Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 151-169).

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.

Cholera toxin (CT) is a classic A-B type protein toxin that has an A subunit (A1 + A2) and a pentameric B subunit. The catalytic A1 domain is linked to the A2 domain via a disulfide linkage. CTA1 must be dissociated from the rest of the toxin to cause a cytopathic effect. Protein disulfide isomerase (PDI) can reduce the CTA1/CTA2 disulfide bond, but disassembly of the reduced toxin requires the partial unfolding of PDI that occurs when it binds to CTA1. This unfolding event allows PDI to push CTA1 away from the rest of the toxin. My research question is whether the efficiency of PDI in disassembling CT would be affected by molecular crowding, where a dense internal cell environment is recreated in vitro by the use of chemical agents such as Ficoll. This will give insight on how CT behaves inside a cell. Our hypothesis was that molecular crowding would make CTA1 disassembly more efficient by recreating the tight packing of macromolecules in cells, which provides an extra nudge to enhance toxin disassembly. We then used enzyme-linked immunosorbent assays (ELISAs), a pull-down assay and a biochemical assay to determine how molecular crowders affect the binding, reduction, and disassembly of CT by PDI. Our results will bring about a deeper understanding of the cellular events that may affect the course of a cholera infection. From the preliminary results, molecular crowders increased PDI's ability to bind to CTA1 and did not prevent PDI from cleaving the CTA1/CTA2 disulfide bond. Based off the disassembly results, molecular crowders reduced PDI's ability to displace CTA1 from the rest of the toxin. This contradicts our original hypothesis. Our new hypothesis is that crowders block PDI unfolding, which is required for CT disassembly. Biophysical experiments using Fourier Transform Infrared Spectroscopy will test this prediction in future work.

Cholera, caused by the secretion of cholera toxin (CT) by Vibrio cholerae within the intestinal lumen, triggers massive secretory diarrhea which may lead to life-threatening dehydration. CT is an AB5-type protein toxin that is comprised of an enzymatically active A1 chain, an A2 linker, and a cell-binding B pentamer. Once secreted, the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) of a host target cell. To cause intoxication, CTA1 must be displaced from CTA2/CTB5 in the ER and is then transferred to the cytosol where it induces a diarrheal response by stimulating the efflux of chloride ions into the intestinal lumen. Protein disulfide isomerase (PDI), a resident ER oxidoreductase and chaperone, is involved in detaching CTA1 from the holotoxin. The PDI domain(s) that binds to CTA1 and precisely how this interaction is involved in CTA1 dissociation from the holotoxin are unknown. The goal of this project is to identify which domain(s) of PDI is responsible for binding to and dislodging CTA1 from the CT holotoxin. Through incorporation of ELISA, surface plasmon resonance (SPR), and Fourier transform infrared (FTIR) spectroscopy techniques in conjunction with a panel of purified PDI deletion constructs, this project aims to provide important molecular insight into a crucial interaction of the CT intoxication process.

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (M.tb), remains a global health problem and although BCG offers some protection against childhood disseminated disease and other mycobacterial infections, its efficacy against pulmonary TB varies between 0 and 80%. Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel TB vaccine designed to boost mycobacterium-specific CD4+ T cell response primed by BCG. MVA85A induces strong interferon (IFN)-γ responses, a cytokine known to be essential for protection following M.tb infection. A strong IFN-γ response is not a correlate of protection and in terms of the adaptive response, interleukin (IL)-17 is emerging as an important cytokine following vaccination as it is thought to help boost IFN-γ production by CD4+ T cells. This thesis shows that MVA85A induces IL-17 in PBMC and whole blood of human BCG – MVA85A vaccinees. This is replicated in mice receiving BCG – MVA85A intranasally. The administration of cholera toxin (CT) with BCG enhances IL-17 and confers improved protection following M.tb challenge, which is partially dependent on IL-17 and on the mucosal route of administration. Since CT is not a suitable adjuvant in humans, an alternative IL-17-inducing pathway was investigated. In human BCG – MVA85A-vaccinated volunteers, blocking the hydrolysing ability of the CD39, an apyrase responsible for hydrolysing pro-inflammatory ATP, enhances IL-17 production. Challenge of BCG – MVA85A-vaccinated CD39-/- mice with M.tb slightly improved the protective capacity of the vaccine, suggesting that a pathway dependent on ATP-driven inflammation may be a target for improving the immunogenicity of a vaccine against M.tb disease. Overall, this thesis has confirmed the important role of IL-17 in vaccine-induced protection against TB disease and identifies a possible target pathway for improvement of a novel vaccine.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce D. Schultz / The first aim of this project is to define the cellular mechanisms that account for the low Na[superscript]+ concentration in human milk. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short circuit current (I[subscript]sc), suggesting activity of the epithelial Na[superscript]+ channel, ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride sensitive I[subscript]sc at all time points tested, an effect that is not reduced with Ctx washout for 12 hours or by cytosolic pathways inhibitors. Ctx increases the abundance of both beta and gamma-ENaC in the apical membrane and increases its monoubiquitination but without changing total protein and mRNA levels. Additionally, Ctx increases the levels of both the phosphorylated and the nonphosphorylated forms of Nedd4-2, a ubiquitin-protein ligase that regulates ENaC degradation. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na[superscript]+ transport. The second project aim is to develop a protocol to isolate mammary gland epithelia for subsequent in vitro culture. Caprine (1[superscript]0CME) and bovine mammary epithelia (1[superscript]0BME) were isolated and cultured on permeable supports to study hormone- and neurotransmitter-sensitive ion transport. Both 1[superscript]0CME and 1[superscript]0BME cells were passed for multiple subcultures and all passages formed electrically tight barriers. 1[superscript]0CME were cultured in the presence of hydrocortisone and exhibited high electrical resistance and amiloride-sensitive I[subscript]sc, suggesting the presence of ENaC-mediated Na[superscript]+ transport. 1[superscript]0BME were grown in a complex media in the presence or absence of dexamethasone. In contrast to 1[superscript]0CME, 1[superscript]0BME exhibited no detectable amiloride-sensitive I[subscript]sc in either culture condition. However, 1[superscript]0BME monolayers responded to an adrenergic agonist, norepinephrine, and a cholinergic agonist, carbamylcholine, with rapid increases in I[subscript]sc. Thus, this protocol for isolation and primary cell culture can be used for future studies that focus on mammary epithelial cell regulation and functions. In conclusion, the results from these projects demonstrate that mammary epithelial cells form electrically tight monolayers and can exhibit neurotransmitter- and/or hormone-induced net ion transport. The mechanisms that regulate Na[superscript]+ transport across mammary gland may provide clues to prevent or treat mastitis.

Shiga toxin and cholera toxin are members of the AB5 family of protein exotoxins. The A subunit is the enzymatic subunit, whereas the pentameric B subunit binds cell surface receptors and carries the A subunit to the endoplasmic reticulum (ER) where it can be released into the cytosol. The B-subunits (STxB or CTxB) mediate toxin traffic along the retrograde pathway from the plasma membrane to the ER via early/recycling endosomes and the Golgi apparatus. It is unknown if STxB requires transport through the Golgi, or if it is just kinetically favorable. It is also unknown if the recycling endosome (RE) plays a role in the retrograde transport of STxB and CTxB. The first goal of this dissertation research was to demonstrate that transport through the Golgi is required for STxB to reach the ER. Using aluminum fluoride treatment, a simple temperature block, and cytoplast studies, I show that Golgi transport is necessary for STxB to reach the ER. The second goal of this dissertation research was to tease apart how STxB and CTxB move through early and recycling endosomes as well as elucidate a mechanism of how STxB exits endosomes en route to the Golgi. The role of the RE in STxB and CTxB transport is unclear. I used transferrin colocalization and temperature block studies to show that STxB and CTxB traffic through the RE. I then used HRP ablation of the RE to show that STxB requires the RE to reach the Golgi. I also examined the role of an RE-specific protein, EHD1, in exit of STxB from the RE. EHD1 has been previously shown to regulate recycling Tfn exit from the RE but its role in STxB transport is unknown. Expression of a dominant negative form of EHD1 arrested STxB at the RE and prevented it from reaching the Golgi. Together, these results suggest that STxB and CTxB transit the RE, STxB requires a functional RE for normal retrograde trafficking, and that STxB exit from the RE is regulated by EHD1.

Diss. (sammanfattning) Göteborg : Univ. , 2009. / Härtill 3 uppsatser.

Cholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host quality control mechanism called ER-associated degradation (ERAD) that facilitates the translocation of misfolded proteins into the cytosol for degradation. Cytosolic CTA1, however, escapes this fate and is then free to activate its target, heterotrimeric G-protein subunit alpha (Gsα), leading to adenlyate cyclase (AC) hyperactivation and increased cAMP concentrations. This causes the secretion of chloride ions and water into the intestinal lumen. The result is severe diarrhea and dehydration which are the major symptoms of cholera. CTA1’s ability to exploit vesicle-mediated transport and ERAD for cytosolic entry demonstrates a potential link between cholera intoxication and a separate quality control mechanism called the unfolded protein response (UPR), which up-regulates vesicle-mediated transport and ERAD during ER stress. Other toxins in the same family such as ricin and Shiga toxin were shown to regulate the UPR, resulting in enhanced intoxication. Here, we show UPR activation by CT, which coincides with a marked increase in cytosolic CTA1 after 4 hours of toxin exposure. Drug induced-UPR activation also increases CTA1 delivery to the cytosol and increases cAMP concentrations during intoxication. We investigated whether CT stimulated UPR activation through Gsα or AC. Chemical activation of Gsα induced the UPR and increased CTA1 delivery to the cytosol. However, AC activation did iv not increase cytosolic CTA1 nor did it activate the UPR. These data provide further insight into the molecular mechanisms that cause cholera intoxication and suggest a novel role for Gsα during intoxication, which is UPR activation via an AC-independent mechanism

Die Struktur der Plasmamembran ist von deren Lipid- und Proteinzusammensetzung abhängig und wird durch die Anbindung an das unterliegende Zytoskelett beeinflusst. Das Ziel der vorliegenden Arbeit war die Untersuchung eines neuen Modellsystems basierend auf po­ren­über­span­nen­den Membranen, welches sowohl die heterogene Lipidzusammensetzung als auch den Einfluss eines unterliegenden Netzwerks berücksichtigt. Lipidmembranen, zusammengesetzt aus der „raft“-ähnlichen Lipidmischung DOPC/Sphingo­myelin/Cho­les­terin (40:40:20), wurden auf porösen, hochgeordneten Siliziumsubstraten mit Po­ren­durch­messern von 0.8, 1.2 und 2.0 µm durch Spreiten und Fusion von Riesenvesikeln (giant unilamellar vesicles, GUVs) präpariert. Die mikroskopische Phasenseparation in koexistierenden flüssig-geordneten (liquid ordered, lo) und flüssig-ungeordneten (liquid disordered, ld) Domänen wurde stark durch das unterliegende poröse Substrat beeinflusst. Die Größe der lo-Domänen konnte durch die Porengröße des Siliziumsubstrats, die Temperatur und den Cholesteringehalt der Membran, welcher durch Zugabe von Methyl-β-Cyclodextrin moduliert wurde, kontrolliert werden. Die Bindung der Shiga Toxin B-Untereinheit (STxB) an po­ren­überspannende Membranen, dotiert mit 5 mol% des Rezeptorlipids Gb3, führte zu einem Anstieg des Anteils der lo-Phase. Außerdem wurde die Bildung von lo-Domänen in nicht-phasenseparierten Membranen, zusammengesetzt aus DOPC/Sphingomyelin/Cholesterin/Gb3 (65:10:20:5), durch die Shiga Toxin-Bindung induziert. Ein Anstieg des Anteils der lo-Phase konnte ebenfalls bei der Bindung der pentameren Cholera Toxin B-Untereinheit (CTxB) an po­ren­überspannende Membranen, dotiert mit 1 mol% des Rezeptorlipids GM1, beobachtet werden. Des Weiteren wurde der Einfluss der chemischen Struktur des Gb3-Moleküls auf die Shiga Toxin-Bindung und die Reorganisation von festkörperunterstützten Membranen (solid supported membranes, SSMs) untersucht. Die STxB-Bindung an α-hydroxyliertes Gb3 erhöhte signifikant den Anteil der lo-Phase, während eine cis-Doppelbindung zur Bildung einer weiteren lo-Phase führte, die vermutlich ungesättigte (Glyko-)Sphingolipide und Cholesterin enthält. Im Falles des ungesättigten Gb3 konnte außerdem eine Kondensation zu größeren Domänen nach der STxB-Bindung beobachtet werden. Die genaue Phasenzuordnung der eingesetzten Glykospingolipide vor der Proteinbindung ist bisher unbekannt. Daher wurde das Phasenverhalten eines fluoreszierenden Polyen-Ga­lac­to­ce­re­bro­sids untersucht, welches bevorzugt in der lo-Phase von GUVs angereichert war. Dieser neue, intrinsische Fluorophor vermag als Grundlage für weitere Studien zum Phasenverhalten von Glykosphingolipiden dienen.