Search

Protonation of key residues in the diphtheria toxin translocation (T)-domain triggered by endosomal acidification is critical for inducing a series of conformational transitions critical for the cellular entry of the toxin. Previous experiments revealed the importance of histidine residues in modulating pH-dependent transitions. They suggested the presence of a "safety latch" preventing premature refolding of the T-domain by a yet poorly understood mechanism. Here, we used constant-pH molecular dynamics simulations to systematically investigate the protonation sequence in the wild-type T-domain and the following mutants: H223Q, H257Q, E259Q, and H223Q/H257Q. Comparison of these computational results with previous experimental data on T-domain stability and activity with the H-to-Q replacements confirms the role of H223 (pK a  = 6.5) in delaying the protonation of the main trigger, H257 (pK a  = 2.2 in the WT and pK a  = 4.9 in H223Q). Our calculations also reveal a very low pK a for a neighboring acidic residue E259, which does not get protonated even during simulations at pH 3. This residue also contributes to the formation of the safety latch, with the pK a of H257 increasing from 2.2 to 5.1 upon E259Q replacement. In contrast, the latter replacement has virtually no effect on the protonation of the H223. Thus, we conclude that the interplay of the protonation in the H223/H257/E259 triad has evolved to prevent triggering the accidental refolding of the T-domain by a fluctuation in the protonation of the main trigger at neutral pH, before the incorporation of the toxin inside the endosome. Subsequent acidification of the endosome overcomes the safety latch and triggers conformational switching via repulsion of H223 + and H257 + . This protonation/conformation relationship corroborates experimental findings and offers a detailed stepwise molecular description of the transition mechanism, which can be instrumental in optimizing the potential applications of the T-domain for targeted delivery of therapies to tumors and other diseased acidic tissues., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α 1 -antitrypsin (α 1 AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α 1 AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α 1 AT. Since α 1 AT is commercially available as a drug for treatment of the α 1 AT deficiency, it could be repurposed for treatment of toxin-mediated diseases., (© 2024. The Author(s).)

Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of therapeutic approaches for lung diseases. We combine mouse models allowing diphtheria toxin-mediated damage of specific epithelial cell types and parallel GFP-labeling of functionally dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncover cell types, including Krt13 + basal and Krt15 + club cells, detect an intermediate cell state between basal and goblet cells, reveal goblet cells as actively dividing progenitor cells, and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also show that diphtheria toxin-expressing cells can persist in the lung, express specific inflammatory factors, and transcriptionally resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to concise epithelial injury, encompassing proliferation, differentiation, and cell-to-cell interactions., (© 2024. The Author(s).)

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases., Competing Interests: Declaration of interests U.B. is employed by Roche Pharma Research and Early Development, and is co-inventor on patent applications that cover assays to detect presence or absence of diphthamide. Roche is interested in targeted therapies and diagnostics. R.S. declares no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor-mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA-mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated., Methods: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays., Results: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells., Conclusion: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

During development of the embryonic mouse lung, the pulmonary mesenchyme differentiates into smooth muscle that wraps around the airway epithelium. Inhibiting smooth muscle differentiation leads to cystic airways, while enhancing it stunts epithelial branching. These findings support a conceptual model wherein the differentiation of smooth muscle sculpts the growing epithelium into branches at precise positions and with stereotyped morphologies. Unfortunately, most approaches to manipulate the differentiation of airway smooth muscle rely on pharmacological or physical perturbations that are conducted ex vivo. Here, we explored the use of diphtheria toxin-based genetic ablation strategies to eliminate airway smooth muscle in the embryonic mouse lung. Surprisingly, neither airway smooth muscle wrapping nor epithelial branching were affected in embryos in which the expression of diphtheria toxin or its receptor were driven by several different smooth muscle-specific Cre lines. Close examination of spatial patterns of Cre activity in the embryonic lung revealed that none of these commonly used Cre lines target embryonic airway smooth muscle robustly or specifically. Our findings demonstrate the need for airway smooth muscle-specific Cre lines that are active in the embryonic lung, and serve as a resource for researchers contemplating the use of these commonly used Cre lines for studying embryonic airway smooth muscle., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre +/- × IL-10 fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (μMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

B-1 lymphocytes exhibit specialized roles in host defense against multiple pathogens. Despite the fact that CD19 + CD93 + B220 lo/- B cells have been identified as B-1 progenitors, the definition for B-1 progenitors remains to be elucidated as CD19 + CD93 + B220 + B cells are capable to give rise to B-1 cells. Given that transcription factor Bhlhe41 is highly and preferentially expressed in B-1 cells and regulates B-1a cell development, we generated a transgenic mouse model, Bhlhe41 dTomato-Cre , for fate mapping and functional analysis of B-1 cells. Bhlhe41 dTomato-Cre mice efficiently traced Bhlhe41 expression, which was mainly restricted to B-1 cells in B-cell lineage. We showed an efficient and specific Cre-mediated DNA recombination in adult B-1 cells and neonatal B-1 progenitors rather than B-2 cells by flow cytometric analysis of Bhlhe41 dTomato-Cre/+ Rosa26 EYFP mice. Treatment of Bhlhe41 dTomato-Cre/+ Rosa26 iDTR mice with diphtheria toxin revealed a robust efficacy of B-1 cell depletion. Interestingly, using Bhlhe41 dTomato-Cre mice, we demonstrated that neonatal B-1 progenitors (CD19 + CD93 + B220 lo/- ) expressed Bhlhe41 and were identical to well-defined transitional B-1a progenitors (CD19 + CD93 + B220 lo/- CD5 + ), which only gave rise to peritoneal B-1a cells. Moreover, we identified a novel population of neonatal splenic CD19 hi dTomato + B220 hi CD43 lo CD5 lo B cells, which differentiated to peritoneal B-1a and B-1b cells. Bhlhe41 deficiency impaired the balance between CD19 hi dTomato + B220 lo/- CD5 hi and CD19 hi dTomato + B220 hi CD5 lo cells. Hence, we identified neonatal CD19 hi dTomato + B220 hi CD43 lo CD5 lo B cells as novel transitional B-1 progenitors. Bhlhe41 dTomato-Cre/+ mouse can be used for fate mapping and functional studies of B-1 cells in host-immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Tang, Ren, Bai, Hu, Jia, Cao, Hong, Xu, Gao, Shi, Pan, Wang, Zheng, Zhao and Wang.)

ADP-ribosyltransferases (ARTs) were among the first identified bacterial virulence factors. Canonical ART toxins are delivered into host cells where they modify essential proteins, thereby inactivating cellular processes and promoting pathogenesis. Our understanding of ARTs has since expanded beyond protein-targeting toxins to include antibiotic inactivation and DNA damage repair. Here, we report the discovery of RhsP2 as an ART toxin delivered between competing bacteria by a type VI secretion system of Pseudomonas aeruginosa. A structure of RhsP2 reveals that it resembles protein-targeting ARTs such as diphtheria toxin. Remarkably, however, RhsP2 ADP-ribosylates 2'-hydroxyl groups of double-stranded RNA, and thus, its activity is highly promiscuous with identified cellular targets including the tRNA pool and the RNA-processing ribozyme, ribonuclease P. Consequently, cell death arises from the inhibition of translation and disruption of tRNA processing. Overall, our data demonstrate a previously undescribed mechanism of bacterial antagonism and uncover an unprecedented activity catalyzed by ART enzymes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Animal models are important to mimic certain pathways or biological aspects of human pathologies including acute and chronic pulmonary diseases. We developed a novel and flexible mouse model of acute epithelial lung injury based on adeno-associated virus (AAV) variant 6.2-mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial and alveolar epithelial cells can be observed. In contrast to other lung injury models, the here described mouse model provides the possibility of targeted injury using specific tropisms of AAV vectors or cell-type-specific promotors to drive the human DTR expression. Also, generation of cell-specific mouse lines is not required. Detailed characterization of the AAV-DTR/DT mouse model including titration of viral genome (vg) load and administered DT amount revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and unspecified cells) and elevation of degenerated cells and infiltrated leukocytes in lung tissue, dependent of vg load and DT dose. Cytokine levels in BAL fluid showed different patterns with higher vg load, e.g., IFNγ, TNFα, and IP10 increasing and IL-5 and IL-6 decreasing, whereas lung function was not affected. In addition, laser-capture microdissection (LCM)-based proteomics of bronchial epithelium and alveolar tissue revealed upregulated immune and inflammatory responses in all regions and extracellular matrix deposition in infiltrated alveoli. Overall, our novel AAV-DTR/DT model allows investigation of repair mechanisms following epithelial injury and resembles specific mechanistic aspects of acute and chronic pulmonary diseases.

Background: Recent studies have established that CCR2 (C-C chemokine receptor type 2) marks proinflammatory subsets of monocytes, macrophages, and dendritic cells that contribute to adverse left ventricle (LV) remodeling and heart failure progression. Elucidation of the effector mechanisms that mediate adverse effects of CCR2 + monocytes, macrophages, and dendritic cells will yield important insights into therapeutic strategies to suppress myocardial inflammation., Methods: We used mouse models of reperfused myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate CCL17 (C-C chemokine ligand 17). We used Ccl17 knockout mice, flow cytometry, RNA sequencing, biochemical assays, cell trafficking studies, and in vivo cell depletion to identify the cell types that generate CCL17, define signaling pathways that controlled its expression, delineate the functional importance of CCL17 in adverse LV remodeling and heart failure progression, and determine the mechanistic basis by which CCL17 exerts its effects., Results: We demonstrated that CCL17 is expressed in CCR2 + macrophages and cluster of differentiation 11b + conventional dendritic cells after myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation. We clarified the transcriptional signature of CCL17 + macrophages and dendritic cells and identified granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling as a key regulator of CCL17 expression through cooperative activation of STAT5 (signal transducer and activator of transcription 5) and canonical NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling. Ccl17 deletion resulted in reduced LV remodeling, decreased myocardial fibrosis and cardiomyocyte hypertrophy, and improved LV systolic function after myocardial infarction and angiotensin II and phenylephrine infusion. We observed increased abundance of regulatory T cells (Tregs) in the myocardium of injured Ccl17 knockout mice. CCL17 inhibited Treg recruitment through biased activation of CCR4. CCL17 activated Gq signaling and CCL22 (C-C chemokine ligand 22) activated both Gq and ARRB (β-arrestin) signaling downstream of CCR4. CCL17 competitively inhibited CCL22 stimulated ARRB signaling and Treg migration. We provide evidence that Tregs mediated the protective effects of Ccl17 deletion on myocardial inflammation and adverse LV remodeling., Conclusions: These findings identify CCL17 as a proinflammatory mediator of CCR2 + macrophages and dendritic cells and suggest that inhibition of CCL17 may serve as an effective strategy to promote Treg recruitment and suppress myocardial inflammation.

Background: Two recently developed novel rodent models have been reported to ablate microglia, either by genetically targeting microglia (via Cx3cr1-creER: iDTR + Dtx) or through pharmacologically targeting the CSF1R receptor with its inhibitor (PLX5622). Both models have been widely used in recent years to define essential functions of microglia and have led to high impact studies that have moved the field forward., Methods: Using either Cx3cr1-iDTR mice in combination with Dtx or via the PLX5622 diet to pharmacologically ablate microglia, we compared the two models via MRI and histology to study the general anatomy of the brain and the CSF/ventricular systems. Additionally, we analyzed the cytokine profile in both microglia ablation models., Results: We discovered that the genetic ablation (Cx3cr1-iDTR + Dtx), but not the pharmacological microglia ablation (PLX5622), displays a surprisingly rapid pathological condition in the brain represented by loss of CSF/ventricles without brain parenchymal swelling. This phenotype was observed both in MRI and histological analysis. To our surprise, we discovered that the iDTR allele alone leads to the loss of CSF/ventricles phenotype following diphtheria toxin (Dtx) treatment independent of cre expression. To examine the underlying mechanism for the loss of CSF in the Cx3cr1-iDTR ablation and iDTR models, we additionally investigated the cytokine profile in the Cx3cr1-iDTR + Dtx, iDTR + Dtx and the PLX models. We found increases of multiple cytokines in the Cx3cr1-iDTR + Dtx but not in the pharmacological ablation model nor the iDTR + Dtx mouse brains at the time of CSF loss (3 days after the first Dtx injection). This result suggests that the upregulation of cytokines is not the cause of the loss of CSF, which is supported by our data indicating that brain parenchyma swelling, or edema are not observed in the Cx3cr1-iDTR + Dtx microglia ablation model. Additionally, pharmacological inhibition of the KC/CXCR2 pathway (the most upregulated cytokine in the Cx3cr1-iDTR + Dtx model) did not resolve the CSF/ventricular loss phenotype in the genetic microglia ablation model. Instead, both the Cx3cr1-iDTR + Dtx ablation and iDTR + Dtx models showed increased activated IBA1 + cells in the choroid plexus (CP), suggesting that CP-related pathology might be the contributing factor for the observed CSF/ventricular shrinkage phenotype., Conclusions: Our data, for the first time, reveal a robust and global CSF/ventricular space shrinkage pathology in the Cx3cr1-iDTR genetic ablation model caused by iDTR allele, but not in the PLX5622 ablation model, and suggest that this pathology is not due to brain edema formation but to CP related pathology. Given the wide utilization of the iDTR allele and the Cx3cr1-iDTR model, it is crucial to fully characterize this pathology to understand the underlying causal mechanisms. Specifically, caution is needed when utilizing this model to interpret subtle neurologic functional changes that are thought to be mediated by microglia but could, instead, be due to CSF/ventricular loss in the genetic ablation model., (© 2021. The Author(s).)

Bacterial protein toxins autonomously enter the cytosol of the target cell where they modify the activities of host components to exert their toxic effects. Many of the toxins enter the host cell by endocytosis followed by endosomal escape. However, their mechanism of endosomal escape remains unresolved. We show herein that diphtheria toxin (DT) and NleC of enteropathogenic Escherichia coli exit the endosome by inducing budding and collapse of small toxin-enriched vesicles from the endosomal membrane.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Conditional ablation of defined cell populations in vivo can be achieved using genetically engineered mice in which the human diphtheria toxin (DT) receptor (DTR) is placed under control of a murine tissue-specific promotor, such that delivery of DT selectively ablates cells expressing this high-affinity human DTR; cells expressing only the endogenous low-affinity mouse DTR are assumed to be unaffected. Surprisingly, we found that systemic administration of DT induced rapid regression of murine lung adenocarcinomas that express human mutant EGFR in the absence of a transgenic allele containing human DTR. DT enzymatic activity was required for tumor regression, and mutant EGFR-expressing tumor cells were the primary target of DT toxicity. In FVB mice, EGFR-mutant tumors upregulated expression of HBEGF, which is the DTR in mice and humans. HBEGF blockade with the enzymatically inactive DT mutant CRM197 partially abrogated tumor regression induced by DT. These results suggest that elevated expression of murine HBEGF, i.e. the low-affinity DTR, confers sensitivity to DT in EGFR-mutant tumors, demonstrating a biological effect of DT in mice lacking transgenic DTR alleles and highlighting a unique vulnerability of EGFR-mutant lung cancers., Competing Interests: Competing interests S.M.K. is Academic Editor at the Journal of Experimental Medicine and has a consultant/advisory board relationship with Celsius Therapeutics and Evolveimmune. K.P. reports receiving commercial research grants (to the Institution) from AstraZeneca, Boehringer Ingelheim and Roche/Genentech; has an ownership interest (including patents) in MSKCC/Molecular MD; and has an unpaid consultant/advisory board relationship with the Lung Cancer Research Foundation. No competing interests are declared by other authors., (© 2021. Published by The Company of Biologists Ltd.)

Polysialic acid, an abundant cell surface component of the developing nervous system, which declines rapidly postnatally to virtual absence in the majority of adult tissues, is highly expressed in some malignant tumors including neuroblastoma. We found that the binding of a noncatalytic endosialidase to polysialic acid causes internalization of the complex from the surface of neuroblastoma kSK-N-SH cells, a subline of SK-N-SH, and leads to a complete relocalization of polysialic acid to the intracellular compartment. The binding and uptake of the endosialidase is polysialic acid-dependent as it is inhibited by free excess ligand or removal of polysialic acid by active endosialidase, and does not happen if catalytic endosialidase is used in place of inactive endosialidase. A fusion protein composed of the noncatalytic endosialidase and the cytotoxic portion of diphtheria toxin was prepared to investigate whether the cellular uptake observed could be used for the specific elimination of polysialic acid-containing cells. The conjugate toxin was found to be toxic to polysialic acid-positive kSK-N-SH with an IC 50 of 1.0 nmol/L. Replacing the noncatalytic endosialidase with active endosialidase decreased the activity to the level of nonconjugated toxin. Normal nonmalignant cells were selectively resistant to the toxin conjugate. The results demonstrate that noncatalytic endosialidase induces a quantitative removal and cellular uptake of polysialic acid from the cell surface which, by conjugation with diphtheria toxin fragment, can be exploited for the selective elimination of polysialic acid-containing tumor cells., (©2021 American Association for Cancer Research.)

The control of axillary bud development after removing the terminal buds (topping) of plants is a research hotspot, and the control of gene expression, like switching on and off, allows us to further study biological traits of interest, such as plant branching and fertility. In this study, a toxin gene control system for plants based on dexamethasone (DEX) induction was constructed, and the positive transgenic tobacco exhibited growth retardation in the application area (axillary bud). The expression level of the lethal Diphtheria toxin A (DTA) gene under different DEX concentrations at different application days was analyzed. The highest expression levels appeared at 5 days after the leaf injection of DEX. The DTA transcripts were induced by 5 µM DEX and peaked in response to 50 µM DEX at 5 days after leaf injection. Here, a chemical induction system, combined with a toxin gene, were used to successfully control the growth of tobacco axillary buds after topping. The DTA expression system under DEX induction was sensitive and efficient, therefore, can be used to control axillary bud growth and development in tobacco., (© 2021. The Author(s).)

Rodent models of Parkinson's disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1 -/- SNCA A53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.

Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation., Competing Interests: SE was employed by Institute of Molecular Biology GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kornstädt, Pierre, Weigert, Ebersberger, Schäufele, Kolbinger, Schmid, Cohnen, Thomas, Ferreirós, Brüne, Ebersberger and Scholich.)

Rationale: Endogenously cycling adult cardiomyocytes increase after myocardial infarction (MI) but remain scarce and are generally thought not to contribute to myocardial function. However, this broadly held assumption has not been tested, mainly because of the lack of transgenic reporters that restrict Cre expression to adult cardiomyocytes that reenter the cell cycle., Objective: We created and validated a new transgenic mouse, αMHC (alpha myosin heavy chain) -MerDreMer-Ki67p-RoxedCre (denoted αDKRC [cardiomyocyte-specific αMHC-MerDreMer-Ki67p-RoxedCre]) that restricts Cre expression to cycling adult cardiomyocytes and uniquely integrates spatial and temporal adult cardiomyocyte cycling events based on the DNA specificities of orthologous Dre and Cre recombinases. We then created αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes and examined the effects of ablating these endogenously cycling cardiomyocytes on myocardial function after ischemic-reperfusion (I/R) MI., Methods and Results: A tandem αDKRC transgene was designed, validated in cultured cells, and used to make transgenic mice. The αDKRC transgene integrated between MYH6 and MYH7 and did not disrupt expression of the surrounding genes. Compared with controls, αDKRC::RLTG (Rox-Lox-tdTomato-eGFP ) mice treated with Tamoxifen expressed tdTomato+ in cardiomyocytes with rare Bromodeoxyuridine+, eGFP+ cardiomyocytes, consistent with reentry of the cell cycle. We then pretreated αDKRC::RLTG mice with Tamoxifen to activate the reporter before sham or reperfusion (I/R) MI surgeries. Compared with Sham surgery, the I/R MI group had increased single and paired eGFP+ (enhanced green fluorescent protein)+ cardiomyocytes predominantly in the border zones (5.8±0.5 versus 3.3±0.3 cardiomyocytes per 10-micron section, N=8-9 mice per group, n=16-24 sections per mouse), indicative of cycled cardiomyocytes. The single to paired eGFP+ cardiomyocyte ratio was ≈9 to 1 (5.2±0.4 single versus 0.6±0.2 paired cardiomyocytes) in the I/R MI group after MI, suggesting that cycling cardiomyocytes were more likely to undergo polyploidy than replication. The ablation of endogenously cycling adult cardiomyocytes in αDKRC::DTA (diphtheria) mice caused progressive worsening left ventricular chamber size and function after I/R MI, compared with controls., Conclusions: Although scarce, endogenously cycling adult cardiomyocytes contribute to myocardial function after injury, suggesting that these cells may be physiologically relevant.

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)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Diphtheria toxin, an exotoxin secreted by Corynebacterium that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5-6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2-3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry.

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing., Competing Interests: Competing interest statement: A patent has been filed that is partially overlapping with the manuscript content. Patent title: Split-immunotoxins for boosting oncolytic virus toxicity. Applicant: Ohio State Innovation Foundation and Research Institute at Nationwide Children’s Hospital. Inventors: V.P., E.K., D.S.K., and Timothy Cripe. Application No.: PCT/US2019/019621. Status of application: PCT pending.

AB toxins with built-in cell targeting and endosomal escape mechanisms are attractive intracellular delivery vehicles. However, their compatibility with nucleic-acid-based therapeutics is not fully explored. Arnold et al. demonstrated the first functional siRNA delivery by diphtheria toxin (DT) in vitro, marking an important step in expanding the utility of AB toxins for nucleic acid delivery., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers., Competing Interests: Competing interest statement: M.B. and K.J.F.S. are authors of a pending patent on use of RRSP as a therapeutic for cancer (WO2016019379A1). K.J.F.S. is author of a published patent on use of cysteine protease domain for autoprocessing of proteins (US20100137563A1). R.A.M. and G.L.B. are authors of a published patent on the use of diphtheria toxin for protein delivery and of a pending patent on RRSP-DTB as a RAS-directed therapeutic (US20180080033A1 and WO2019104433A1, respectively). K.J.F.S. has a significant financial interest in Situ Biosciences LLC, which conducts contract research unrelated to this work.

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 ( ITGB1 )-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b ( eIF-3b )-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b 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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

DAB 389 IL-2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T-cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta-gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6-tag fused at the N-terminus of DAB 389 IL-2 was used to express the soluble immunotoxin in E. coli Rosetta-gami (DE3). After the purification of the soluble protein by two-step column chromatographies, the structure of DAB 389 IL-2 was analyzed using the Native-PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB 389 IL-2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta-gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

BackgroundDiphtheria is a potentially fatal disease caused by toxigenic strains of Corynebacterium diphtheriae, C. ulcerans or C. pseudotuberculosis. AimOur objective was to review the epidemiology of diphtheria in the United Kingdom (UK) and the impact of recent changes in public health management and surveillance.MethodsPutative human toxigenic diphtheria isolates in the UK are sent for species confirmation and toxigenicity testing to the National Reference Laboratory. Clinical, epidemiological and microbiological information for toxigenic cases between 2009 and 2017 are described in this population-based prospective surveillance study.ResultsThere were 33 toxigenic cases of diphtheria aged 4 to 82 years. Causative species were C. diphtheriae (n = 18) and C. ulcerans (n = 15). Most C. diphtheriae cases were cutaneous (14/18) while more than half of C. ulcerans cases had respiratory presentations (8/15). Two thirds (23/33) of cases were inadequately immunised. Two cases with C. ulcerans infections died, both inadequately immunised. The major risk factor for C. diphtheriae aquisition was travel to an endemic area and for C. ulcerans, contact with a companion animal. Most confirmed C. diphtheriae or C. ulcerans isolates (441/507; 87%) submitted for toxigenicity testing were non-toxigenic , however, toxin positivity rates were higher (15/23) for C. ulcerans than C. diphtheriae (18/469). Ten non-toxigenic toxin gene-bearing (NTTB) C. diphtheriae were also detected.ConclusionDiphtheria is a rare disease in the UK. In the last decade, milder cutaneous C. diphtheriae cases have become more frequent. Incomplete vaccination status was strongly associated with the risk of hospitalisation and death.

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh-Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α-smooth muscle actin-labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β-HSD double-positive fetal LCs could be observed in Amh-Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β-HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs' central role in fetal testis development., (© 2020 Wiley Periodicals, Inc.)

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

In Western Europe, the incidence of both respiratory and cutaneous diphtheria, caused by toxin-producing Corynebacterium diphtheriae , Corynebacterium ulcerans or Corynebacterium pseudotuberculosis , has been low over the past few decades thanks to the use of an effective vaccine and a high level of vaccination coverage. However, the disease has still not been eradicated and continues to occur in all of Europe. In order to prevent sequelae or a fatal outcome, diphtheria antitoxin (DAT) should be administered to suspected diphtheria patients as soon as possible, but economic factors and issues concerning regulations have led to poor availability of DAT in many countries. The European Centre for Disease Prevention and Control and World Health Organization have called for European Union-wide solutions to this DAT-shortage. In order to illustrate the importance of these efforts and underline the need for continued diphtheria surveillance, we present data on all registered cases of toxigenic and non-toxigenic C. diphtheriae , C. ulcerans and C. pseudotuberculosis in Belgium during the past decade, up to and including 2017.

Diphtheria is one of the most well studied of all the bacterial infectious diseases. These milestone studies of toxigenic Corynebacterium diphtheriae along with its primary virulence determinant, diphtheria toxin, have established the paradigm for the study of other related bacterial protein toxins. This review highlights those studies that have contributed to our current understanding of the structure-function relationships of diphtheria toxin, the molecular mechanism of its entry into the eukaryotic cell cytosol, the regulation of diphtheria tox expression by holo-DtxR, and the molecular basis of transition metal ion activation of apo-DtxR itself. These seminal studies have laid the foundation for the protein engineering of diphtheria toxin and the development of highly potent eukaryotic cell-surface receptor-targeted fusion protein toxins for the treatment of human diseases that range from T cell malignancies to steroid-resistant graft-versus-host disease to metastatic melanoma. This deeper scientific understanding of diphtheria toxin and the regulation of its expression have metamorphosed the third-most-potent bacterial toxin known into a life-saving targeted protein therapeutic, thereby at least partially fulfilling Paul Erlich's concept of a magic bullet-"a chemical that binds to and specifically kills microbes or tumor cells."

Outer hair cells (OHCs) are responsible for the amplification of sound, and the death of these cells leads to hearing loss. Although the mechanisms for sound amplification and OHC death have been well investigated, the effects on the cochlea after OHC death are poorly understood. To study the consequences of OHC death, we established an OHC knockout system using a novel mouse model, Prestin-hDTR, which uses the prestin promoter to express the human diphtheria toxin (DT) receptor gene (hDTR). Administration of DT to adult Prestin-hDTR mice results in the depletion of almost all OHCs without significant damage to other cochlear and vestibular cells, suggesting that this system is an effective tool for the analysis of how other cells in the cochlea and vestibula are affected after OHC death. To evaluate the changes in the cochlea after OHC death, we performed differential gene expression analysis between the untreated and DT-treated groups of wild-type and Prestin-hDTR mice. This analysis revealed that genes associated with inflammatory/immune responses were significantly upregulated. Moreover, we found that several genes linked to hearing loss were strongly downregulated by OHC death. Together, these results suggest that this OHC knockout system is a useful tool to identify biomarkers associated with OHC death.

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

The protein translation elongation factor eEF2 undergoes a unique posttranslational modification called diphthamidation. eEF2 is an essential factor in protein translation, and the diphthamide modification has been a famous target of the diphtheria toxin for a long time. On the other hand, the physiological function of this rare modification in vivo remains unknown. Recent studies have suggested that diphthamide has specific functions for the cellular stress response and active proliferation. In this review, we summarize the history and findings of diphthamide obtained to date and discuss the possibility of a specific function for diphthamide in regulating protein translation.

Senescent cells accumulate in tissues during aging and are considered to underlie several aging-associated phenotypes and diseases. We recently reported that the elimination of p19 ARF -expressing senescent cells from lung tissue restored tissue function and gene expression in middle-aged (12-month-old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19 ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19 ARF -expressing cells prevented lung tissue from elastase-induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema-associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure-function relationships in membranous systems. The resulting structural heterogeneity is especially evident for peripheral and spontaneously inserting membrane proteins, which are not constrained by the well-defined transmembrane topology and exert their action in the context of intimate interaction with lipids. Here, we propose a concerted approach combining depth-dependent fluorescence quenching with Molecular Dynamics simulation to decipher dynamic interactions of membrane proteins with the lipid bilayers. We apply this approach to characterize membrane-mediated action of the diphtheria toxin translocation domain. First, we use a combination of the steady-state and time-resolved fluorescence spectroscopy to characterize bilayer penetration of the NBD probe selectively attached to different sites of the protein into membranes containing lipid-attached nitroxyl quenching groups. The constructed quenching profiles are analyzed with the Distribution Analysis methodology allowing for accurate determination of transverse distribution of the probe. The results obtained for 12 NBD-labeled single-Cys mutants are consistent with the so-called Open-Channel topology model. The experimentally determined quenching profiles for labeling sites corresponding to L350, N373, and P378 were used as initial constraints for positioning TH8-9 hairpin into the lipid bilayer for Molecular Dynamics simulation. Finally, we used alchemical free energy calculations to characterize protonation of E362 in soluble translocation domain and membrane-inserted conformation of its TH8-9 fragment. Our results indicate that membrane partitioning of the neutral E362 is more favorable energetically (by ~ 6 kcal/mol), but causes stronger perturbation of the bilayer, than the charged E362.

The homologous recombination (HR) pathway is a promising target for cancer therapy as it is frequently upregulated in tumors. One such strategy is to target tumors with cancer-specific, hyperactive promoters of HR genes including RAD51 and RAD51C. However, the promoter size and the delivery method have limited its potential clinical applications. Here we identified the ~2.1 kb promoter of XRCC2, similar to ~6.5 kb RAD51 promoter, as also hyperactivated in cancer cells. We found that XRCC2 expression is upregulated in nearly all types of cancers, to a degree comparable to RAD51 while much higher than RAD51C. Further study demonstrated that XRCC2 promoter is hyperactivated in cancer cell lines, and diphtheria toxin A (DTA) gene driven by XRCC2 promoter specifically eliminates cancer cells. Moreover, lentiviral vectors containing XRCC2 promoter driving firefly luciferase or DTA were created and applied to subcutaneous HeLa xenograft mice. We demonstrated that the pXRCC2-luciferase lentivirus is an effective tool for in vivo cancer visualization. Most importantly, pXRCC2-DTA lentivirus significantly inhibited the growth of HeLa xenografts in comparison to the control group. In summary, our results strongly indicate that virus-mediated delivery of constructs built upon the XRCC2 promoter holds great potential for tumor diagnosis and therapy.

Prematurely born children often develop neurodevelopmental delay that has been correlated with reduced growth and microstructural alterations in the cerebral cortex. Much research has focused on apoptotic neuronal cell death as a key neuropathological features following preterm brain injuries. How scattered apoptotic death of neurons may contribute to microstructural alterations remains unknown. The present study investigated in a rat model the effects of targeted neuronal apoptosis on cortical microstructure using in vivo MRI imaging combined with neuronal reconstruction and histological analysis. We describe that mild, targeted death of layer IV neurons in the developing rat cortex induces MRI-defined metabolic and microstructural alterations including increased cortical fractional anisotropy. Delayed architectural modifications in cortical gray matter and myelin abnormalities in the subcortical white matter such as hypomyelination and microglia activation follow the acute phase of neuronal death and axonal degeneration. These results establish the link between mild cortical apoptosis and MRI-defined microstructure changes that are reminiscent to those previously observed in preterm babies., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The human pathogen Corynebacterium diphtheriae is the causative agent of diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced more than a decade ago, not much is known about its transcriptome. Our aim was to use transcriptome sequencing (RNA-Seq) to close this knowledge gap and gain insights into the transcriptional landscape of a C. diphtheriae tox + strain., Results: We applied two different RNA-Seq techniques, one to retrieve 5'-ends of primary transcripts and the other to characterize the whole transcriptional landscape in order to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1656 transcription start sites (TSS), of which 1202 were assigned to genes and 454 to putative novel transcripts. By using the TSS data promoter regions recognized by the housekeeping sigma factor σ A and its motifs were analyzed in detail, revealing a well conserved -10 but an only weakly conserved -35 motif, respectively. Furthermore, with the TSS data 5'-UTR lengths were explored. The observed 5'-UTRs range from zero length (leaderless transcripts), which make up 20% of all genes, up to over 450 nt long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 471 operons which are further divided into 167 sub-operon structures. In a differential expression analysis approach, we discovered that genetic disruption of the iron-sensing transcription regulator DtxR, which controls expression of diphtheria toxin (DT), causes a strong influence on general gene expression. Nearly 15% of the genome is differentially transcribed, indicating that DtxR might have other regulatory functions in addition to regulation of iron metabolism and DT. Furthermore, our findings shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for two tox antisense RNAs, which point to a new way of transcriptional regulation of toxin production., Conclusions: This study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding gene characterization, transcriptional regulatory networks, and regulation of the tox gene in particular.

Background/aims: The abnormally activated EGFR promotes tumor growth, invasion and metastasis. Current therapeutics targeting EGFR have markedly improved the clinical outcome, but they are limited in use due to transient efficacy, frequent administration, high cost and significant toxicity., Methods: We rationally designed a multiepitope immunogen against EGFR, named as DEGFRm. The immunogen is composed of an epitope peptide (EGFR265-283) and the extracellular domain III (EGFR334-505) of mouse EGFR. EGFR265-283 is grafted onto the translocation domain of diphtheria toxin (DTT), and EGFR334-505 is fused to C-terminal of DTT. Next, the immunogenicity and anti-tumor efficacies of DEGFRm vaccine were examined in mouse tumor models., Results: When formulated with Alum and CpG, DEGFRm vaccine elicits Th 1 immune responses and inhibits tumor growth in both prophylactic and therapeutic mouse tumor models. Moreover, the tumor microvasculature is markedly reduced and the tumor infiltration of CD8+ T lymphocytes is greatly enhanced., Conclusions: These data suggest that active immunization with DEGFRm vaccine is a promising strategy for therapy of various EGFR+ cancers., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Recombinase responsive mouse lines expressing diphtheria toxin subunit A (DTA) are well established tools for targeted ablation of genetically defined cell populations. Here we describe a new knock-in allele at the Gt(Rosa)26Sor locus that retains the best features of previously described DTA alleles-including a CAG promoter, attenuated mutant DTA cDNA, and ubiquitous EGFP labeling-with the addition of a Cre-dependent FLEx switch for tight control of expression. The FLEx switch consists of two pairs of antiparallel lox sites requiring Cre-mediated recombination for inversion of the DTA to the proper orientation for transcription. We demonstrate its utility by Cre-dependent ablation of both a broad domain in the embryonic nervous system and a discrete population of cells in the fetal gonads. We conclude that this new DTA line is useful for targeted ablation of genetically-defined cell populations., (© 2017 Wiley Periodicals, Inc.)

Over the course of a 4-day period of metamorphosis, the Drosophila larval nervous system is remodeled to prepare for adult-specific behaviors. One example is the reorganization of peripheral nerves in the abdomen, where five pairs of abdominal nerves (A4-A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re-appear on the third day of metamorphosis. Perineurial glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub-Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT-1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT-1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. The studies will help to establish the groundwork for further work on cellular and molecular processes that underlie the co-ordinated remodeling of glia and the peripheral nerves they ensheath. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1144-1160, 2017., (© 2017 Wiley Periodicals, Inc.)