Ken Teter

Cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are highly related AB5-type protein toxins that contain a catalytic A1 subunit, an A2 linker, and a cell-binding homopentamer of B subunits. The two proteins bind the same GM1 receptor and have similar levels of enzymatic activity toward their shared Gsα target, yet CT is more potent than LT. This difference has been mapped to a sequence of 11 amino acids (residues 226–236) near the C-terminus of the A2 linker. We now identify a single amino acid, aspartic acid at position 229, as a key contributor to the function of CT: substituting it for glutamic acid 229 from LT reduced the toxicity of CT to the level of LT. Toxin disassembly by protein disulfide isomerase (PDI), which is an essential event in the intoxication process, was less efficient for CTD229E and LT than for wild-type CT. This was further confirmed by a CT variant, in which four residues were converted into the LT sequence (D229E, I230V, T232I, and H233Y). To test our previous hypothesis that the positioning of the A1 subunit over the B pentamer critically determines toxin disassembly and thus potency, we determined crystal structures of both toxin variants to 1.6 Å resolution and calculated the angle at which the A2 linker exits the pore. The conformations of the two toxin chimeras lie between CT and LT, with the A2 C-terminus of the CT quadruple variant adopting essentially the same fold as LT. The A subunits of the toxin variants as well as that of LT showed a higher conformational freedom than CT as determined by molecular dynamics simulations, suggesting that a more rigid positioning of the A subunit drives toxin disassembly and potency. Our collective data uncover the critical role of D229 in toxin folding and structural dynamics, which in turn impacts toxin potency through effects on the efficiency of PDI-driven toxin disassembly.

Chronic infection is important for the transmission of Salmonella enterica serovar Typhi ( S . Typhi), an obligate human pathogen responsible for typhoid fever. The immunomodulatory action of typhoid toxin helps establish an initial stealth invasion of the intestinal tract that can lead to systemic spread, colonization of the gallbladder, and persistent shedding. Typhoid toxin contains a cell‐binding pentamer and two catalytic subunits: cytolethal distending toxin B (CdtB, a DNase) and pertussis‐like toxin A (PltA, an ADP‐ribosyltransferase). It undergoes endocytosis and transport to the endoplasmic reticulum (ER) where toxin disassembly occurs. This allows the catalytic subunits to exit the ER for interaction with their nuclear or cytosolic targets. However, all known cellular and physiological effects of typhoid toxin are attributed to CdtB. These effects reduce the severity of intestinal inflammation, promoting host survival and long‐term infections. PltA has in vitro enzymatic activity but does not affect cells challenged with typhoid toxin. It is thought that the cytosolic action of PltA will be identified in future work. We instead propose that PltA never reaches the cytosol: it is retained in the ER to limit toxin potency, thereby allowing the anti‐inflammatory effects of CdtB to promote intestinal colonization. The absence of Salmonella ADP‐ribosylating toxin AB (ArtAB) from all strains of S . Typhi may serve a similar purpose. We accordingly propose a model of balanced pathogenicity in which the lack of toxin ADP‐ribosyltransferase activity is essential for S . Typhi pathogenesis.

Aggregated amyloid beta peptide (Aβ) contributes to Alzheimer's disease through neurotoxic effects and a prion‐like mode of transmission. We report that protein disulfide isomerase (PDI) exhibits disaggregase activity against oligomeric but not fibrillar forms of Aβ. PDI did not bind monomeric Aβ, indicating its highly effective inhibition of fibril formation occurs through reversal of early‐stage oligomers rather than prevention of the initial aggregate. Cells exposed to both PDI and oligomeric Aβ were protected from Aβ‐induced toxicity. An S‐nitrosylated form of PDI that is associated with neurodegeneration could not bind to oligomeric Aβ, thereby eliminating its neuroprotective disaggregase activity. Our observations suggest PDI could be used both physiologically and therapeutically to dissolve the oligomeric forms of Aβ.

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.

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.

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 ^TM 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.

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.