Generation of a synthetic binary plasmid that confers resistance to nourseothricin for genetic engineering of Sporothrix schenckii

Tamez-Castrellón, Alma K.; Romo-Lucio, Ricardo; Martínez-Duncker, Iván; Mora-Montes, Héctor M.

doi:10.1016/j.plasmid.2018.09.006

Cited by 5 publications

(7 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sorbitol was added as an osmotic stabilizer of protoplasts. In addition, we tested three different concentrations of antibiotics for two selection markers previously utilized in S. schenckii transformation (Lozoya-Perez et al, 2019; Tamez-Castrellon et al, 2018): hygromycin B (80, 140 and 180 μg/ml) and nourseothricin (50, 100 and 200 μg/ml). The concentrations of 140 μg/ml hygromycin B and 50 μg/ml nourseothricin were sufficient to inhibit mycelial growth of all strains tested (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Genetic engineering is a key tool for dissecting the molecular mechanisms that underlie virulence and for facilitating the search for more efficient antimicrobial therapies. Sporothrix species have been considered challenging for genetic manipulation (Mora-Montes et al ., 2015) and only limited tools have been available for applying reverse genetics to resolve molecular mechanisms critical for sporotrichosis development (Lozoya-Perez et al, 2019; Lozoya-Perez et al ., 2018; Romero-Martinez et al ., 2000; Song et al ., 2021; Tamez-Castrellon et al ., 2018; Tamez-Castrellon et al ., 2021). RNA interference-mediated gene silencing using Agrobacterium -mediated transformation has been used for studying the Golgi α-1,6-mannosyltransferase ( Och1 ) and protein rhamnosylation ( rmlD ) genes in S. schenckii (Lozoya-Perez et al ., 2019; Tamez-Castrellon et al ., 2021).…”

Section: Discussionmentioning

confidence: 99%

“…In the last two decades, there has been an increased effort to develop new tools for diagnosis, epidemiologic and virulence studies, but few studies have focused on new approaches to improve the molecular and genetic manipulation of the Sporothrix species complex (de Carvalho et al, 2022; Rodrigues et al ., 2022). Currently, the genetic manipulation of Sporothrix species has focused on gene silencing using vectors delivered to fungal cells via Agrobacterium tumefaciens-mediated transformation, or by random UV-induced mutation of S. schenckii and S. globosa (Lozoya-Perez et al, 2019; Lozoya-Perez et al, 2018; Romero-Martinez et al ., 2000; Song et al, 2021; Tamez-Castrellon et al, 2018; Tamez-Castrellon et al, 2021). Sporothrix species have represented a challenge for the application of universal genetic engineering tools (Mora-Montes et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

CRISPR-based tools for genetic manipulation in pathogenicSporothrixspecies

Hatinguais

Leaves

Brown

et al. 2022

Preprint

View full text Add to dashboard Cite

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis. Although certain virulence factors have been proposed as potential sporotrichosis determinants, the scarcity of molecular tools for reverse genetics studies on 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 expression in S. brasiliensis, S. schenckii and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol allowed 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 KU80 homologue that is critical for non-homologous end-joining DNA repair. The use of S. brasiliensis delta ku80 strains enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR-based tools for genetic manipulation in pathogenicSporothrixspecies

Hatinguais

Leaves

Brown

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Our group has previously generated genetically modified S. schenckii strains to either validate new tools for the genetic and molecular analysis of this organism, 33 , 59 or to assess the contribution of particular genes in the S. schenckii -host interaction. 32 The strains HSS2, HSS3, HSS4, HSS12, and HSS13 harbor one copy of pBGgHg plasmid, which confers resistance to hygromycin B and expresses the green fluorescent protein; while strains HSS5 andHSS6 contain three copies of this plasmid integrated into the genome.…”

Section: Resultsmentioning

confidence: 99%

Tenebrio molitor as an Alternative Model to Analyze the Sporothrix Species Virulence

Lozoya-Pérez¹,

García-Carnero²,

Martínez-Álvarez³

et al. 2021

IDR

Self Cite

View full text Add to dashboard Cite

Background Sporotrichosis is an increasing threat for humans, affecting mainly skin and subcutaneous tissues but that can cause disseminated infection in immunocompromised patients. Sporothrix schenckii, Sporothrix brasiliensis , and Sporothrix globosa are the main etiological agents of this mycosis, and each species show different virulence levels. The gold standard to assess fungal virulence is the mouse model that is expensive and time-consuming. Thus, invertebrate models have been reported as an alternative for the evaluation of fungal virulence. Here, we assessed whether Tenebrio molitor larvae could be a new alternative to study Sporothrix spp. virulence. Methods T. molitor larvae were inoculated with different doses of S. schenckii, S. brasiliensis , and S. globosa , and animal mortality, cytotoxicity, and immunological parameters were analyzed, including the ability to stimulate immunological priming. Results Mortality curves demonstrated that yeast-like cells were the best fungal morphology to kill larvae and showed a similar ranking in virulence than that reported in other animal models, ie, being S. brasiliensis and S. globosa the species with the highest and lowest virulence, respectively. The usefulness of this model was validated with the analysis of several S. schenckii strains with different virulence degrees, and changes in cytotoxicity, humoral and cellular immunological parameters. Low-virulence strains stimulated low levels of cytotoxicity, phenoloxidase activity, and hemocyte countings, and these immunological cells poorly uptake fungi. Moreover, using recombinant Gp70 from S. schenckii immunological priming was stimulated in larvae and this protected against a lethal dose of fungal cells from any of the three species under study. Conclusion The study demonstrated that T. molitor larvae are an appropriate alternative invertebrate model to analyze the virulence of S. schenckii, S. brasiliensis , and S. globosa . Additionally, hemocyte levels, phenoloxidase activity, cytotoxicity, uptake by hemocytes, and immunological priming are biological parameters that can be used to study the Sporothrix–T. molitor interaction.

show abstract

“…In the last two decades, there has been an increased effort to develop new tools for diagnosis, epidemiologic, and virulence studies, but few studies have focused on new approaches to improve the molecular and genetic manipulation of the Sporothrix species complex ( 2 , 18 ). Currently, the genetic manipulation of Sporothrix species has focused on gene silencing using vectors delivered to fungal cells via Agrobacterium tumefaciens -mediated transformation or by random UV-induced mutation of S. schenckii and S. globosa ( 15 , 19 – 23 ). Sporothrix species have represented a challenge for the application of universal genetic engineering tools ( 24 ).…”

Section: Introductionmentioning

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

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.

show abstract

Generation of a synthetic binary plasmid that confers resistance to nourseothricin for genetic engineering of Sporothrix schenckii

Cited by 5 publications

References 19 publications

CRISPR-based tools for genetic manipulation in pathogenicSporothrixspecies

CRISPR-based tools for genetic manipulation in pathogenicSporothrixspecies

Tenebrio molitor as an Alternative Model to Analyze the Sporothrix Species Virulence

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

Contact Info

Product

Resources

About