Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration.

Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine-binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.

Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration.

Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens appeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax™ X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein deuteration using deuterated minimal media with deuterium oxide as solvent, and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news since isotopic labeling is expensive and often ineffective, but represents a necessary prerequisite for some structural techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein deuteration in amounts suitable for structural biology studies.

Reduction is sufficient for the disassembly of ricin and Shiga toxin 1 but not Escherichia coli heat-labile enterotoxin.

Many AB toxins contain an enzymatic A moiety that is anchored to a cell-binding B moiety by a disulfide bridge. After receptor-mediated endocytosis, some AB toxins undergo retrograde transport to the endoplasmic reticulum (ER) where reduction of the disulfide bond occurs. The reduced A subunit then dissociates from the holotoxin and enters the cytosol to alter its cellular target. Intoxication requires A chain separation from the holotoxin, but, for many toxins, it is unclear if reduction alone is sufficient for toxin disassembly. Here, we examined the link between reduction and disassembly for several ER-translocating toxins. We found disassembly of the reduced Escherichia coli heat-labile enterotoxin (Ltx) required an interaction with one specific ER-localized oxidoreductase: protein disulfide isomerase (PDI). In contrast, the reduction and disassembly of ricin toxin (Rtx) and Shiga toxin 1 (Stx1) were coupled events that did not require PDI and could be triggered by reductant alone. PDI-deficient cells accordingly exhibited high resistance to Ltx with continued sensitivity to Rtx and Stx1. The distinct structural organization of each AB toxin thus appears to determine whether holotoxin disassembly occurs spontaneously upon disulfide reduction or requires the additional input of PDI.

Science communication from a course-based undergraduate research experience.

Course-based undergraduate research experiences (CUREs) provide an efficient mechanism to provide many students with an original research project. CUREs often culminate in a capstone poster presentation, but reports on these classes usually focus on the preparation and execution of the project rather than communication of the results. This article summarizes a CURE-associated research seminar that focuses on developing the communication and interpersonal skills required for the production and presentation of a conference poster. The class is designed to provide students with the tools and confidence for effective communication of their research. From the two class offerings to date, the 18 participating students have received five awards from 19 conference presentations.

The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin.

After binding to the surface of a target cell, cholera toxin (CT) moves to the endoplasmic reticulum (ER) by retrograde transport. In the ER, the catalytic CTA1 subunit dissociates from the rest of the toxin and is transferred to the cytosol where it is degraded by a ubiquitin-independent proteasomal mechanism. However, CTA1 persists long enough to induce excessive cAMP production through the activation of Gsα. It is generally believed that only one or a few molecules of cytosolic CTA1 are necessary to elicit a cytopathic effect, yet no study has directly correlated the levels of cytosolic toxin to the extent of intoxication. Here, we used the technology of surface plasmon resonance to quantify the cytosolic pool of CTA1. Our data demonstrate that only 4% of surface-bound CTA1 is found in the cytosol after 2 h of intoxication. This represented around 2600 molecules of cytosolic toxin per cell, and it was sufficient to produce a robust cAMP response. However, we did not detect elevated cAMP levels in cells containing less than 700 molecules of cytosolic toxin. Thus, a threshold quantity of cytosolic CTA1 is required to elicit a cytopathic effect. When translocation to the cytosol was blocked soon after toxin exposure, the pool of CTA1 already in the cytosol was degraded and was not replenished. The cytosolic pool of CTA1 thus remained below its functional threshold, preventing the initiation of a cAMP response. These observations challenge the paradigm that extremely low levels of cytosolic toxin are sufficient for toxicity, and they provide experimental support for the development of post-intoxication therapeutic strategies.

Acid-induced disassembly of the Haemophilus ducreyi cytolethal distending toxin.

The cytolethal distending toxins (CDTs) produced by many Gram-negative pathogens are tripartite genotoxins with a single catalytic subunit (CdtB) and two cell-binding subunits (CdtA + CdtC). CDT moves by vesicle carriers from the cell surface to the endosomes and through the Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi apparatus, and CdtB separates from CdtC in the ER. The free CdtB subunit, which is only active after holotoxin disassembly, then crosses the ER membrane and enters the nucleus where it generates DNA breaks. We hypothesized that the acidified lumen of the endosomes is responsible for separating CdtA from the CdtB/CdtC heterodimer. To test this prediction, possible acid-induced disruptions to the CDT holotoxin were monitored by size exclusion chromatography and surface plasmon resonance. We found that CDT could not efficiently assemble from its individual subunits at the early endosome pH of 6.3. Partial disassembly of the CDT holotoxin also occurred at pH 6.3, with complete separation of CdtA from an intact CdtB/CdtC heterodimer occurring at both pH 6.0 and the late endosome pH of 5.6. Acidification caused the precipitation of CdtA at pH 6.5 and below, but neither CdtB nor CdtC were affected by a pH as low as 5.2. Circular dichroism further showed that the individual CdtB subunit adopts a different secondary structure as compared to its structure in the holotoxin. We conclude the first stage of CDT disassembly occurs in the early endosomes, where an acid-induced alteration to CdtA releases it from the CdtB/CdtC heterodimer.

The manipulation of cell signaling and host cell biology by cholera toxin.

Vibrio cholerae colonizes the small intestine and releases cholera toxin into the extracellular space. The toxin binds to the apical surface of the epithelium, is internalized into the host endomembrane system, and escapes into the cytosol where it activates the stimulatory alpha subunit of the heterotrimeric G protein by ADP-ribosylation. This initiates a cAMP-dependent signaling pathway that stimulates chloride efflux into the gut, with diarrhea resulting from the accompanying osmotic movement of water into the intestinal lumen. G protein signaling is not the only host system manipulated by cholera toxin, however. Other cellular mechanisms and signaling pathways active in the intoxication process include endocytosis through lipid rafts, retrograde transport to the endoplasmic reticulum, the endoplasmic reticulum-associated degradation system for protein delivery to the cytosol, the unfolded protein response, and G protein de-activation through degradation or the function of ADP-ribosyl hydrolases. Although toxin-induced chloride efflux is thought to be an irreversible event, alterations to these processes could facilitate cellular recovery from intoxication. This review will highlight how cholera toxin exploits signaling pathways and other cell biology events to elicit a diarrheal response from the host.

Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin.

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally similar AB5-type protein toxins. They move from the cell surface to the endoplasmic reticulum where the A1 catalytic subunit is separated from its holotoxin by protein disulfide isomerase (PDI), thus allowing the dissociated A1 subunit to enter the cytosol for a toxic effect. Despite similar mechanisms of toxicity, CT is more potent than LT. The difference has been attributed to a more stable domain assembly for CT as compared to LT, but this explanation has not been directly tested and is arguable as toxin disassembly is an indispensable step in the cellular action of these toxins. We show here that PDI disassembles CT more efficiently than LT, which provides a possible explanation for the greater potency of the former toxin. Furthermore, direct examination of CT and LT domain assemblies found no difference in toxin stability. Using novel analytic geometry approaches, we provide a detailed characterization of the positioning of the A subunit with respect to the B pentamer and demonstrate significant differences in the interdomain architecture of CT and LT. Protein docking analysis further suggests that these global structural differences result in distinct modes of PDI-toxin interactions. Our results highlight previously overlooked structural differences between CT and LT that provide a new model for the PDI-assisted disassembly and differential potency of these toxins.

Proline Isomerization as a Key Determinant for Hsp90-Toxin Interactions.

The A chains of ADP-ribosylating toxins exploit Hsp90 for translocation into the host cytosol. Here, we hypothesize that cis proline residues play a key role in toxin recognition by Hsp90. Our model is largely derived from studies on the unusual interplay between Hsp90 and the catalytic A1 subunit of cholera toxin (CTA1), including the recent identification of an RPPDEI-like binding motif for Hsp90 in CTA1 and several other bacterial toxins. Cis/trans proline isomerization is known to influence protein-protein interactions and protein structure/function, but it has not yet been proposed to affect Hsp90-toxin interactions. Our model thus provides a new framework to understand the molecular basis for Hsp90 chaperone function and Hsp90-driven toxin translocation.

Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin.

Many Gram-negative pathogens produce a cytolethal distending toxin (CDT) with two cell-binding subunits (CdtA + CdtC) and a catalytic CdtB subunit. After adhesion to the plasma membrane of a target cell, CDT moves by retrograde transport to endoplasmic reticulum. CdtB then enters the nucleus where it generates DNA breaks that lead to cell cycle arrest and apoptosis or senescence. CdtA anchors the CDT holotoxin to the plasma membrane and is thought to remain on the cell surface after endocytosis of the CdtB/CdtC heterodimer. Here, we re-examined the potential endocytosis and intracellular transport of CdtA from the Haemophilus ducreyi CDT. We recorded the endocytosis of holotoxin-associated CdtA with a cell-based enzyme-linked immunoabsorbent assay (CELISA) and visualised its presence in the early endosomes by confocal microscopy 10 min after CDT binding to the cell surface. Western blot analysis documented the rapid degradation of internalised CdtA. Most of internalised CdtB and CdtC were degraded as well. The rapid rate of CDT internalisation and turnover, which could explain why CdtA endocytosis was not detected in previous studies, suggests only a minor pool of cell-associated CdtB reaches the nucleus. Our work demonstrates that CDT is internalised as an intact holotoxin and identifies the endosomes as the site of CdtA dissociation from CdtB/CdtC. TAKE AWAYS: During the endocytosis of CDT, CdtA is thought to remain at the cell surface. A cell-based ELISA documented the rapid endocytosis of CdtA. CdtA was visualised in the early endosomes by confocal microscopy. Intracellular CdtA was rapidly degraded, along with most of CdtB and CdtC.

cAMP-Independent Activation of the Unfolded Protein Response by Cholera Toxin.

Cholera toxin (CT) is an AB5 protein toxin that activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα) through ADP-ribosylation. Activation of Gsα produces a cytopathic effect by stimulating adenylate cyclase and the production of cAMP. To reach its cytosolic Gsα target, CT binds to the plasma membrane of a host cell and travels by vesicle carriers to the endoplasmic reticulum (ER). The catalytic CTA1 subunit then exploits the quality control mechanism of ER-associated degradation to move from the ER to the cytosol. ER-associated degradation is functionally linked to another quality control system, the unfolded protein response (UPR). However, the role of the UPR in cholera intoxication is unclear. We report here that CT triggers the UPR after 4 h of toxin exposure. A functional toxin was required to induce the UPR, but, surprisingly, activation of the adenylate cyclase signaling pathway was not sufficient to trigger the process. Toxin-induced activation of the UPR coincided with increased toxin accumulation in the cytosol. Chemical activation of the heterotrimeric G protein or the UPR also enhanced the onset of CTA1 delivery to the cytosol, thus producing a toxin-sensitive phenotype. These results indicate there is a cAMP-independent response to CT that activates the UPR and thereby enhances the efficiency of intoxication.

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase.

Aggregates of α-synuclein contribute to the etiology of Parkinson's Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson's Disease. The protective role of PDI in Parkinson's Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

Bimodal Response to Shiga Toxin 2 Subtypes Results from Relatively Weak Binding to the Target Cell.

There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.

Stability and Conformational Resilience of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an abb'xa' structural organization in which the a and a' domains have CGHC active sites, the b and b' domains are involved with substrate binding, and x is a flexible linker. PDI exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether this unfolding-refolding cycle is a substrate-induced process or an intrinsic physical property of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal denaturation. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54 °C unfolding transition temperature (Tm) and regained 85% of its native structure after nearly complete thermal denaturation. Oxidized PDI had a lower Tm of 48-50 °C and regained 70% of its native conformation after 75% denaturation. Both reduced PDI and oxidized PDI were functional after refolding from these denatured states. Additional studies documented increased stability of a PDI construct lacking the a' domain and decreased thermal stability of a construct lacking the a domain. Furthermore, oxidation of the a domain limited the ability of PDI to refold. The stability and conformational resilience of PDI are thus linked to both redox-dependent and domain-specific effects. These findings document previously unrecognized properties of PDI and provide insight into the physical foundation of its biological function.

Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT). In the ER, PDI physically displaces the reduced CTA1 subunit from its non-covalent assembly in the CT holotoxin. This is followed by CTA1 dislocation from the ER to the cytosol where the toxin interacts with its G protein target for a cytopathic effect. Q3R blocked the conformational change in PDI that accompanies its binding to CTA1, which, in turn, prevented PDI from displacing CTA1 from its holotoxin and generated a toxin-resistant phenotype. Other steps of the CT intoxication process were not affected by Q3R, including PDI binding to CTA1 and CT reduction by PDI. Additional experiments with the B chain of ricin toxin found that Q3R could also disrupt PDI function through the loss of substrate binding. Q3R can thus inhibit PDI function through distinct mechanisms in a substrate-dependent manner.

Intracellular Trafficking and Translocation of Pertussis Toxin.

Pertussis toxin (PT) is a multimeric complex of six proteins. The PTS1 subunit is an ADP-ribosyltransferase that inactivates the alpha subunit of heterotrimeric Gi/o proteins. The remaining PT subunits form a pentamer that positions PTS1 in and above the central cavity of the triangular structure. Adhesion of this pentamer to glycoprotein or glycolipid conjugates on the surface of a target cell leads to endocytosis of the PT holotoxin. Vesicle carriers then deliver the holotoxin to the endoplasmic reticulum (ER) where PTS1 dissociates from the rest of the toxin, unfolds, and exploits the ER-associated degradation pathway for export to the cytosol. Refolding of the cytosolic toxin allows it to regain an active conformation for the disruption of cAMP-dependent signaling events. This review will consider the intracellular trafficking of PT and the order-disorder-order transitions of PTS1 that are essential for its cellular activity.

A binding motif for Hsp90 in the A chains of ADP-ribosylating toxins that move from the endoplasmic reticulum to the cytosol.

Cholera toxin (Ctx) is an AB-type protein toxin that acts as an adenosine diphosphate (ADP)-ribosyltransferase to disrupt intracellular signalling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER-associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognises two peptide sequences from CtxA1: an N-terminal RPPDEI sequence (residues 11-16) and an LDIAPA sequence in the C-terminal region (residues 153-158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full-length CtxA1. Both sequences were necessary for the ER-to-cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI-like motif at the N- or C-termini of the A chains from four other ER-translocating toxins that act as ADP-ribosyltransferases: pertussis toxin, Escherichia coli heat-labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP-ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER-to-cytosol export of CtxA1 and possibly other toxin A chains as well.

HSC70 and HSP90 chaperones perform complementary roles in translocation of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol.

Cholera toxin (CT) travels by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where the catalytic A1 subunit of CT (CTA1) dissociates from the rest of the toxin, unfolds, and moves through a membrane-spanning translocon pore to reach the cytosol. Heat shock protein 90 (HSP90) binds to the N-terminal region of CTA1 and facilitates its ER-to-cytosol export by refolding the toxin as it emerges at the cytosolic face of the ER membrane. HSP90 also refolds some endogenous cytosolic proteins as part of a foldosome complex containing heat shock cognate 71-kDa protein (HSC70) and the HSC70/HSP90-organizing protein (HOP) linker that anchors HSP90 to HSC70. We accordingly predicted that HSC70 and HOP also function in CTA1 translocation. Inactivation of HSC70 by drug treatment disrupted CTA1 translocation to the cytosol and generated a toxin-resistant phenotype. In contrast, the depletion of HOP did not disrupt CT activity against cultured cells. HSC70 and HSP90 could bind independently to disordered CTA1, even in the absence of HOP. This indicated HSP90 and HSC70 recognize distinct regions of CTA1, which was confirmed by the identification of a YYIYVI-binding motif for HSC70 that spans residues 83-88 of the 192-amino acid CTA1 polypeptide. Refolding of disordered CTA1 occurred in the presence of HSC70 alone, indicating that HSC70 and HSP90 can each independently refold CTA1. Our work suggests a novel translocation mechanism in which sequential interactions with HSP90 and HSC70 drive the N- to C-terminal extraction of CTA1 from the ER.

Protein disulfide isomerase does not act as an unfoldase in the disassembly of cholera toxin.

Cholera toxin (CT) is composed of a disulfide-linked A1/A2 heterodimer and a ring-like, cell-binding B homopentamer. The catalytic A1 subunit must dissociate from CTA2/CTB5 to manifest its cellular activity. Reduction of the A1/A2 disulfide bond is required for holotoxin disassembly, but reduced CTA1 does not spontaneously separate from CTA2/CTB5: protein disulfide isomerase (PDI) is responsible for displacing CTA1 from its non-covalent assembly in the CT holotoxin. Contact with PDI shifts CTA1 from a protease-resistant conformation to a protease-sensitive conformation, which is thought to represent the PDI-mediated unfolding of CTA1. Based solely on this finding, PDI is widely viewed as an 'unfoldase' that triggers toxin disassembly by unfolding the holotoxin-associated A1 subunit. In contrast with this unfoldase model of PDI function, we report the ability of PDI to render CTA1 protease-sensitive is unrelated to its role in toxin disassembly. Multiple conditions that promoted PDI-induced protease sensitivity in CTA1 did not support PDI-mediated disassembly of the CT holotoxin. Moreover, preventing the PDI-induced shift in CTA1 protease sensitivity did not affect PDI-mediated disassembly of the CT holotoxin. Denatured PDI could still convert CTA1 into a protease-sensitive state, and equal or excess molar fractions of PDI were required for both efficient conversion of CTA1 into a protease-sensitive state and efficient disassembly of the CT holotoxin. These observations indicate the 'unfoldase' property of PDI does not play a functional role in CT disassembly and does not represent an enzymatic activity.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis.

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.