PMID:22412978

Citation	Ngamskulrungroj, P, Chang, Y, Hansen, B, Bugge, C, Fischer, E and Kwon-Chung, KJ (2012) Characterization of the chromosome 4 genes that affect fluconazole-induced disomy formation in Cryptococcus neoformans. PLoS ONE 7:e33022
Abstract	Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1Δglo3Δ did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1Δ but without any discernable alteration in gcs2Δ and glo3Δ under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3Δ and gcs2Δ strains. In sey1Δ, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C. neoformans under azole stress.
Links	PubMed PMC3296764 Online version:10.1371/journal.pone.0033022
Keywords	Antifungal Agents/toxicity; Chromosome Duplication; Chromosomes, Fungal; Cryptococcus neoformans/drug effects; Cryptococcus neoformans/genetics; Drug Resistance, Fungal/genetics; Drug Tolerance/genetics; Endoplasmic Reticulum/genetics; Endoplasmic Reticulum/metabolism; Endoplasmic Reticulum/ultrastructure; Fluconazole/toxicity; Gene Deletion; Genes, Fungal; Translocation, Genetic; Uniparental Disomy/chemically induced
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CRYNH:J9VP90		GO:0007029: endoplasmic reticulum organization	ECO:0000315:		P		Figure 8 shows reticular ER morphology was barely detectable and formed mostly expanded cisternae.	complete CACAO 11534
CRYNJ:SEY1		GO:0071783: endoplasmic reticulum cisternal network organization	ECO:0000315:		P		Figure 8 shows reticular ER morphology was barely detectable and formed mostly expanded cisternae.	complete CACAO 11322
CRYNH:J9VRH3		GO:0007029: endoplasmic reticulum organization	ECO:0000315:		P		Figure 8 shows reticular ER morphology was barely detectable and formed mostly expanded cisternae.	complete CACAO 11719
CRYNH:J9VMB5		GO:0007029: endoplasmic reticulum organization	ECO:0000315:		P		Figure 8 shows reticular ER morphology was barely detectable and formed mostly expanded cisternae.	complete CACAO 11720

References

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