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.

SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation.

The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.

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.

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.

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.

A Cell-Based Fluorescent Assay to Detect the Activity of AB Toxins that Inhibit Protein Synthesis.

Many AB toxins elicit a cytotoxic effect involving the inhibition of protein synthesis. In this chapter, we describe a simple cell-based fluorescent assay to detect and quantify the inhibition of protein synthesis. The assay can also identify and characterize toxin inhibitors.

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds.

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

A simple and fast method to study the hydrodynamic size difference of protein disulfide isomerase in oxidized and reduced form using gold nanoparticles and dynamic light scattering.

The hydrodynamic dimension of a protein is a reflection of both its molecular weight and its tertiary structures. Studying the hydrodynamic dimensions of proteins in solutions can help elucidate the structural properties of proteins. Here we report a simple and fast method to measure the hydrodyamic size of a relatively small protein, protein disulfide isomerase (PDI), using gold nanoparticle probes combined with dynamic light scattering. Proteins can readily adsorb to citrate-capped gold nanoparticles to form a protein corona. By measuring the average diameter of the gold nanoparticles before and after protein corona formation, the hydrodynamic diameter of the protein can be deduced from the net particle size increase of the assay solution. This study found that when the disulfide bonds in PDI are reduced to thiols, the reduced PDI exhibits a smaller hydrodynamic diameter than the oxided PDI. This finding is in good agreement with the X-ray diffraction analysis of PDI in single crystals. In comparison with other techniques that are used for protein hydrodynamic size analysis, the current method is easy to use, requires a trace amount of protein samples, with results obtained in minutes instead of hours.

Grape extracts inhibit multiple events in the cell biology of cholera intoxication.

Vibrio cholerae produces cholera toxin (CT), an AB5 protein toxin that is primarily responsible for the profuse watery diarrhea of cholera. CT is secreted into the extracellular milieu, but the toxin attacks its Gsα target within the cytosol of a host cell. Thus, CT must cross a cellular membrane barrier in order to function. This event only occurs after the toxin travels by retrograde vesicular transport from the cell surface to the endoplasmic reticulum (ER). The catalytic A1 polypeptide then dissociates from the rest of the toxin and assumes an unfolded conformation that facilitates its transfer to the cytosol by a process involving the quality control system of ER-associated degradation. Productive intoxication is blocked by alterations to the vesicular transport of CT and/or the ER-to-cytosol translocation of CTA1. Various plant compounds have been reported to inhibit the cytopathic activity of CT, so in this work we evaluated the potential anti-CT properties of grape extract. Two grape extracts currently sold as nutritional supplements inhibited CT and Escherichia coli heat-labile toxin activity against cultured cells and intestinal loops. CT intoxication was blocked even when the extracts were added an hour after the initial toxin exposure. A specific subset of host-toxin interactions involving both the catalytic CTA1 subunit and the cell-binding CTB pentamer were affected. The extracts blocked toxin binding to the cell surface, prevented unfolding of the isolated CTA1 subunit, inhibited CTA1 translocation to the cytosol, and disrupted the catalytic activity of CTA1. Grape extract could thus potentially serve as a novel therapeutic to prevent or possibly treat cholera.