Single-cell division tracing and transcriptomics reveal cell types and differentiation paths in the regenerating lung.

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.

Diphthamide - a conserved modification of eEF2 with clinical relevance.

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.

Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination.

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.

Analysis of Cre lines for targeting embryonic airway smooth muscle.

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.

Identification of novel B-1 transitional progenitors by B-1 lymphocyte fate-mapping transgenic mouse model Bhlhe41 dTomato-Cre.

B-1 lymphocytes exhibit specialized roles in host defense against multiple pathogens. Despite the fact that CD19+CD93+B220lo/- 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, Bhlhe41dTomato-Cre , for fate mapping and functional analysis of B-1 cells. Bhlhe41dTomato-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+B220lo/-) expressed Bhlhe41 and were identical to well-defined transitional B-1a progenitors (CD19+CD93+B220lo/-CD5+), which only gave rise to peritoneal B-1a cells. Moreover, we identified a novel population of neonatal splenic CD19hidTomato+B220hiCD43loCD5lo B cells, which differentiated to peritoneal B-1a and B-1b cells. Bhlhe41 deficiency impaired the balance between CD19hidTomato+B220lo/-CD5hi and CD19hidTomato+B220hiCD5lo cells. Hence, we identified neonatal CD19hidTomato+B220hiCD43loCD5lo 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.

An ADP-ribosyltransferase toxin kills bacterial cells by modifying structured non-coding RNAs.

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.

Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

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-10fl/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.

Structure-Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin.

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.

Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury.

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.

CCL17 Aggravates Myocardial Injury by Suppressing Recruitment of Regulatory T Cells.

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 (ß-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.

Diphtheria toxin induced but not CSF1R inhibitor mediated microglia ablation model leads to the loss of CSF/ventricular spaces in vivo that is independent of cytokine upregulation.

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.

Elevated murine HB-EGF confers sensitivity to diphtheria toxin in EGFR-mutant lung adenocarcinoma.

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.

Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice.

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-/-SNCAA53T 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.

Bacterial Toxins Escape the Endosome by Inducing Vesicle Budding and Collapse.

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.

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

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.

Control of axillary bud growth in tobacco through toxin gene expression system.

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.

Design of a Cytotoxic Neuroblastoma-Targeting Agent Using an Enzyme Acting on Polysialic Acid Fused to a Toxin.

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 IC50 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.

Conformational switching, refolding and membrane insertion of the diphtheria toxin translocation domain.

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).

Loss of Endogenously Cycling Adult Cardiomyocytes Worsens Myocardial Function.

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.

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy.

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.