Dakshina M. Jandhyala

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

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 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 eIF2α, 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 α‐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‐α‐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, ZAKα and/or β 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.

CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies.

ABSTRACT The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has Km and Vmax values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B. pumilus (strain 8A3) was cloned and expressed. This suggests that cynD, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B. pumilus and is not a reflection of its origin at a mining waste site.

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-1β, IL-6 and CXCL1 RNA and the production of these proteins. Co-administration of nilotinib and doxorubicin to mice decreased the expression of IL-1β 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.

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.

The Operon Encoding SubAB, a Novel Cytotoxin, Is Present in Shiga toxin-producing Escherichia coli Isolates from the United States

Diarrhea-associated hemolytic uremic syndrome (D+HUS) is thought to result from endothelial cell damage following gastrointestinal infection with Shiga toxin-producing Escherichia coli (STEC) ([10][1]); Shiga toxin (Stx) is regarded as the main virulence factor causing these microangiopathic

Shiga Toxin 2 and Flagellin from Shiga-Toxigenic Escherichia coli Superinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation

ABSTRACT 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 κB (NF-κB) 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-κB 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.

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 Gb3. 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.

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.

Prevalence of the Operon Encoding Subtilase Cytotoxin in Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Humans in the United States

Shiga toxin-producing Escherichia coli (STEC) causes diarrhea and can lead to hemolytic uremic syndrome (HUS). In addition to Shiga toxin (Stx), gene products encoded by the l ocus of e nterocyte e ffacement (LEE) pathogenicity island are well-known virulence factors. This gene cluster is present in