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Characterization of the chromosome 4 genes that affect fluconazole-induced disomy formation in Cryptococcus
Popchai Ngamskulrungroj1, Yun Chang, Bryan Hansen
1Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
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.
Insights
Cryptococcus neoformans develops azole resistance through chromosome disomy. Genes maintaining endoplasmic reticulum integrity are crucial for this disomy formation under fluconazole stress.
Area of Science:
- Microbiology
- Genetics
- Cell Biology
Background:
- Heteroresistance in Cryptococcus neoformans is an adaptive resistance to azoles, often linked to chromosome disomy.
- Chromosome 1 (Chr1) disomy, involving ERG11 and AFR1, is a known mechanism for fluconazole resistance.
- The role of other chromosomes and genes in this adaptive resistance remains to be fully elucidated.
Purpose of the Study:
- To identify additional chromosomal regions and genes involved in azole heteroresistance in Cryptococcus neoformans.
- To investigate the role of specific Chr4 genes in the formation of chromosome disomy under fluconazole stress.
- To explore the connection between endoplasmic reticulum (ER) integrity and chromosome disomy in azole-resistant fungal strains.
Main Methods:
- Fluconazole (FLC) stress was applied to Cryptococcus neoformans to induce heteroresistance and chromosome disomy.
- Nine candidate genes on Chromosome 4 (Chr4) were deleted to assess their role in disomy formation.
- Fluorescence microscopy, transmission electron microscopy (TEM), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) were used to analyze ER morphology in mutant strains.
Main Results:
- Chromosome 4 (Chr4) was identified as the second most frequent site of disomy under high fluconazole concentrations.
- Disruption of three Chr4 genes (homologous to Sey1, Glo3, and Gcs2) significantly reduced Chr4 disomy frequency.
- Mutations in these genes led to aberrant endoplasmic reticulum morphology, suggesting a link between ER integrity and chromosome disomy.
- FLC-resistant clones lacking Chr4 or Chr1 disomy showed increased copy numbers of ERG11-proximal genes on Chr1.
Conclusions:
- Genes essential for maintaining endoplasmic reticulum integrity play a significant role in the formation of disomic chromosomes in Cryptococcus neoformans under azole stress.
- Targeting ER integrity pathways could be a potential strategy to overcome azole resistance in fungal pathogens.
- This study expands the understanding of the genetic basis of heteroresistance and chromosome instability in Cryptococcus neoformans.
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