Biology subjects

Baldin, C.

Publications and source records attributed to Baldin, C..

3 recordsLinked to original sources

bioRxiv2025

Simply cut out -Combining CRISPR/Cas9 RNPs and transiently selected telomere vectors for marker free-gene deletion in Trichoderma atroviride

Trichoderma atroviride is a well-known mycoparasitic fungus widely used for the biological control of fungal plant pathogens. Expanding its genetic toolbox is essential to facilitate efficient genetic manipulation, including successive transformations and multiple or reusable selection markers for consecutive gene deletions. We applied CRISPR/Cas9 via ribonucleoprotein (RNP) complexes for gene editing in T. atroviride and successfully deleted three target genes, i.e., pks4 (involved in spore pigment production), pyr4 (pyrimidine biosynthesis), and pex5 (receptor for peroxisomal matrix protein import). Although double-strand breaks induced by Cas9 can be repaired via homology-directed repair (HDR), using donor templates, the most effective gene deletions in our case were achieved via non-homologous end joining (NHEJ), by co-transforming a transiently stable telomere vector carrying the hygromycin-resistance gene (hph), which was rapidly lost under non-selective conditions. This strategy promoted NHEJ repair and resulted in the efficient deletion of open reading frames between two Cas9 target sites. Our results demonstrate that combining CRISPR/Cas9 RNP delivery with transient telomere vectors provides a fast and reliable method for marker-free gene deletion and vector recycling in T. atroviride, advancing the reverse genetic toolkit available for this important biocontrol fungus.

microbiology↗

bioRxiv2022

The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR.

molecular biology↗

bioRxiv2021

Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We now demonstrate that Mucorales fungi produce a toxin that plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro, and prevent hypovolemic shock, organ necrosis, and death in mice with mucormycosis. RNAi inhibition of the toxin in Rhizopus delemar, compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin, ricin, including the ability to inhibit protein synthesis by its N-glycosylase activity, the existence of a motif that mediates vascular leak, and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross-react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name "mucoricin" for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin- like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

microbiology↗