Development of a Versatile Toolbox for Genetic Manipulation of
            <i>Sporothrix brasiliensis</i>

Tavares, Matheus Augusto Avelino; Sousa-Filho, Jorge Carlos; Machado, Ian Alves; Gonçales, Relber Aguiar; Antunes, Daniela; Mendes-Frias, Ana; Silvestre, Ricardo; Carvalho, Agostinho; Torrado, Egídio; Cunha, Cristina; Rodrigues, Fernando

doi:10.1128/spectrum.04564-22

Cited by 1 publication

(1 citation statement)

References 36 publications

(57 reference statements)

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“…In addition, the functional expression of a red-shifted luciferase enables real-time monitoring of in vivo of disease progression as shown for other fungal species such as Aspergillus fumigatus , Aspergillus terreus , Candida albicans, and Cryptococcus neoformans ( 38 – 46 ). Our developments on genetic tools to study Sporothrix species complex been reinforced by a parallel study that also describes approaches for heterologous gene expression in S. brasiliensis ( 47 ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species

Hatinguais,

Leaves,

Brown

et al. 2023

Microbiol Spectr

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Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis . Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1 , which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU 80 homolog that is critical for non-homologous end-joining DNA repair. The use of Δ ku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

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Section: Discussionmentioning

confidence: 99%

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species

Hatinguais,

Leaves,

Brown

et al. 2023

Microbiol Spectr

View full text Add to dashboard Cite

show abstract

Development of a Versatile Toolbox for Genetic Manipulation of Sporothrix brasiliensis

Abstract: Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and has recently become a public health concern. Although immunocompetent hosts are also prone to sporotrichosis, immunodeficient hosts often develop a more severe and disseminated form of disease.

Cited by 1 publication

References 36 publications

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species

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