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      Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii

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          Abstract

          Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients . Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

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          Current knowledge of Trichosporon spp. and Trichosporonosis.

          Trichosporon spp. are basidiomycetous yeast-like fungi found widely in nature. Clinical isolates are generally related to superficial infections. However, this fungus has been recognized as an opportunistic agent of invasive infections, mostly in cancer patients and those exposed to invasive medical procedures. It is possible that the ability of Trichosporon strains to form biofilms on implanted devices, the presence of glucuronoxylomannan in their cell walls, and the ability to produce proteases and lipases are all factors likely related to the virulence of this genus and therefore may account for the progress of invasive trichosporonosis. Disseminated trichosporonosis has been increasingly reported worldwide and represents a challenge for both diagnosis and species identification. Phenotypic identification methods are useful for Trichosporon sp. screening, but only molecular methods, such as IGS region sequencing, allow the complete identification of Trichosporon isolates at the species level. Methods for the diagnosis of invasive trichosporonosis include PCR-based methods, Luminex xMAP technology, and, more recently, proteomics. Treating patients with trichosporonosis remains a challenge because of limited data on the in vitro and in vivo activities of antifungal drugs against clinically relevant species of the genus. Despite the mentioned limitations, the use of antifungal regimens containing triazoles appears to be the best therapeutic approach.
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            The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus.

            To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB(KU80)). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuB(KU80) mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB(KU80) had no influence on pathogenicity in a low-dose murine infection model.
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              Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining.

              Gene disruption and overexpression play central roles in the analysis of gene function. Homologous recombination is, in principle, the most efficient method of disrupting, modifying, or replacing a target gene. Although homologous integration of exogenous DNA into the genome occurs readily in Saccharomyces cerevisiae, it is rare in many other organisms. We identified and disrupted Neurospora crassa genes homologous to human KU70 and KU80, which encode proteins that function in nonhomologous end-joining of double-stranded DNA breaks. The resulting mutants, named mus-51 and mus-52, showed higher sensitivity to methyl methanesulfonate, ethyl methanesulfonate, and bleomycin than wild type, but not to UV, 4-nitroquinoline 1-oxide, camptothecin, or hydroxyurea. Vegetative growth, conidiation, and ascospore production in homozygous crosses were normal. The frequency of integration of exogenous DNA into homologous sequences of the genome in the KU disruption strains of N. crassa was compared with that in wild type, mei-3, and mus-11. In mei-3 and mus-11, which are defective in homologous recombination, none or few homologous integration events were observed under any conditions. When mtr target DNA with approximately 2-kb 5' and 3' flanking regions was used for transformation of the KU disruption strains, 100% of transformants exhibited integration at the homologous site, compared to 10 to 30% for a wild-type recipient. Similar results were obtained when the ad-3A gene was targeted for disruption. These results indicate that KU disruption strains are efficient recipients for gene targeting.
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                Author and article information

                Contributors
                ymatsumoto@my-pharm.ac.jp
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                14 September 2021
                14 September 2021
                2021
                : 11
                : 18270
                Affiliations
                [1 ]GRID grid.411763.6, ISNI 0000 0001 0508 5056, Department of Microbiology, , Meiji Pharmaceutical University, ; 2-522-1, Noshio, Kiyose, Tokyo 204-8588 Japan
                [2 ]GRID grid.264706.1, ISNI 0000 0000 9239 9995, Teikyo University Institute of Medical Mycology, ; 359 Otsuka, Hachioji, Tokyo 192-0395 Japan
                [3 ]GRID grid.264706.1, ISNI 0000 0000 9239 9995, Asia International Institute of Infectious Disease Control, Teikyo University, ; 2-11-1, Kaga, Itabashi-ku, Tokyo, 173-8605 Japan
                Article
                97287
                10.1038/s41598-021-97287-3
                8440527
                34521867
                33595ee6-fcf9-4aea-badd-be50f00f956b
                © The Author(s) 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 18 June 2021
                : 24 August 2021
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;
                Award ID: JP20K07022
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s) 2021

                Uncategorized
                gene targeting,microbiology techniques,fungal genetics,fungal pathogenesis
                Uncategorized
                gene targeting, microbiology techniques, fungal genetics, fungal pathogenesis

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