Structure Of Holotoxin Research Articles

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin–toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection

ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin–toxin monomer (FaeG-FedF-LTA2–B) elicited protective antibodies. In this study, we constructed a new adhesin–toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT192A2:5LTB) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT192A2:5LTB fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.

A Single-Step Purification and Molecular Characterization of Functional Shiga Toxin 2 Variants from Pathogenic Escherichia coli

A one-step affinity chromatography method was developed to purify Shiga toxin 2 variants (Stx2) Stx2a, Stx2c, Stx2d and Stx2g from bacterial culture supernatants. Analysis of the purified Stx2 variants by denaturing gel electrophoresis revealed 32 kDa and 7 kDa protein bands, corresponding to the Stx2A- and B-subunits, respectively. However, native gel electrophoresis indicated that purified Stx2c and Stx2d were significantly higher in molecular weight than Stx2a and Stx2g. In a cytotoxicity assay with Hela cells, the 50% cytotoxic dose of Stx2a and Stx2g were 100 pg and 10 pg, respectively, but 1 ng each for Stx2c and Stx2d. Interestingly, analysis of the 50% inhibitory dose in a cell-free translational system from rabbit reticulocyte lysates indicated that Stx2g had a lower capacity to inhibit protein synthesis than the other Stx2 variants. The cytotoxicities in Hela cells were neutralized with an anti-Stx2B antibody and were denatured at 80 °C for 1 h. These findings demonstrated that Stx2 variants exhibited different toxicities, holotoxin structure, and stabilities using distinct systems for assessing toxin activities. The development of a simple method for purification of Stx2 variants will enable further studies of Stx2-mediated toxicity in various model systems.

The A Subunit of Escherichia coli Heat-Labile Enterotoxin Functions as a Mucosal Adjuvant and Promotes IgG2a, IgA, and Th17 Responses to Vaccine Antigens

Enterotoxigenic Escherichia coli (ETEC) produces both heat-labile (LT) and heat-stable (ST) enterotoxins and is a major cause of diarrhea in infants in developing countries and in travelers to those regions. In addition to inducing fluid secretion, LT is a powerful mucosal adjuvant capable of promoting immune responses to coadministered antigens. In this study, we examined purified A subunit to further understand the toxicity and adjuvanticity of LT. Purified A subunit was enzymatically active but sensitive to proteolytic degradation and unable to bind gangliosides, and even in the presence of admixed B subunit, it displayed low cyclic AMP (cAMP) induction and no enterotoxicity. Thus, the AB5 structure plays a key role in protecting the A subunit from proteolytic degradation and in delivering the enzymatic signals required for secretion. In contrast, the A subunit alone was capable of activating dendritic cells and enhanced immune responses to multiple antigens following intranasal immunization; therefore, unlike toxicity, LT adjuvanticity is not dependent on the AB5 holotoxin structure or the presence of the B subunit. However, immune responses were maximal when signals were received from both subunits either in an AB5 structure or with A and B admixed. Furthermore, the quality of the immune response (i.e., IgG1/IgG2 balance and mucosal IgA and IL-17 secretion) was determined by the presence of an A subunit, revealing for the first time induction of Th17 responses with the A subunit alone. These results have important implications for understanding ETEC pathogenesis, unraveling immunologic responses induced by LT-based adjuvants, and developing new mucosal vaccines.

Design and in silico screening of inhibitors of the cholera toxin

Background: Cholera toxin (CT) is the causative agent for cholera, which is responsible for the death of millions of people. The holotoxin structure of CT in conjunction with its mechanism of action reveals four potential target areas to design CT therapeutic agents: i) the inhibition of adenylate cyclase; ii) the blockage of the active site of the enzyme located in the A subunit; iii) the disruption of the assembly of the holotoxin by interrupting the A2–B interaction; and iv) the interception of the receptor binding to the bottom of the B pentamer. Objective/method: In this review, some of the most exciting and successful CT inhibitors and their design strategies are highlighted. These include transition state analogues and substrate analogues as CTA inhibitors, and pentavalent ligand, sugar-containing dendrimers and sugar mimics as CTB inhibitors. Some recent progress in in silico screening methods is also summarized. Conclusion: The most potent inhibitor demonstrated an IC50 of 50 pM.

Structure and solution dynamics of CdtB, the biologically active subunit of Cytolethal Distending Toxin

Cytolethal Distending Toxin is a DNA-damaging bacterial toxin that induces cell cycle arrest in eukaryotic cells. Following cell-surface binding and cellular entry, CdtB, a type I DNase homolog, traffics to the nucleus and cleaves chromosomal DNA. Here we combine X-ray crystallography and multidimensional NMR to examine the molecular structure and solution dynamics of the free Escherichia coli CdtB (EcCdtB) subunit. Comparison of the EcCdtB crystal structure and NMR solution state structure and dynamics data reveals distinct differences in two key regions of CdtB. Most notably, residues 186-210 (region1) adopt a well-defined tertiary structure in the crystallographic model, in contrast to the solution state that has “random coil” structural and dynamics characteristics. CdtB residues R235-I239 (region 2) are also highly mobile in solution. Regions 1 and 2 are either flanked by or encompass two bipartite nuclear localization sequences (NLS), respectively. Region 2 also makes contacts in the CdtB interface with CdtA and CdtC subunits as defined by the holotoxin structure of the Haemophilus ducreyi CDT. With the exception of regions 1 and 2, EcCdtB and the CdtB component of H. ducreyi holotoxin have very similar structures (Cα r.m.s.d. of 0.98 Å). Overall, these comparisons of solution and crystal forms of CdtB provide new insights into holotoxin assembly and CdtB nuclear localization.

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.

Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues.

The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.

Saponin and sapogenol. XV. Antifungal glycosides from the sea cucumber Stichopus japonicus selenka. (2). Structures of holotoxin A and holotoxin B.

The structure of holotoxin A, the major antifungal glycoside isolated from the Far Eastern sea cucumber Stichopus japonicus SELENKA, has been elucidated as 4 on the bases of chemical and physicochemical evidence and enzymatic hydrolysis. Holotoxin B (14), a minor antifungal glycoside of the same origin, has been suggested to be the glucoside of holotoxin A (4) on the similar basis. Holotoxin C, another minor glycoside, has been suggested to be the similar type of glycoside as holotoxin A and B. Holotoxin A and B are distinctive by the possession of two α-linkages of D-xylose and D-quinovose in the oligosaccharide portion. Finally, comparison of the anti-microbial activity of holotoxins with the various kinds of saponins isolated from the plant materials has been presented.