Maize Transposable Elements Ac/Ds as Insertion Mutagenesis Tools in Candida albicans

Mielich, Kevin; Shtifman-Segal, Ella; Golz, Julia C.; Zeng, Guisheng; Wang, Yan Ming; Berman, Judith; Kunze, Reinhard

doi:10.1534/g3.117.300388

Cited by 24 publications

(25 citation statements)

References 45 publications

(59 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Also, their use in diploid C. albicans creates heterozygous mutations that do not result in readily detectable phenotypes except for a small number of haploinsufficient genes 40 , 41 . Mielich et al recently introduced the Activator / Dissociation transposon of maize into haploid C. albicans 42 , but the system is not very useful at its current state because of frequent autodiploidization.…”

Section: Discussionmentioning

confidence: 99%

Candida albicans gains azole resistance by altering sphingolipid composition

Gao

Wang²,

et al. 2018

Nat Commun

Self Cite

101

124

View full text Add to dashboard Cite

Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen’s diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.

show abstract

Section: Discussionmentioning

confidence: 99%

Candida albicans gains azole resistance by altering sphingolipid composition

Gao

Wang²,

et al. 2018

Nat Commun

Self Cite

101

124

View full text Add to dashboard Cite

show abstract

“…We constructed transposon insertion libraries in a haploid C. albicans isolate (YJB-T900) by sequential insertion of a codon-optimized Ac transposase ( Ac TPase4xCa) at the neutral NEUT5L locus and a Ds-NAT1 transposon such that it interrupted the ADE2 promoter region ( 37 ). After insertion of the Ac transposase, single colonies were checked for DNA content by flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

“…We leveraged two critical resources—a highly stable C. albicans haploid strain that does not autodiploidize and in vivo transposition using a modified Ac transposase/ Ds-NAT1 two-element system. A strength of the Ac / Ds transposon is that, unlike Hermes or PiggyBac ( 67 , 68 ), it does not have a strong insertion site preference either in maize or in other organisms, including S. cerevisiae ( 34 , 37 , 40 , 69 ).…”

Section: Discussionmentioning

confidence: 99%

“…Strain YJB-T1792 was constructed by directly transforming yeast with a fragment containing the Ac TPase4xCa expression cassette together with a URA3 marker and flanking sequences from the C. albicans NEUT5L locus ( 86 ) from NaeI -digested BJB-T135/pKM300 ( 37 ) into strain YJB-T900. To integrate Ds-NAT1 into the ADE2 promoter, strain YJB-T1792 was transformed with NotI-digested BJB-T133/pRK402 ( Table S4B ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans

Segal

Gritsenko

Levitan

et al. 2018

mBio

Self Cite

122

147

View full text Add to dashboard Cite

Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

show abstract

“…It was applied to identify 96 loci crucial for the viability of A. fumigatus [133]. Meilich et al also developed a two-element transposon tagging system known as Ac/Ds in C. albicans to allow in vivo insertion mutagenesis [134].…”

Section: Transposable Elementsmentioning

confidence: 99%

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

2020

View full text Add to dashboard Cite

Pathogenic fungi represent an increasing infectious disease threat to humans, especially with an increasing challenge of antifungal drug resistance. Over the decades, numerous tools have been developed to expedite the study of pathogenicity, initiation of disease, drug resistance and host-pathogen interactions. In this review, we highlight advances that have been made in the use of molecular tools using CRISPR technologies, RNA interference and transposon targeted mutagenesis. We also discuss the use of animal models in modelling disease of human fungal pathogens, focusing on zebrafish, the silkworm, Galleria mellonella and the murine model.

show abstract

Maize Transposable Elements Ac/Ds as Insertion Mutagenesis Tools in Candida albicans

Cited by 24 publications

References 45 publications

Candida albicans gains azole resistance by altering sphingolipid composition

Candida albicans gains azole resistance by altering sphingolipid composition

Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis

Contact Info

Product

Resources

About