A Role for Topoisomerase I in Fusarium graminearum and F. culmorum Pathogenesis and Sporulation

Fusarium graminearum and F. culmorum are the causal agents of Fusarium ear blight (FEB) in wheat. A forward genetics approach was taken to discover novel pathogenicity genes in the genome of F. graminearum. A library of transformants created by random plasmid insertional mutagenesis was screened on wheat ears for virulence defects. Plasmid rescue on one of the reduced-virulence mutants revealed a single-copy plasmid insertion in the gene coding for the DNA interacting enzyme, topoisomerase I. Targeted topoisomerase I gene-deletion mutants were created in strains of both F. graminearum and F. culmorum. The top1 mutants of both species exhibited greatly reduced virulence in wheat ear infection assays (GO:0009405 and GO:0044145). Detailed microscopy analyses revealed that top1 hyphal growth was restricted to palea tissue whereas host responses were discernable 1,000 μm further away in the rachis node. Asexual sporulation was reduced in the F. graminearum mutants and was absent from the F. culmorum mutants. The F. graminearum mutant did not develop sexual spores when subjected to an in vitro perithecia production assay. During in vitro growth, the top1 mutants of both species were still able to produce the trichothecene mycotoxin, deoxynivalenol.

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Aspergillus fumigatus drives tissue damage via iterative assaults upon mucosal integrity and immune homeostasis

10.1101/2021.11.09.468003 ◽

2021 ◽

Author(s):

Uju Joy Okaa ◽

Margherita Bertuzzi ◽

Rachael Fortune-Grant ◽

Darren D Thomson ◽

David L Moyes ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Tissue Damage ◽

Host Response ◽

Nuclear Translocation ◽

Hyphal Growth ◽

Host Responses ◽

Significant Feature ◽

Pulmonary Tissue ◽

Mechanistic Basis

The human lung is constantly exposed to Aspergillus fumigatus spores, the most prevalent worldwide cause of fungal respiratory disease. Pulmonary tissue damage is a unifying feature of Aspergillus-related diseases; however, the mechanistic basis of damage is not understood. In the lungs of susceptible hosts A. fumigatus undergoes an obligatory morphological switch involving spore germination and hyphal growth. We modelled A. fumigatus infection in cultured A549 human pneumocytes, capturing phosphoactivation status of five host signalling pathways, nuclear translocation & DNA binding of eight host transcription factors, and expression of nine host response proteins over six time points encompassing exposures to live fungus and the secretome thereof. The resulting dataset, comprised of more than 1000 data points, reveals that pneumocytes mount differential responses to A. fumigatus spores, hyphae and soluble secreted products via the NF-kB, JNK, and JNK + p38 pathways respectively. Importantly, via selective degradation of host pro-inflammatory (IL-6 and IL-8) cytokines and growth factors (FGF-2), fungal secreted products reorchestrate the host response to fungal challenge as well as driving multiparametric epithelial damage, culminating in cytolysis. Dysregulation of NF-kB signalling, involving iterative stimulation of canonical and non-canonical signalling, was identified as a significant feature of host damage both in vitro and in a mouse model of invasive aspergillosis. Our data demonstrate that composite tissue damage results from iterative exposures to different fungal morphotypes and secreted products and suggest that modulation of host responses to fungal challenge might represent a unified strategy for therapeutic control of pathologically distinct types of Aspergillus-related disease.

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Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

International Journal of Molecular Sciences ◽

10.3390/ijms22020687 ◽

2021 ◽

Vol 22 (2) ◽

pp. 687

Author(s):

Tong Zhou ◽

Bolan Zhou ◽

Yasong Zhao ◽

Qing Li ◽

Guili Song ◽

...

Keyword(s):

Antiviral Activity ◽

Grass Carp ◽

Single Copy ◽

Type I ◽

Western Blots ◽

Paramisgurnus Dabryanus ◽

Expression Activity ◽

Transgenic Construct ◽

Mucus Gland

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

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Internalization of Foldamer-Based DNA Mimics through a Site-Specific Antibody Conjugate to Target HER2-Positive Cancer Cells

Pharmaceuticals ◽

10.3390/ph14070624 ◽

2021 ◽

Vol 14 (7) ◽

pp. 624

Author(s):

Valentina Corvaglia ◽

Imène Ait Mohamed Amar ◽

Véronique Garambois ◽

Stéphanie Letast ◽

Aurélie Garcin ◽

...

Keyword(s):

Cancer Cells ◽

Topoisomerase I ◽

Ovarian Cancer Cells ◽

Cellular Functions ◽

Antibody Drug Conjugate ◽

Her2 Positive ◽

Dna Topoisomerase ◽

Antibody Conjugate ◽

A Site

Inhibition of protein–DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells.

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DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)49889-x ◽

1992 ◽

Vol 267 (16) ◽

pp. 11156-11162

Author(s):

D.S. Samuels ◽

N Shimizu

Keyword(s):

Protein Kinase ◽

Topoisomerase I ◽

Dna Topoisomerase I ◽

Dna Topoisomerase ◽

Murine Fibroblasts

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Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

Cells ◽

10.3390/cells10051192 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1192

Author(s):

Francesco Tini ◽

Giovanni Beccari ◽

Gianpiero Marconi ◽

Andrea Porceddu ◽

Micheal Sulyok ◽

...

Keyword(s):

Dna Methylation ◽

Fusarium Graminearum ◽

Environmental Changes ◽

Virulent Strain ◽

Fungal Virulence ◽

Wheat Seedlings ◽

Wide Range ◽

Conidia Production ◽

Natural Hosts

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

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The Culturable Mycobiome of Mesophotic Agelas oroides: Constituents and Changes Following Sponge Transplantation to Shallow Water

Journal of Fungi ◽

10.3390/jof7070567 ◽

2021 ◽

Vol 7 (7) ◽

pp. 567

Author(s):

Eyal Ben-Dor Cohen ◽

Micha Ilan ◽

Oded Yarden

Keyword(s):

Microbial Community ◽

Shallow Water ◽

Mediterranean Sea ◽

Hyphal Growth ◽

Marine Sponges ◽

The Core ◽

The Mediterranean ◽

Dynamic Components ◽

In Vitro Interactions

Marine sponges harbor a diverse array of microorganisms and the composition of the microbial community has been suggested to be linked to holo-biont health. Most of the attention concerning sponge mycobiomes has been given to sponges present in shallow depths. Here, we describe the presence of 146 culturable mycobiome taxa isolated from mesophotic niche (100 m depth)-inhabiting samples of Agelas oroides, in the Mediterranean Sea. We identify some potential in vitro interactions between several A. oroides-associated fungi and show that sponge meso-hyl extract, but not its predominantly collagen-rich part, is sufficient to support hyphal growth. We demonstrate that changes in the diversity of culturable mycobiome constituents occur following sponge transplantation from its original mesophotic habitat to shallow (10 m) waters, where historically (60 years ago) this species was found. We conclude that among the 30 fungal genera identified as associated with A. oroides, Aspergillus, Penicillium and Trichoderma constitute the core mycobiome of A. oroides, and that they persist even when the sponge is transplanted to a suboptimal environment, indicative of the presence of constant, as well as dynamic, components of the sponge mycobiome. Other genera seemed more depth-related and appeared or disappeared upon host’s transfer from 100 to 10 m.

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The DNA damage inducible lncRNA SCAT7 regulates genomic integrity and topoisomerase 1 turnover in lung adenocarcinoma

NAR Cancer ◽

10.1093/narcan/zcab002 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Luisa Statello ◽

Mohamad M Ali ◽

Silke Reischl ◽

Sagar Mahale ◽

Subazini Thankaswamy Kosalai ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cancer Biology ◽

Topoisomerase I ◽

Cell Cycle Checkpoints ◽

Topoisomerase 1 ◽

Damage Response ◽

Promote Cell Survival

Abstract Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1–cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

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Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽

10.1186/s13100-021-00237-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Justin M. Waldern ◽

Dorie Smith ◽

Carol Lyn Piazza ◽

E. Jake Bailey ◽

Nicholas J. Schiraldi ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Fold Increase ◽

Group Ii Intron ◽

In Vivo Experiments ◽

Cellular Factors ◽

Group Ii ◽

Self Splicing ◽

Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

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Modulation of IGF2 Expression in the Murine Thymus and Thymic Epithelial Cells Following Coxsackievirus-B4 Infection

Microorganisms ◽

10.3390/microorganisms9020402 ◽

2021 ◽

Vol 9 (2) ◽

pp. 402

Author(s):

Hélène Michaux ◽

Aymen Halouani ◽

Charlotte Trussart ◽

Chantal Renard ◽

Hela Jaïdane ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Line ◽

Thymic Epithelial Cells ◽

Altered Expression ◽

Coxsackievirus B4 ◽

Gene Coding ◽

Self Tolerance ◽

Stat3 Phosphorylation ◽

The Stability

Coxsackievirus B4 (CV-B4) can infect human and murine thymic epithelial cells (TECs). In a murine TEC cell line, CV-B4 can downregulate the transcription of the insulin-like growth factor 2 (Igf2) gene coding for the self-peptide of the insulin family. In this study, we show that CV-B4 infections of a murine TEC cell line decreased Igf2 P3 promoter activity by targeting a region near the transcription start site; however, the stability of Igf2 transcripts remained unchanged, indicating a regulation of Igf2 transcription. Furthermore, CV-B4 infections decreased STAT3 phosphorylation in vitro. We also showed that mice infected with CV-B4 had an altered expression of Igf2 isoforms as detected in TECs, followed by a decrease in the pro-IGF2 precursor in the thymus. Our study sheds new light on the intrathymic regulation of Igf2 transcription during CV-B4 infections and supports the hypothesis that a viral infection can disrupt central self-tolerance to insulin by decreasing Igf2 transcription in the thymic epithelium.

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