Roger Neill

Role of shiga-like toxin I in bacterial enteritis: Comparison between isogenic Escherichia coli strains induced in rabbits

BACKGROUND/AIMS Enteroadherent Escherichia coli that produce Shiga-like toxins are important causes of human disease, including enterohemorrhagic E. coli-induced colitis (EHEC). The role of Shiga-like toxins in these illnesses is unclear. The aim of this study was to establish an animal model for human EHEC and to determine the role of Shiga-like toxin I (SLT-I) in this model. METHODS E. coli strain RDEC-1 is an enteroadherent rabbit diarrheal pathogen. An isogenic variant of RDEC-1 (termed RDEC-H19A) producing high levels of SLT-I was obtained by infecting RDEC-1 with an SLT-I-converting bacteriophage. The effects of in vivo enteric infection produced in rabbits by RDEC-H19A were compared with those in uninfected and RDEC-1-infected animals. RESULTS SLT-I-producing RDEC-H19A induced a severe, noninvasive, enteroadherent infection in rabbits. Clinically, infection with RDEC-H19A was more severe than infection with RDEC-1 and caused more serious histological lesions including vascular changes, edema, and more severe inflammation. Interleukin 1 and platelet-activating factor appear to be important inflammatory mediators to this infection. CONCLUSIONS The illness induced by RDEC-H19A in rabbits resembled enterohemorrhagic E. coli-induced colitis of humans. SLT-I is an important virulence factor in the pathogenesis of EHEC.

The staphylococcal enterotoxins.

Abstract : Staphylococcal enterotoxins (SEs) constitute a family of related proteins whose biological toxicities include staphylococcal food poisoning (SFP), various skin disorders toxic shock syndrome (TSS), and possible involvement in autoimmune disorders. The SEs (named sequentially by letter) include SEA, B, C1, C2, C3, D, E, G, H, I, J and toxic shock syndrome toxin (TSST-1); their properties will be compared in throughout this discussion . They have been thoroughly characterized as super antigens, due to their massive impact on the immune system of the host, and their interaction with lymphoid cells has been widely studied. SE also have been shown to exert effects on endothelial cells, kidney proximal tubule cells, synovial cells, and human platelets.

TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY‐5Y cells

The light chain (LC) of botulinum neurotoxin B (BoNT/B) is unable to enter target neuronal cells by itself. It is brought into the cell in association with the BoNT/B heavy chain (HC) through endocytosis. The BoNT HC‐LC subunits are held together by a single disulfide bond. Intracellular reduction of this bond and separation of the two subunits activates the endopeptidase activity of the LC. This requirement suggests a strategy to prevent uptake by prophylactic reduction to disrupt the disulfide bond prior to endocytosis of the complex. We examined the utility of tris‐(2‐carboxyethyl)‐phosphine hydrochloride (TCEP), a relatively non‐toxic, non‐sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercapto‐containing reducing agents. We found that TCEP was as effective as DTT with maximal LC endopeptidase activation occurring at 1 mM, a concentration not toxic to the human neuronal cell line, SHSY‐5Y. In these cells, 1 mM TCEP maximally protected against BoNT/B inhibition of [3H]‐NA release, achieving 72% of the release from un‐intoxicated controls. This effect appears to be due to the sparing of SNARE proteins as the levels of VAMP‐2, the specific target of BoNT/B, were protected. These results show that TCEP disrupts the structure of BoNT/B by reduction of the LC and HC bridging disulfide bond and prevents neuronal intoxication. Since disulfide bond coupling between toxin subunits is a general motif for many toxins, e.g., ricin, snake venom, and all BoNT serotypes, this suggests that TCEP is a promising means to protect against these toxins by preventing cell penetration. J. Cell. Biochem. 107: 1021–1030, 2009. Published 2009 Wiley‐Liss, Inc.

The cellular and molecular immune response of the weanling piglet to staphylococcal enterotoxin B.

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.

Identification of staphylococcal enterotoxin B domains involved in binding to cultured human kidney proximal tubular cells: imparting proliferation and death.

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 [125I]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.

Early indicators of exposure to biological threat agents using host gene profiles in peripheral blood mononuclear cells

BackgroundEffective prophylaxis and treatment for infections caused by biological threat agents (BTA) rely upon early diagnosis and rapid initiation of therapy. Most methods for identifying pathogens in body fluids and tissues require that the pathogen proliferate to detectable and dangerous levels, thereby delaying diagnosis and treatment, especially during the prelatent stages when symptoms for most BTA are indistinguishable flu-like signs.MethodsTo detect exposures to the various pathogens more rapidly, especially during these early stages, we evaluated a suite of host responses to biological threat agents using global gene expression profiling on complementary DNA arrays.ResultsWe found that certain gene expression patterns were unique to each pathogen and that other gene changes occurred in response to multiple agents, perhaps relating to the eventual course of illness. Nonhuman primates were exposed to some pathogens and the in vitro and in vivo findings were compared. We found major gene expression changes at the earliest times tested post exposure to aerosolized B. anthracis spores and 30 min post exposure to a bacterial toxin.ConclusionHost gene expression patterns have the potential to serve as diagnostic markers or predict the course of impending illness and may lead to new stage-appropriate therapeutic strategies to ameliorate the devastating effects of exposure to biothreat agents.