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The Journal of Experimental Medicine Oct 2023The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate...
The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate immunity by activating the human NLRP1 inflammasome. Here, we report that ZAKα and NLRP1 sense bacterial exotoxins that target ribosome elongation factors. One such toxin, diphtheria toxin (DT), the causative agent for human diphtheria, triggers RSR-dependent inflammasome activation in primary human keratinocytes. This process requires iron-mediated DT production in the bacteria, as well as diphthamide synthesis and ZAKα/p38-driven NLRP1 phosphorylation in host cells. NLRP1 deletion abrogates IL-1β and IL-18 secretion by DT-intoxicated keratinocytes, while ZAKα deletion or inhibition additionally limits both pyroptotic and inflammasome-independent non-pyroptotic cell death. Consequently, pharmacologic inhibition of ZAKα is more effective than caspase-1 inhibition at protecting the epidermal barrier in a 3D skin model of cutaneous diphtheria. In summary, these findings implicate ZAKα-driven RSR and the NLRP1 inflammasome in antibacterial immunity and might explain certain aspects of diphtheria pathogenesis.
Topics: Humans; Diphtheria Toxin; Diphtheria; Inflammasomes; Pyroptosis; Immunity, Innate; NLR Proteins
PubMed: 37642997
DOI: 10.1084/jem.20230105 -
Science Advances May 2020Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took...
Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 ()-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b ()-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.
Topics: Diphtheria Toxin; Endocytosis; Endosomes; Humans; Lysosomes; RNA, Small Interfering
PubMed: 32917630
DOI: 10.1126/sciadv.aaz4848 -
Methods in Enzymology 2021Diphtheria toxin is among many bacterial toxins that utilize the endosomal pathway of cellular entry, which is ensured by the bridging of the endosomal membrane by the...
Diphtheria toxin is among many bacterial toxins that utilize the endosomal pathway of cellular entry, which is ensured by the bridging of the endosomal membrane by the toxin's translocation (T) domain. Endosomal acidification triggers a series of conformational changes of the T-domain, that take place first in aqueous and subsequently in membranous milieu. These rearrangements ultimately result in establishing membrane-inserted conformation(s) and translocation of the catalytic moiety of the toxin into the cytoplasm. We discuss here the strategy for combining site-selective labeling with various spectroscopic methods to characterize structural and thermodynamic aspects of protonation-dependent conformational switching and membrane insertion of the diphtheria toxin T-domain. Among the discussed methods are FRET, FCS and depth-dependent fluorescence quenching with lipid-attached bromine atoms and spin probes. The membrane-insertion pathway of the T-domain contains multiple intermediates and is governed by staggered pH-dependent transitions involving protonation of histidines and acidic residues. Presented data demonstrate that the lipid bilayer plays an active part in T-domain functioning and that the so-called Open-Channel State does not constitute the translocation pathway, but is likely to be a byproduct of the translocation. The spectroscopic approaches presented here are broadly applicable to many other systems of physiological and biomedical interest for which conformational changes can lead to membrane insertion (e.g., other bacterial toxins, host defense peptides, tumor-targeting pHLIP peptides and members of Bcl-2 family of apoptotic regulators).
Topics: Diphtheria Toxin; Hydrogen-Ion Concentration; Lipid Bilayers; Molecular Conformation; Protein Conformation; Thermodynamics
PubMed: 33712192
DOI: 10.1016/bs.mie.2020.12.016 -
Scientific Reports Jan 2020Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways and the produced diphtheria toxin...
Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (DT), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against DT (diphtheria antitoxin; DAT). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three DT domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. These recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAT.
Topics: Animals; Antibodies, Neutralizing; Corynebacterium diphtheriae; Diphtheria Toxin; Epitope Mapping; Guinea Pigs; Humans; Immunoglobulin G; Injections, Intradermal; Models, Molecular; Peptide Elongation Factor 2; Peptide Library; Protein Conformation; Single-Chain Antibodies
PubMed: 31953428
DOI: 10.1038/s41598-019-57103-5 -
Toxins Jun 2023Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for...
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pK values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKs ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pK values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.
Topics: Diphtheria Toxin; Histidine; Molecular Dynamics Simulation; Catalytic Domain; Protein Transport; Hydrogen-Ion Concentration; Protein Conformation
PubMed: 37505680
DOI: 10.3390/toxins15070410 -
Emerging Infectious Diseases Nov 2019Corynebacterium ulcerans infection is emerging in humans. We conducted phylogenetic analyses of C. ulcerans and C. diptheriae, which revealed diverse diphtheria toxin in...
Corynebacterium ulcerans infection is emerging in humans. We conducted phylogenetic analyses of C. ulcerans and C. diptheriae, which revealed diverse diphtheria toxin in C. ulcerans. Diphtheria toxin diversification could decrease effectiveness of diphtheria toxoid vaccine and diphtheria antitoxin for preventing and treating illnesses caused by this bacterium.
Topics: Amino Acid Sequence; Corynebacterium; Diphtheria; Diphtheria Toxin; Diphtheria Toxoid; Genetic Variation; Humans; Japan; Mutation; Phylogeny; RNA, Ribosomal, 16S
PubMed: 31625851
DOI: 10.3201/eid2511.181455 -
Journal of Pharmaceutical Sciences Jan 2020Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of...
Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH, (2) formaldehyde, (3) formaldehyde and NaCNBH followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatography-electrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4 T-cell epitopes were affected to some extent, one universal CD4 T-cell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine.
Topics: Borohydrides; Chromatography, Reverse-Phase; Diphtheria Toxin; Diphtheria Toxoid; Drug Compounding; Electrophoresis, Polyacrylamide Gel; Epitopes, T-Lymphocyte; Formaldehyde; Glycine; Models, Molecular; Protein Conformation; Spectrometry, Mass, Electrospray Ionization; Structure-Activity Relationship
PubMed: 31678246
DOI: 10.1016/j.xphs.2019.10.047 -
Journal of Clinical Microbiology Sep 2020Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of , with similar illness produced occasionally by...
Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of , with similar illness produced occasionally by toxigenic or, rarely, While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (). Nontoxigenic -bearing (NTTB) isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect and distinguish from the closely related species and Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for , Diph_, and CUP_ targets, respectively. Twenty-nine NTTB isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in or the promoter region. This new RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.
Topics: Corynebacterium; Corynebacterium diphtheriae; Diphtheria; Diphtheria Toxin; Humans; Real-Time Polymerase Chain Reaction
PubMed: 32727830
DOI: 10.1128/JCM.00639-20 -
International Journal of Molecular... Mar 2022, the etiological agent of diphtheria, is a re-emerging pathogen, responsible for several thousand deaths per year. In addition to diphtheria, systemic infections, often... (Review)
Review
, the etiological agent of diphtheria, is a re-emerging pathogen, responsible for several thousand deaths per year. In addition to diphtheria, systemic infections, often by non-toxigenic strains, are increasingly observed. This indicates that besides the well-studied and highly potent diphtheria toxin, various other virulence factors may influence the progression of the infection. This review focuses on the known components of responsible for adhesion, invasion, inflammation, and cell death, as well as on the cellular signaling pathways activated upon infection.
Topics: Corynebacterium; Corynebacterium diphtheriae; Diphtheria; Diphtheria Toxin; Humans; Virulence Factors
PubMed: 35328715
DOI: 10.3390/ijms23063298 -
Biomolecules Aug 2022Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like...
Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart's molecular characteristics.
Topics: Antineoplastic Agents; Cytotoxins; Diphtheria Toxin; Disulfides; Macromolecular Substances; Structure-Activity Relationship
PubMed: 36009005
DOI: 10.3390/biom12081111