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Factors influencing the nitrogen-source dependent flucytosine resistance in Cryptococcus species

ABSTRACT: Flucytosine (5-FC) is an antifungal drug commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungal species, cytosine permease and cytosine deaminase are known to play major roles in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs of cytosine permease (FCY2, FCY3, and FCY4) and three paralogs of cytosine deaminase (FCY1, FCY5, and FCY6). Like in other fungi, we found Fcy1 and Fcy2 to be the primary cytosine deaminase and permease respectively in C. neoformans H99 (VNI), C. gattii R265 (VGIIa), and WM276 (VGI). However, when different amino acids were used as the sole nitrogen source, Fcy3 and Fcy6 functioned as the secondary cytosine permease and deaminase respectively in C. gattii. Transcriptome analysis using RNA-seq under three different nitrogen sources (NH4, Asn, and Pro) revealed that these permease and deaminase genes as well as two GATA family transcription factor genes, GAT1 and CIR1, were differentially expressed in WM276. In addition, deletion studies of GAT1 and CIR1 demonstrated that the expression of each permease and deaminase genes was under the regulation of GAT1 and CIR1 individually. Furthermore, the expression levels of FCY3 and FCY6 regulated by GAT1 and CIR1 contributed to the 5-FC susceptibility in fcy2Δ or fcy1Δ background under different amino acid conditions. This study offers an insight into the mechanism of 5-FC resistance in C. gattii which is orchestrated by cytosine permease and deaminase genes, and two GATA family transcription factor genes, GAT1 and CIR1, under diverse nitrogen conditions.

ORGANISM(S): Cryptococcus gattii

PROVIDER: GSE215311 | GEO | 2023/01/11

REPOSITORIES: GEO

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Project description:We used cDNA microarray technology to compare the genome-wide expression profiles of a wild type strain (BY4700) (E02, E04, E06) or the isogenic strain deleted of GLN3 and GAT1 genes (E01, E03, E05) grown in YNB medium with glutamine as nitrogen source (M.Gln) against the wild type strain grown in M.Gln after addition of rapamacyn (20 min) (E01, E02), or M.proline (M.Pro) (E03, E04) or after a two hours shift from M.Gln to M.Pro (E05, E06), all growth conditions known to modify the expression of genes involved in nitrogen utilization. These microarrays allowed to identify the set of genes that were up or down-regulated in response to the quality of the nitrogen source. To evaluate whether the majority of genes responding to the nitrogen source were dependent on Gln3 and Gat1, we compared the expression profiles of the wild type strain and of the isogenic strain deleted of GLN3 and GAT1 genes (03167b: ura3, gln3â, gat1â), when both strains were grown on M.Gln + rapamycin (E07), or M.Pro (E08) or after a shift from M.Gln to M.Pro (E09). We also used an independent means of identifying Gln3-Gat1 regulated genes by comparing the expression profiles in wild type and ure2â (4709âURE2) strains on M.Gln medium (E10). The hybridization signal was measured using a GSM418 laser scanner. Image analysis for each array was processed using the GenePix Pro 4.0 (Axon Instruments, Inc.) software package, which measures fluorescence intensity pairs for each gene. Following image acquisition, a visual inspection of the individual spots on each microarray (size, signal-to-noise ratio, background level, and spot uniformity) completed the flagging (present/not present, good/bad) of the data. To maximize sensitivity two scans were made, one at high laser power and high PMT (Photo Multiplier Tube) gain to detect the faintest spots, and a second one using low laser power and a PMT gain avoiding saturation. The values for spots presenting â¥ 5% saturation in the first scan were calculated based on an extrapolation after linear regression analysis of the intensities from both scans. These data were then imported in the GeneSpring 7.1 (Silicon Genetics) software package, applying a per spot per chip intensity-dependent (Lowess) normalization for further analysis.

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Factors influencing the nitrogen-source dependent flucytosine resistance in Cryptococcus species

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