A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Tat-dependent conditionally replicating adenoviruses expressing diphtheria toxin A for specifically killing HIV-1-infected cells.

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effects and drive the emergence of drug-resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1-infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1-infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1-infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1-negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. The findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.

Neutralizing anti-diphtheria toxin scFv produced by phage display.

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. MATERIALS AND METHODS: The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. RESULTS: The size of the constructed scFv library was calculated to be 1.3 × 106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107 M-1. CONCLUSION: This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

Whole lung proteome of an acute epithelial injury mouse model in comparison to spatially resolved proteomes.

Epithelial injury is one of the major drivers of acute pulmonary diseases. Recurring injury followed by aberrant repair is considered as the primary cause of chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Preclinical in vivo models allow studying early disease-driving mechanisms like the recently established adeno-associated virus-diphtheria toxin receptor (AAV-DTR) mouse model of acute epithelial lung injury, which utilises AAV mediated expression of the human DTR. We performed quantitative proteomics of homogenised lung samples from this model and compared the results to spatially resolved proteomics data of epithelial cell regions from the same animals. In whole lung tissue proteins involved in cGAS-STING and interferon pathways, proliferation, DNA replication and the composition of the provisional extracellular matrix were upregulated upon injury. Besides epithelial cell markers SP-A, SP-C and Scgb1a1, proteins involved in cilium assembly, lipid metabolism and redox pathways were among downregulated proteins. Comparison of the bulk to spatially resolved proteomics data revealed a large overlap of protein changes and striking differences. Together our study underpins the broad usability of bulk proteomics and pinpoints to the benefit of sophisticated proteomic analyses of specific tissue regions or single cell types.

A collection of genetic mouse lines and related tools for inducible and reversible intersectional mis-expression.

Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches using two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach uses available lines expressing tTA/rTA to control the timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf), and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms.

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

The molecular mechanisms of the bacterial iron sensor IdeR.

Life came to depend on iron as a cofactor for many essential enzymatic reactions. However, once the atmosphere was oxygenated, iron became both scarce and toxic. Therefore, complex mechanisms have evolved to scavenge iron from an environment in which it is poorly bioavailable, and to tightly regulate intracellular iron contents. In bacteria, this is typically accomplished with the help of one key regulator, an iron-sensing transcription factor. While Gram-negative bacteria and Gram-positive species with low guanine-cytosine (GC) content generally use Fur (ferric uptake regulator) proteins to regulate iron homeostasis, Gram-positive species with high GC content use the functional homolog IdeR (iron-dependent regulator). IdeR controls the expression of iron acquisition and storage genes, repressing the former, and activating the latter in an iron-dependent manner. In bacterial pathogens such as Corynebacterium diphtheriae and Mycobacterium tuberculosis, IdeR is also involved in virulence, whereas in non-pathogenic species such as Streptomyces, it regulates secondary metabolism as well. Although in recent years the focus of research on IdeR has shifted towards drug development, there is much left to learn about the molecular mechanisms of IdeR. Here, we summarize our current understanding of how this important bacterial transcriptional regulator represses and activates transcription, how it is allosterically activated by iron binding, and how it recognizes its DNA target sites, highlighting the open questions that remain to be addressed.

Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts.

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.

Genomic Analysis of Corynebacterium diphtheriae Strains Isolated in the Years 2007-2022 with a Report on the Identification of the First Non-Toxigenic tox Gene-Bearing Strain in Poland.

Infections caused by non-toxigenic Corynebacterium diphtheriae have been reported every year in Poland since 2004, with the ST8 biovar gravis strains being most commonly isolated. This study analyzed thirty strains isolated between 2017 and 2022 and six previously isolated strains. All the strains were characterized using classic methods in terms of species, biovar level, and diphtheria toxin production, as well as by means of whole genome sequencing. The phylogenetic relationship based on SNP analysis was determined. The number of C. diphtheriae infections has been rising in Poland every year with a maximum of 22 cases in the year 2019. Since 2022, only the non-toxigenic gravis ST8 (most common) and mitis ST439 (less common) strains have been isolated. An analysis of the genomes of the ST8 strains showed that they had many potential virulence factors, such as adhesins and iron-uptake systems. The situation rapidly changed in 2022 and strains from different STs were isolated (ST32, 40, and 819). The ST40 biovar mitis strain was found to be non-toxigenic tox gene-bearing (NTTB), with the tox gene inactivated due to a single nucleotide deletion. Such strains were previously isolated in Belarus. The sudden appearance of new C. diphtheriae strains with different STs and the isolation of the first NTTB strain in Poland indicate that C. diphtheriae should be classified as a pathogen of special public health concern.

In Silico Identification of 1-DTP Inhibitors of Corynebacterium diphtheriae Using Phytochemicals from Andrographis paniculata.

A number of phytochemicals have been identified as promising drug molecules against a variety of diseases using an in-silico approach. The current research uses this approach to identify the phyto-derived drugs from Andrographis paniculata (Burm. f.) Wall. ex Nees (AP) for the treatment of diphtheria. In the present study, 18 bioactive molecules from Andrographis paniculata (obtained from the PubChem database) were docked against the diphtheria toxin using the AutoDock vina tool. Visualization of the top four molecules with the best dockscore, namely bisandrographolide (-10.4), andrographiside (-9.5), isoandrographolide (-9.4), and neoandrographolide (-9.1), helps gain a better understanding of the molecular interactions. Further screening using molecular dynamics simulation studies led to the identification of bisandrographolide and andrographiside as hit compounds. Investigation of pharmacokinetic properties, mainly ADMET, along with Lipinski's rule and binding affinity considerations, narrowed down the search for a potent drug to bisandrographolide, which was the only molecule to be negative for AMES toxicity. Thus, further modification of this compound followed by in vitro and in vivo studies can be used to examine itseffectiveness against diphtheria.

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.

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.

An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion.

BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Detection of diphtheria toxin production by toxigenic corynebacteria using an optimized Elek test.

PURPOSE: Diphtheria, still present in many countries of the world, is caused by toxigenic strains of species of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and the emerging zoonotic pathogen C. ulcerans. The immunoprecipitation test according to Elek is the gold standard for detection of the major virulence factor diphtheria toxin (DT) in toxigenic corynebacteria. Due to its sophisticated methodological requirements, the classical Elek test is performed mainly by specialized reference laboratories. It was revealed that the current modification of the Elek test does not detect the toxin in weakly toxigenic isolates. Therefore, a more robust method for detecting free DT is urgently needed, especially for toxigenic C. ulcerans strains which are known to produce often much lower amounts of DT than C. diphtheriae. METHODS: Thirty-one tox-positive C. ulcerans isolates with a negative standard Elek test result previously determined as NTTB (non-toxigenic tox bearing) were re-analyzed in this study using a modified immunoprecipitation method optimized regarding different parameters including type and concentration of antitoxin, medium volume, inoculum distance from the antitoxin disk and position of controls. RESULTS: All 31 C. ulcerans strains tested positive in the optimized Elek test. CONCLUSION: Only with a reliable and easy-to-handle method for detecting the toxigenicity of C. ulcerans, it is possible to assess the etiological role of this emerging zoonotic bacterium in human pathology.

Interactions between the Re-Emerging Pathogen Corynebacterium diphtheriae and Host Cells.

Corynebacterium diphtheriae, the etiological agent of diphtheria, is a re-emerging pathogen, responsible for several thousand deaths per year. In addition to diphtheria, systemic infections, often by non-toxigenic strains, are increasingly observed. This indicates that besides the well-studied and highly potent diphtheria toxin, various other virulence factors may influence the progression of the infection. This review focuses on the known components of C. diphtheriae responsible for adhesion, invasion, inflammation, and cell death, as well as on the cellular signaling pathways activated upon infection.

Advances on Delivery of Cytotoxic Enzymes as Anticancer Agents.

Cancer is one of the most serious human diseases, causing millions of deaths worldwide annually, and, therefore, it is one of the most investigated research disciplines. Developing efficient anticancer tools includes studying the effects of different natural enzymes of plant and microbial origin on tumor cells. The development of various smart delivery systems based on enzyme drugs has been conducted for more than two decades. Some of these delivery systems have been developed to the point that they have reached clinical stages, and a few have even found application in selected cancer treatments. Various biological, chemical, and physical approaches have been utilized to enhance their efficiencies by improving their delivery and targeting. In this paper, we review advanced delivery systems for enzyme drugs for use in cancer therapy. Their structure-based functions, mechanisms of action, fused forms with other peptides in terms of targeting and penetration, and other main results from in vivo and clinical studies of these advanced delivery systems are highlighted.

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice.

AIMS/HYPOTHESIS: Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. METHODS: We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. RESULTS: Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12-15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. CONCLUSIONS/INTERPRETATION: Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes. DATA AVAILABILITY: The single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164 ).

Development and patterning of rib primordia are dependent on associated musculature.

The importance of skeletal muscle for rib development and patterning in the mouse embryo has not been resolved, largely because different experimental approaches have yielded disparate results. In this study, we utilize both gene knockouts and muscle cell ablation approaches to re-visit the extent to which rib growth and patterning are dependent on developing musculature. Consistent with previous studies, we show that rib formation is highly dependent on the MYOD family of myogenic regulatory factors (MRFs), and demonstrate that the extent of rib formation is gene-, allele-, and dosage-dependent. In the absence of Myf5 and MyoD, one allele of Mrf4 is sufficient for extensive rib growth, although patterning is abnormal. Under conditions of limiting MRF dosage, MyoD is identified as a positive regulator of rib patterning, presumably due to improved intercostal muscle development. In contrast to previous muscle ablation studies, we show that diphtheria toxin subunit A (DTA)-mediated ablation of muscle progenitors or differentiated muscle, using MyoDiCre or HSA-Cre drivers, respectively, profoundly disrupts rib development. Further, a comparison of three independently derived Rosa26-based DTA knockin alleles demonstrates that the degree of rib perturbations in MyoDiCre/+/DTA embryos is markedly dependent on the DTA allele used, and may in part explain discrepancies with previous findings. The results support the conclusion that the extent and quality of rib formation is largely dependent on the dosage of Myf5 and Mrf4, and that both early myotome-sclerotome interactions, as well as later muscle-rib interactions, are important for proper rib growth and patterning.

Production of IgY polyclonal antibody against diphtheria toxin and evaluation of its neutralization effect by Vero cell assay.

BACKGROUND: Diphtheria is a bacterial disease which is caused by Corynebacterium diphtheriae. The symptoms are due to the diphtheria toxin produced by the bacteria. Antibiotic therapy and the use of diphtheria antitoxin is a recommended strategy to control diphtheria. Although mammalian antibodies are used to treat patients, IgY antibody has advantages over mammalian ones, including cost-effectiveness and production through non-invasive means. Moreover, in contrast to mammalian antibodies, IgY does not bind to the rheumatoid factor and does not activate the complement system. The objective of this study was to evaluate the in vitro neutralizing effect of IgY against diphtheria toxin. RESULTS: Anti-DT IgY was produced by immunization of the laying white leghorn chickens. Indirect enzyme-linked immunosorbent assay revealed successful immunization of the animals, and the IgY was purified with a purity of 93% via polyethylene glycol precipitation method. The neutralizing activity of the purified IgY was evaluated by Vero cell viability assay. This assay confirmed that 1.95 µg (8.6 µg/ml of culture medium) of anti-DT IgY would neutralize 10 fold of cytotoxic dose 99% of DT, which was 0.3 ng (1.33 ng/ml of culture medium). CONCLUSION: This anti-DT IgY may be applicable for diphtheria treatment and quality controls in vaccine production.

Molecular and Epidemiological Characterization of Toxigenic and Nontoxigenic Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, and Corynebacterium ulcerans Isolates Identified in Spain from 2014 to 2019.

This study examines the microbiological and epidemiological characteristics of toxigenic and nontoxigenic Corynebacterium isolates submitted to the national reference laboratory in Spain, between 2014 and 2019, in order to describe the current situation and improve our knowledge regarding these emerging pathogens. Epidemiological information was extracted from the Spanish Surveillance System. Microbiological and molecular characterization was carried out using phenotypic methods, multilocus sequence typing (MLST), whole-genome sequencing (WGS), and core genome MLST (cgMLST). Thirty-nine isolates were analyzed. Twenty-one isolates were identified as Corynebacterium diphtheriae (6 toxigenic), 14 as C. belfantii, 4 as C. ulcerans (3 toxigenic), and 1 as C. rouxii One C. diphtheriae isolate was identified as nontoxigenic tox gene bearing (NTTB). Ages of patients ranged from 1 to 89 years, with 10% (3/30) of nontoxigenic and 22% (2/9) of toxigenic isolates collected from children less than 15 years. Twenty-five of the patients were males (17/30 in nontoxigenic; 8/9 in toxigenic). MLST identified 28 sequence types (STs), of which 7 were described for the first time in Spain. WGS analysis showed that 10 isolates, including 3 toxigenic isolates, harbored a variety of antibiotic resistance genes in addition to the high prevalence of penicillin resistance phenotypically demonstrated. Phylogenetic analysis revealed one cluster of isolates from family members. Risk information was available for toxigenic isolates (9/39); 3 patients reported recent travels to countries of endemicity and 3 had contact with cats/dogs. One unvaccinated child with respiratory diphtheria had a fatal outcome. Including nontoxigenic Corynebacterium infections in disease surveillance and using WGS could further improve current surveillance.