtxA from A. hydrophila catalyzed the covalent cross-linking of the host cellular actin, whereas the ACD truncated on the NH(2), COOH or both ends did not exhibit such actin cross-linking characteristics. Further, we showed that the full-length ACD of A. hydrophila RtxA disrupted the actin cytoskeleton of HeLa cells, resulting in their rounding phenotype. Finally, our data provided evidence that the full-length ACD of RtxA induced host cell apoptosis. Our study is the first to report that A. hydrophila possesses a functional RtxA having an ACD that contributes to the host cell apoptosis, and hence could represent a potential virulence factor of this emerging human pathogen.