Journal: Microorganisms

Article Title: Exploring the Toxin-Mediated Mechanisms in Clostridioides difficile Infection

doi: 10.3390/microorganisms12051004

Figure Lengend Snippet: ( A ) Pathogenicity Locus (PaLoc). The genes tcdA and tcdB (pink arrows) encode the toxins TcdA and TcdB, respectively. The regulatory genes tcdR (positive) and tcdC (negative) modulate the transcription of tcdA and tcdB and are presented with green arrows. Genes tcdE and tcdL (grey arrows) encode a holin and an endolysin, respectively, which are involved in toxin secretion. The direction of the arrows represents the direction of transcription of the genes. ( B ) TcdA and TcdB are divided into four domains: the glycosyltransferase domain (GTD; red), the autoprotease domain (APD; blue), the delivery and receptor-binding domain (DRBD; yellow), and the combined repetitive oligopeptides (CROPs; green).

Article Snippet: The second vaccine, developed by Pfizer, was based on full-length recombinant molecules of TcdA and TcdB.

Techniques: Binding Assay

Journal: Microorganisms

Article Title: Exploring the Toxin-Mediated Mechanisms in Clostridioides difficile Infection

doi: 10.3390/microorganisms12051004

Figure Lengend Snippet: Mechanism of action of TcdA and TcdB. Toxins bind surface receptors on the colonic epithelium and are endocytosed in acidic endosomes. Low pH triggers a conformational change in the toxins resulting in pore formation and translocation of GTD and APD in the cytosol. The activation of APD results in the cleavage and release of the GTD. The GTD blocks the function of Rho and Ras GTPases by transferring the UDP-glycose to GTPases, resulting in the induction of cytoskeletal damage.

Article Snippet: The second vaccine, developed by Pfizer, was based on full-length recombinant molecules of TcdA and TcdB.

Techniques: Translocation Assay, Activation Assay, Transferring

Journal: Microorganisms

Article Title: Exploring the Toxin-Mediated Mechanisms in Clostridioides difficile Infection

doi: 10.3390/microorganisms12051004

Figure Lengend Snippet: Representation of CDI-mediated inflammatory host response. Once TcdA and TcdB destroy the intestinal epithelium, they cause damage to deeper layers of tissue such as the destruction of the myofibroblasts. The presence of toxins triggers the release of dendritic cells, neutrophils, monocytes, and macrophages from the blood vessels. IL-1β and IL-8 produced by the intestinal epithelial cells enhance inflammation and attract neutrophils to the lumen of the colon. Within, neutrophils form pseudomembranes. At the same time, toxins induce the degranulation of mast cells and the release of substance P (SP) and Calcitonin gene-related peptide (CGRP) from neurons of the enteric nervous system.

Article Snippet: The second vaccine, developed by Pfizer, was based on full-length recombinant molecules of TcdA and TcdB.

Techniques: Produced

Journal: Microorganisms

Article Title: Exploring the Toxin-Mediated Mechanisms in Clostridioides difficile Infection

doi: 10.3390/microorganisms12051004

Figure Lengend Snippet: During CDI, C. difficile toxins TcdA and TcdB can breach the intestinal barrier and enter the bloodstream, resulting in systemic toxemia. Elevated levels of these toxins in the bloodstream can cause damage outside the colon, leading to dysfunction in multiple organs such as the heart, thymus, kidneys, and brain.

Article Snippet: The second vaccine, developed by Pfizer, was based on full-length recombinant molecules of TcdA and TcdB.

Techniques:

Journal: Antibiotics

Article Title: High Serum Levels of Toxin A Correlate with Disease Severity in Patients with Clostridioides difficile Infection

doi: 10.3390/antibiotics10091093

Figure Lengend Snippet: Representative images and the standard curve of ( A ) TcdA and ( B ) TcdB dot plots necessary for determining the concentration of the toxins in the blood of CDI patients. Toxin concentrations are reported as the mean value of triplicate dots ± standard deviation (SD) normalized to control (i.e., 0 pg/mL); a.d.u., arbitrary densitometric unit.

Article Snippet: Recombinant TcdA (BML-G140) and TcdB (BML-G150) were obtained from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Concentration Assay, Standard Deviation

Journal: The Journal of Infectious Diseases

Article Title: A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

doi: 10.1093/infdis/jiu196

Figure Lengend Snippet: Binding of specific heavy-chain-only antibody (VHH) to TcdA and TcdB. A, Diagram illustrates specific binding of individual VHHs to the glucosyltransferase domain (GTD), cysteine protease domain (CPD), translocation domain (TD), or receptor-binding domain (RBD) in TcdA or TcdB. B and C, Enzyme-linked immunosorbent assay analysis of serially diluted anti-TcdA (B) or anti-TcdB (C) VHHs in microtiter plates coated with 0.5 µg/mL of TcdA or TcdB, respectively.

Article Snippet: For pull-down selection, the recombinant TcdA and TcdB [ 22 ] proteins were biotinylated using the Pierce EZ-Link NHS-PEG4 Biotin kit (Pierce Biotechnology, Rockford, IL) per the manufacturer's instructions.

Techniques: Binding Assay, Translocation Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Infectious Diseases

Article Title: A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

doi: 10.1093/infdis/jiu196

Figure Lengend Snippet: Neutralizing activities of anti-Tcd heavy-chain-only antibodies (VHHs). CT26 cells were exposed to either 10 ng/mL TcdA (A) or 0.2 ng/mL TcdB (B) premixed with a serial dilution of each anti-TcdA or anti-TcdB VHH, respectively, and then added to cells for 24 hours. Morphological changes in cells were observed under a phase-contrast microscope, and the percentage of cell rounding was assessed.

Article Snippet: For pull-down selection, the recombinant TcdA and TcdB [ 22 ] proteins were biotinylated using the Pierce EZ-Link NHS-PEG4 Biotin kit (Pierce Biotechnology, Rockford, IL) per the manufacturer's instructions.

Techniques: Serial Dilution, Microscopy

Journal: The Journal of Infectious Diseases

Article Title: A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

doi: 10.1093/infdis/jiu196

Figure Lengend Snippet: Generation of bispecific ABA against TcdA and TcdB. A, Diagram of the ABA fusion protein. AH3/E3/E3/AA6 heavy-chain-only antibody (VHH) subunits were separated by a flexible linker sequence (FS). A His(6)-tag and E-tag were genetically fused at the N-terminal and C-terminal, respectively, of the ABA molecule. B, Checkerboard Enzyme-linked immunosorbent assay analysis of ABA competition with individual components. Serially diluted ABA (ng/mL) was mixed with different serially diluted thioredoxin/VHH fusion proteins targeting TcdA (AH3, AA6, or AC1) or TcdB (E3 or B12) and added to microplates coated with TcdA and TcdB, respectively. Biotinylated anti-thioredoxin VHH was used for detection.

Article Snippet: For pull-down selection, the recombinant TcdA and TcdB [ 22 ] proteins were biotinylated using the Pierce EZ-Link NHS-PEG4 Biotin kit (Pierce Biotechnology, Rockford, IL) per the manufacturer's instructions.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Infectious Diseases

Article Title: A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

doi: 10.1093/infdis/jiu196

Figure Lengend Snippet: ABA binds to TcdA and TcdB simultaneously. Serially diluted ABA (ng/mL) was added to microplate wells coated with TcdA (A) or TcdB (B). After incubation and washes, serially diluted TcdB (A) or TcdA (B) were added to the plates. The binding of toxins was determined by detecting TcdB (A) or TcdA (B), respectively, using toxin-specific mouse monoclonal antibodies.

Article Snippet: For pull-down selection, the recombinant TcdA and TcdB [ 22 ] proteins were biotinylated using the Pierce EZ-Link NHS-PEG4 Biotin kit (Pierce Biotechnology, Rockford, IL) per the manufacturer's instructions.

Techniques: Incubation, Binding Assay

Journal: The Journal of Infectious Diseases

Article Title: A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

doi: 10.1093/infdis/jiu196

Figure Lengend Snippet: In vitro and in vivo neutralization of TcdA and TcdB due to ABA. A and B, CT26 cells were exposed to serially diluted E3, AH3, AA6, or ABA together with either 10 ng/mL TcdA (A) or 0.2 ng/mL TcdB (B) for 24 hours. Morphological changes in cells were observed under a phase-contrast microscope, and the percentage of cell rounding was assessed. C, For in vivo neutralization, mice were injected intraperitoneally with the indicated doses of ABA or a mixture of the individual heavy-chain-only antibody (VHH) components, followed 1 hour later by intraperitoneal inoculation with a mixture of TcdA and TcdB (25 ng each/mouse). Overall mouse survival was analyzed by Kaplan–Meier survival curves. Abbreviation: PBS, phosphate-buffered saline.

Article Snippet: For pull-down selection, the recombinant TcdA and TcdB [ 22 ] proteins were biotinylated using the Pierce EZ-Link NHS-PEG4 Biotin kit (Pierce Biotechnology, Rockford, IL) per the manufacturer's instructions.

Techniques: In Vitro, In Vivo, Neutralization, Microscopy, Injection