A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response.

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak-/- mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak-/- and zak+/+ bone marrow-derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak-/- mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak-/- mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak-/- strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak-/- mice have decreased duodenal damage following in vivo ricin challenge. zak-/- mice demonstrate a distinct ribotoxic stress-associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling.

Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation.

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.

Shiga toxin type-2 (Stx2) induces glutamate release via phosphoinositide 3-kinase (PI3K) pathway in murine neurons.

Shiga toxin-producing Escherichia coli (STEC) can cause central nervous system (CNS) damage resulting in paralysis, seizures, and coma. The key STEC virulence factors associated with systemic illness resulting in CNS impairment are Shiga toxins (Stx). While neurons express the Stx receptor globotriaosylceramide (Gb3) in vivo, direct toxicity to neurons by Stx has not been studied. We used murine neonatal neuron cultures to study the interaction of Shiga toxin type 2 (Stx2) with cell surface expressed Gb3. Single molecule imaging three dimensional STochastic Optical Reconstruction Microscopy-Total Internal Reflection Fluorescence (3D STORM-TIRF) allowed visualization and quantification of Stx2-Gb3 interactions. Furthermore, we demonstrate that Stx2 increases neuronal cytosolic Ca(2+), and NMDA-receptor inhibition blocks Stx2-induced Ca(2+) influx, suggesting that Stx2-mediates glutamate release. Phosphoinositide 3-kinase (PI3K)-specific inhibition by Wortmannin reduces Stx2-induced intracellular Ca(2+) indicating that the PI3K signaling pathway may be involved in Stx2-associated glutamate release, and that these pathways may contribute to CNS impairment associated with STEC infection.

Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

Effects of Shiga toxin type 2 on a bioengineered three-dimensional model of human renal tissue.

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy, following infections by Shiga toxin-producing Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood renal failure worldwide. There are currently no specific therapies for STEC-associated HUS, and the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully representative animal models and an inability to monitor disease progression on a molecular or cellular level in humans at early stages. Three-dimensional (3D) tissue models have been shown to be more in vivo-like in their phenotype and physiology than 2D cultures for numerous disease models, including cancer and polycystic kidney disease. It is unknown whether exposure of a 3D renal tissue model to Stx will yield a more in vivo-like response than 2D cell culture. In this study, we characterized Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine interleukin-8 compared to the 2D cell cultures. This study represents the first application of 3D tissues for the study of Stx-mediated kidney injury.

Shiga toxin-producing Escherichia coli O104:H4: an emerging pathogen with enhanced virulence.

Pathogenic Escherichia coli are genetically diverse and encompass a broad variety of pathotypes, such as enteroaggregative E. coli (EAEC) or enterohemorrhagic E. coli (EHEC), which cause distinct clinical syndromes. The historically large 2011 German outbreak of hemolytic uremic syndrome (HUS), caused by a Shiga-toxin producing E. coli (STEC) of the serotype O104:H4, illustrated the emerging importance of non-O157 STEC. STEC O104:H4, with features characteristic of both enteroaggregative E. coli and enterohemorrhagic E. coli, represents a unique and highly virulent pathotype. The German outbreak both allowed for the evaluation of several potential therapeutic approaches to STEC-induced HUS and emphasizes the importance of early and specific detection of both O157 and non-O157 STEC.

Small molecule kinase inhibitors block the ZAK-dependent inflammatory effects of doxorubicin.

The adverse side effects of doxorubicin, including cardiotoxicity and cancer treatment-related fatigue, have been associated with inflammatory cytokines, many of which are regulated by mitogen-activated protein kinases (MAPKs). ZAK is an upstream kinase of the MAPK cascade. Using mouse primary macrophages cultured from ZAK-deficient mice, we demonstrated that ZAK is required for the activation of JNK and p38 MAPK by doxorubicin. Nilotinib, ponatinib and sorafenib strongly suppressed doxorubicin-mediated phosphorylation of JNK and p38 MAPK. In addition, these small molecule kinase inhibitors blocked the expression of IL-1ß, IL-6 and CXCL1 RNA and the production of these proteins. Co-administration of nilotinib and doxorubicin to mice decreased the expression of IL-1ß RNA in the liver and suppressed the level of IL-6 protein in the serum compared with mice that were injected with doxorubicin alone. Therefore, by reducing the production of inflammatory mediators, the inhibitors identified in the current study may be useful in minimizing the side effects of doxorubicin and potentially other chemotherapeutic drugs.

Shiga toxin 2-induced intestinal pathology in infant rabbits is A-subunit dependent and responsive to the tyrosine kinase and potential ZAK inhibitor imatinib.

Shiga toxin producing Escherichia coli (STEC) are a major cause of food-borne illness worldwide. However, a consensus regarding the role Shiga toxins play in the onset of diarrhea and hemorrhagic colitis (HC) is lacking. One of the obstacles to understanding the role of Shiga toxins to STEC-mediated intestinal pathology is a deficit in small animal models that perfectly mimic human disease. Infant rabbits have been previously used to study STEC and/or Shiga toxin-mediated intestinal inflammation and diarrhea. We demonstrate using infant rabbits that Shiga toxin-mediated intestinal damage requires A-subunit activity, and like the human colon, that of the infant rabbit expresses the Shiga toxin receptor Gb(3). We also demonstrate that Shiga toxin treatment of the infant rabbit results in apoptosis and activation of p38 within colonic tissues. Finally we demonstrate that the infant rabbit model may be used to test candidate therapeutics against Shiga toxin-mediated intestinal damage. While the p38 inhibitor SB203580 and the ZAK inhibitor DHP-2 were ineffective at preventing Shiga toxin-mediated damage to the colon, pretreatment of infant rabbits with the drug imatinib resulted in a decrease of Shiga toxin-mediated heterophil infiltration of the colon. Therefore, we propose that this model may be useful in elucidating mechanisms by which Shiga toxins could contribute to intestinal damage in the human.

Ricin and Shiga toxins: effects on host cell signal transduction.

Shiga toxins and ricin are potent inhibitors of protein synthesis. In addition to causing inhibition of protein synthesis, these toxins activate proinflammatory signaling cascades that may contribute to the severe diseases associated with toxin exposure. Treatment of cells with Shiga toxins and ricin have been shown to activate a number of signaling pathways including those associated with the ribotoxic stress response, Nuclear factor kappa B activation, inflammasome activation, the unfolded protein response, mTOR signaling, hemostasis, and retrograde trafficking. In this chapter, we review our current understanding of these signaling pathways as they pertain to intoxication by Shiga toxins and ricin.

Shiga toxin 2 and flagellin from shiga-toxigenic Escherichia coli superinduce interleukin-8 through synergistic effects on host stress-activated protein kinase activation.

Shiga toxins expressed in the intestinal lumen during infection with Shiga-toxigenic Escherichia coli must translocate across the epithelium and enter the systemic circulation to cause systemic (pathological) effects, including hemolytic uremic syndrome. The transepithelial migration of polymorphonuclear leukocytes in response to chemokine expression by intestinal epithelial cells is thought to promote uptake of Stx from the intestinal lumen by compromising the epithelial barrier. In the present study, we investigated the hypothesis that flagellin acts in conjunction with Shiga toxin to augment this chemokine expression. We investigated the relative contributions of nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling to transcription and translation of interleukin-8. Using reporter gene constructs, we showed that flagellin-mediated interleukin-8 gene transcription is heavily dependent on both NF-kappaB and extracellular signal-regulated kinase 1 and 2 (ERK-1/2) activation. In contrast, inhibition of p38 has no detectable effect on interleukin-8 gene transcription, even though flagellin-mediated activation of host p38 is critical for maximal interleukin-8 protein expression. Inhibition of MAPK-interacting kinase 1 suggests that p38 signaling affects the posttranscriptional regulation of interleukin-8 protein expression induced by flagellin. Cotreatment with Stx2 and flagellin results in a synergistic upregulation of c-Jun N-terminal protein kinases (JNKs), p38 activation, and a superinduction of interleukin-8 mRNA. This synergism was also evident at the protein level, with increased interleukin-8 protein detectable following cotreatment with flagellin and Stx2. We propose that flagellin, in conjunction with Shiga toxin, synergistically upregulates stress-activated protein kinases, resulting in superinduction of interleukin-8 and, ultimately, absorption of Stx into the systemic circulation.

Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways.

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum (ER) chaperone BiP (GRP78). In the present study, we have examined its capacity to trigger the three ER stress-signalling pathways in Vero cells. Activation of PKR-like ER kinase was demonstrated by phosphorylation of eIF2alpha, which occurred within 30 min of toxin treatment, and correlated with inhibition of global protein synthesis. Activation of inositol-requiring enzyme 1 was demonstrated by splicing of X-box-binding protein 1 mRNA, while activating transcription factor 6 activation was demonstrated by depletion of the 90 kDa uncleaved form, and appearance of the 50 kDa cleaved form. The rapidity with which ER stress-signalling responses are triggered by exposure of cells to SubAB is consistent with the hypothesis that cleavage by the toxin causes BiP to dissociate from the signalling molecules.

ZAK: a MAP3Kinase that transduces Shiga toxin- and ricin-induced proinflammatory cytokine expression.

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the alpha-sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen-activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP-2 (7-[3-fluoro-4-aminophenyl-(4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl))]-quinoline), a zipper sterile-alpha-motif kinase (ZAK)-specific inhibitor, blocks Stx2/ricin-induced SAPKinase activation. Treatment of cells with DHP-2 also blocks Stx2/ricin-mediated upregulation of the proinflammatory cytokine interleukin-8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N-terminus of ZAK diminishes Stx2/Ricin-induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKalpha and/or beta isoforms may act as potential therapeutic target(s) for treating Stx/ricin-associated illnesses. Furthermore, a small molecule inhibitor like DHP-2 may prove valuable in preventing the Stx/ricin-induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx-producing Escherichia coli and ricin.

Humoral and cellular immune responses in sheep immunized with a 22 kilodalton exported protein of Mycobacterium avium subspecies paratuberculosis.

An immunogenic 22 kilodalton exported Mycobacterium avium subspecies paratuberculosis (MAP) lipoprotein (P22) was previously identified, and found to belong to the LppX/LprAFG family of mycobacterial lipoproteins. N-terminal polyhistidine-tagged P22 was produced and purified from Escherichia coli. Antibody recognition of P22, and interferon-gamma (IFN-gamma) responses in vitro using blood from a sheep vaccinated with Neoparasec, confirmed its immunogenicity. To evaluate the immunogenicity of P22 in vivo, five sheep were immunized with a single dose containing 0.8 mg recombinant P22 protein in adjuvant. Blood was collected at 4, 13 and 29 weeks post-immunization (p.i.) and tested for anti-P22 antibodies and P22-specific IFN-gamma production. P22-specific antibodies were detected by Western blot analysis in all five Neoparasec-immunized sheep at the three time points. Three out of five P22-immunized sheep produced P22-specific antibodies for up to 13 weeks p.i., and two gave a response at 29 weeks p.i. Recombinant P22 was able to stimulate significant IFN-gamma production in blood of P22-immunized sheep at 13 and 29 weeks p.i. Recombinant P22 also elicited an IFN-gamma response in blood of sheep immunized with Neoparasec.

Comparison of cyanide-degrading nitrilases.

Recombinant forms of three cyanide-degrading nitrilases, CynD from Bacillus pumilus C1, CynD from Pseudomonas stutzeri, and CHT from Gloeocercospora sorghi, were prepared after their genes were cloned with C-terminal hexahistidine purification tags and expressed in Escherichia coli, and the enzymes purified using nickel-chelate affinity chromatography. The enzymes were compared with respect to their pH stability, thermostability, metal tolerance, and kinetic constants. The two bacterial genes, both cyanide dihydratases, were similar with respect to pH range, retaining greater than 50% activity between pH 5.2 and pH 8 and kinetic properties, having similar K(m) (6-7 mM) and V(max) (0.1 mmol min(-1) mg(-1)). They also exhibited similar metal tolerances. However, the fungal CHT enzyme had notably higher K(m) (90 mM) and V(max) (4 mmol min(-1) mg(-1)) values. Its pH range was slightly more alkaline (retaining nearly full activity above 8.5), but exhibited a lower thermal tolerance. CHT was less sensitive to Hg(2+) and more sensitive to Pb(2+) than the CynD enzymes. These data describe, in part, the current limits that exist for using nitrilases as agents in the bioremediation of cyanide-containing waste effluent, and may help serve to determine where and under what conditions these nitrilases may be applied.