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Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Cytosolic and organelle-based heat-shock protein (HSP) chaperones ensure proper folding and function of nascent and injured polypeptides to support cell growth. Under conditions of cellular stress, including oncogenic transformation, proteostasis components maintain homeostasis and prevent apoptosis. Although this cancer-relevant function has provided a rationale for therapeutically targeting proteostasis regulators (e.g., HSP90), cancer-subtype dependencies upon particular proteostasis components are relatively undefined. Here, we show that human rhabdomyosarcoma (RMS) cells, but not several other cancer cell types, depend upon heat-shock protein 70 kDA (HSP70) for survival. HSP70-targeted therapy (but not chemotherapeutic agents) promoted apoptosis in RMS cells by triggering an unfolded protein response (UPR) that induced PRKR-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor α (eIF2α)-CEBP homologous protein (CHOP) signaling and CHOP-mediated cell death. Intriguingly, inhibition of only cytosolic HSP70 induced the UPR, suggesting that the essential activity of HSP70 in RMS cells lies at the endoplasmic reticulum-cytosol interface. We also found that increased CHOP mRNA in clinical specimens was a biomarker for poor outcomes in chemotherapy-treated RMS patients. The data suggest that, like human epidermal growth factor receptor 2 (HER2) amplification in breast cancer, increased CHOP in RMS is a biomarker of decreased response to chemotherapy but enhanced response to targeted therapy. Our findings identify the cytosolic HSP70-UPR axis as an unexpected regulator of RMS pathogenesis, revealing HSP70-targeted therapy as a promising strategy to engage CHOP-mediated apoptosis and improve RMS treatment. Our study highlights the utility of dissecting cancer subtype-specific dependencies on proteostasis networks to uncover unanticipated cancer vulnerabilities.

Protein folding by the endoplasmic reticulum (ER) is physiologically critical; its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here, we report that unmitigated ER stress promoted apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, which allowed time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2010.02.003 Byline: Travis Beddoe (1), Adrienne W. Paton (2), JerA[acute accent]me Le Nours (1), Jamie Rossjohn (1), James C. Paton (2) Abstract: AB.sub.5 toxins are important virulence factors for several major bacterial pathogens, including Bordetella pertussis, Vibrio cholerae, Shigella dysenteriae and at least two distinct pathotypes of Escherichia coli. The AB.sub.5 toxins are so named because they comprise a catalytic A-subunit, which is responsible for disruption of essential host functions, and a pentameric B-subunit that binds to specific glycan receptors on the target cell surface. The molecular mechanisms by which the AB.sub.5 toxins cause disease have been largely unravelled, including recent insights into a novel AB.sub.5 toxin family, subtilase cytotoxin (SubAB). Furthermore, AB.sub.5 toxins have become a valuable tool for studying fundamental cellular functions, and are now being investigated for potential applications in the clinical treatment of human diseases. Author Affiliation: (1) The Protein Crystallography Unit, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia (2) Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia

Shiga-toxigenic Escherichia coli (STEC) colonizing the bowel are exposed to a variety of shortchain fatty acids (SCFAs), including acetate, propionate and butyrate, produced by gut microfiora. However, the total concentrations and relative amounts of SOFAs in the lumen vary with intestinal niche. Here we report that conditions simulating SCFA concentrations present in the human gut trigger expression of the iha gene, which encodes an adherence-conferring outer-membrane protein of pathogenic E. coli. We show that growth under conditions simulating colonic, but not ileal, SCFA concentrations increases iha expression in three tested STEC strains, with the strongest expression detected in LEE-negative STEC O113:H21 strain 98NK2. Expression of iha is known to be subject to Fur-mediated iron repression in O157:H7 STEC, and the same occurs in 98NK2. However, exogenous iron did not repress iha expression in the presence of colonic SCFAs in either 98NK2 or the O157:H7 strain EDL933. Moreover, exposure to the iron chelator 2,2'-dipyridyl caused no further enhancement of iha expression over that induced by colonic SCFAs. These findings indicate that SOFAs regulate iha expression in STEC independently of iron. Increased expression of iha under colonic but not ileal SCFA conditions possibly may contribute to preferential colonization of the human colon by STEC. DOI 10.1099/mic.0.029454-0

Most Shiga toxin-producing Escherichia coli (STEC) infections that are associated with severe sequelae such as hemolytic uremic syndrome (HUS) are caused by attaching and effacing pathogens that carry the locus [...]

We found that, in murine podocytes, expression of monocyte chemoattractant protein 1 (MCP-1) in response to TNF-[alpha] was suppressed by indomethacin but not by ibuprofen. This anti-inflammatory potential was correlated with induction of 78-kDa glucose-regulated protein (GRP78), a marker of unfolded protein response (UPR). Indomethacin, but not ibuprofen, also triggered expression of CHOP, another endogenous indicator of UPR, as well as repression of endoplasmic reticulum stress-responsive alkaline phosphatase, an exogenous indicator of UPR. Like ibuprofen, other nonsteroidal anti-inflammatory drugs including aspirin and sulindac also did not induce UPR, indicating that the induction of UPR by indomethacin was independent of cyclooxygenase inhibition. The induction of UPR by indomethacin was observed similarly in other cells including mesangial cells and tubular epithelial cells. In tumor necrosis factor (TNF)-[alpha]-treated cells, suppression of MCP-1 by indomethacin was inversely correlated with induction of UPR, and other inducers of UPR including tunicamycin, thapsigargin, and A23187 reproduced the suppressive effect. Reporter assays showed that indomethacin as well as thapsigargin attenuated activation of NF-[kappa]B by TNF-[alpha] and it was associated with enhanced degradation of TNF receptor-associated factor 2 (TRAF2) and blunted degradation of I[kappa]B[beta]. Subsequent experiments revealed that acute ablation of GRP78 protein by AB5 subtilase cytotoxin caused reinforcement of MCP-1 induction and NF-[kappa]B activation by TNF-[alpha] and that transfection with GRP78 significantly suppressed the cytokine-induced activation of NF-[kappa]B. These results suggested that indomethacin suppressed the response of podocytes to TNF-[alpha] via UPR and that UPR-triggered induction of GRP78 and degradation of TRAF2 may be responsible, at least in part, for the suppressive effect of indomethacin. monocyte chemoattractant protein 1; nuclear factor-[kappa]B; tumor necrosis factor-[alpha] unfolded protein response; 78-kDa glucose-regulated protein

Author(s): Adrienne W. Paton (corresponding author) [1]; Travis Beddoe [2]; Cheleste M. Thorpe [3]; James C. Whisstock [2]; Matthew C. J. Wilce [2]; Jamie Rossjohn [2]; Ursula M. Talbot [1]; [...]