A joint PCR-based gene-targeting method using electroporation in the pathogenic fungus Trichosporon asahii

Matsumoto, Yasuhiko; Nagamachi, Tae; Yoshikawa, Asami; Yamada, Tsuyoshi; Sugita, Takashi

doi:10.1186/s13568-022-01431-9

Cited by 7 publications

(6 citation statements)

References 25 publications

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“…Gene transfer using electroporation was performed according to a previous report 33 . The 5'-UTR (hog1) -NAT1-3'-UTR (hog1) or 5'-UTR (hog1) -hog1-hph-3'-UTR (hog1) fragments were ampli ed by PCR with the primers shown in Supplementary Table 3.…”

Section: Methodsmentioning

confidence: 99%

“…The virulence of clinical isolates of T. asahii can be evaluated quantitatively by calculating the half-maximal lethal dose (LD 50 ), which is the dose of a pathogen required to kill half the animals in a group 29 . An e cient gene-targeting system was developed to generate gene-de cient mutants of T. asahii 30,33 . Genede cient T. asahii mutants of calcineurin, a calcium-calmodulin-activated phosphatase, exhibit decreased virulence against silkworms 34 .…”

Section: Introductionmentioning

confidence: 99%

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Hog1-mediated stress tolerance in the pathogenic fungus Trichosporon asahii

Matsumoto

Nagamachi

et al. 2023

Preprint

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Trichosporon asahii is a conditional pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients.Hog1, a mitogen-activated protein kinase, is known to regulate the stress resistance of some pathogenic fungi, but its role in T. asahii has not been investigated. Here, we demonstrated that the hog1 gene-deficient T. asahii mutant is sensitive to high temperature, cell-membrane stress, oxidative stress, and antifungal drugs. The growth of the hog1 gene-deficient T. asahii mutant was delayed at 40˚C. The hog1 gene-deficient T. asahii mutant also exhibited sensitivity to sodium dodecyl sulfate, hydrogen peroxide, menadione, methyl methanesulfonate, UV exposure, and antifungal drugs such as amphotericin B under a glucose-rich condition. Under a glucose-restricted condition, the hog1 gene-deficient mutant exhibited sensitivity to NaCl and KCl. The virulence of the hog1gene-deficient mutant against silkworms was attenuated. Moreover, the cell viability of the hog1 gene-deficient mutant was decreased in the silkworm hemolymph. These phenotypes were restored by re-introducing the hog1 gene into the gene-deficient mutant. Our findings suggest that Hog1 has a critical role in regulating the cellular stress responses of T. asahii.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hog1-mediated stress tolerance in the pathogenic fungus Trichosporon asahii

Matsumoto

Nagamachi

et al. 2023

Preprint

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“…Genotyping PCR was performed according to a previous report (Matsumoto et al 2022a;Matsumoto et al 2021). To generate the ku70 gene-de cient mutant, the transformants were grown on SDA containing G418 (300 µg/ml).…”

Section: Genotyping Pcrmentioning

confidence: 99%

A gene-targeting system in Trichosporon asahii JCM2466 by Agrobacterium tumefaciens-mediated transformation

Matsumoto,

Nakayama,

Shimizu

et al. 2024

Preprint

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Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. The complete genome information of T. asahii JCM2466 (CBS2479), a standard type strain widely used in research, is publicly available, but a gene-targeting system for T. asahii JCM2466 has not yet been developed. Here we generated gene-deficient mutants of T. asahii JCM2466 by Agrobacterium tumefaciens-mediated transformation (ATMT). A gene-deficient mutant lacking the ku70 gene, which encodes the Ku70 protein involved in non-homologous end-joining repair of DNA double-strand breaks, was generated by ATMT. The ku70 gene-deficient mutant exhibited higher gene-targeting efficiency than the wild-type strain for generating a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. In the ku70 gene-deficient mutant used as a parent strain, cnb1 gene-deficient mutants were generated by ATMT. The cnb1 gene-deficient mutants exhibited sensitivities to high temperature and several stress-inducing compounds. These results suggest that the combination of the ku70 gene-deficient mutant and ATMT is a useful method for generating gene-deficient mutants of T. asahii JCM2466.

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“…DNA isolation was performed according to the Quick DNA fungal/bacterial miniprep protocol provided by Zymo Research [39].…”

Section: Dna Isolation and Genome Sequencingmentioning

confidence: 99%

Indole Diketopiperazine Alkaloids from the Marine Sediment-Derived Fungus Aspergillus chevalieri against Pancreatic Ductal Adenocarcinoma

El-Kashef,

Obidake,

Schiedlauske

et al. 2023

Marine Drugs

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A new prenylated indole diketopiperazine alkaloid, rubrumline P (1), was isolated along with six more analogues and characterized from the fermentation culture of a marine sediment-derived fungus, Aspergillus chevalieri, collected at a depth of 15 m near the lighthouse in Dahab, Red Sea, Egypt. In the current study, a bioassay-guided fractionation allowed for the identification of an active fraction displaying significant cytotoxic activity against the human pancreatic adenocarcinoma cell line PANC-1 from the EtOAc extract of the investigated fungus compared to the standard paclitaxel. The structures of the isolated compounds from the active fraction were established using 1D/2D NMR spectroscopy and mass spectrometry, together with comparisons with the literature. The absolute configuration of the obtained indole diketopiperazines was established based on single-crystal X-ray diffraction analyses of rubrumline I (2) and comparisons of optical rotations and NMR data, as well as on biogenetic considerations. Genome sequencing indicated the formation of prenyltransferases, which was subsequently confirmed by the isolation of mono-, di-, tri-, and tetraprenylated compounds. Compounds rubrumline P (1) and neoechinulin D (4) confirmed preferential cytotoxic activity against PANC-1 cancer cells with IC50 values of 25.8 and 23.4 µM, respectively. Although the underlying mechanism-of-action remains elusive in this study, cell cycle analysis indicated a slight increase in the sub-G1 peak after treatment with compounds 1 and 4.

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A joint PCR-based gene-targeting method using electroporation in the pathogenic fungus Trichosporon asahii

Cited by 7 publications

References 25 publications

Hog1-mediated stress tolerance in the pathogenic fungus Trichosporon asahii

Hog1-mediated stress tolerance in the pathogenic fungus Trichosporon asahii

A gene-targeting system in Trichosporon asahii JCM2466 by Agrobacterium tumefaciens-mediated transformation

Indole Diketopiperazine Alkaloids from the Marine Sediment-Derived Fungus Aspergillus chevalieri against Pancreatic Ductal Adenocarcinoma

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