Abstract
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.
Highlights
Some haploid C. albicans strains were isolated[14]
We replaced all CUG codons with UUG because CUG encodes serine instead of the conventional leucine in C. albicans, added a 3×SV40 nuclear localization signal to its N-terminus, and controlled the expression using the tetracyclineinducible (Tet-On) promoter (Fig. 1a)
To test whether PB transposition would occur in C. albicans, we transformed the donor plasmid pPB[URA3] carrying the ITRURA3-inverted terminal repeats (ITRs) cassette into YW01 cells that had been grown in the absence or presence of doxycycline (Dox) for 48 h and spread the cells onto uridine-deficient (-Uri) plates
Summary
Some haploid C. albicans strains were isolated[14]. The haploids have opened up opportunities for scientists to explore large-scale, genome-wide genetic screening strategies in this pathogen. Transposon-mediated mutagenesis is a widely used genetic tool. Transposons are movable genetic units and have two essential components, a transposase and a pair of transposonspecific inverted terminal repeats (ITRs). PB transposon inserts randomly throughout the genome[20]. We develop a highly efficient, PB-based mutagenesis system using stable haploid C. albicans strains. We apply this technology to conduct genome-wide genetic screens for genes involved in resistance to the antifungal drug fluconazole and discover a new resistance mechanism
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