Characterization of the Chromosome 4 Genes That Affect Fluconazole-Induced Disomy Formation in <em>Cryptococcus neoformans</em>

<div><p>Heteroresistance in <em>Cryptococcus neoformans</em> is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, <em>ERG11</em>, the fluconazole target, and <em>AFR1</em>, 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 <em>sey1Δglo3Δ</em> did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to <em>ERG11</em> locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in <em>Saccharomyces cerevisiae</em>, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in <em>sey1Δ</em> but without any discernable alteration in <em>gcs2Δ</em> and <em>glo3Δ</em> 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 <em>glo3Δ</em> and <em>gcs2Δ</em> strains. In <em>sey1Δ</em>, 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 <em>C. neoformans</em> under azole stress.</p> </div

Frequency of Chr4 disomy formation in deletants of Chr4 genes.

<p>Clones resistant to 3LHF derived from each deletant were isolated and the copy number of Chr1 and Chr4 in the genome was inferred by assessing the copy number of the genes specific to each chromosome by qPCR. The figure displays the percentage of each type of disomy in the FLC resistant strains. Type1 denotes strains that contain Chr1 and Chr4 disomy, Type2 denotes strains that contain Chr1 disomy, Type3 denotes strains that contain no disomic Chr1 or Chr4.</p

List of strains and their levels of heteroresistance.

*<p>The <i>GCS1</i> homolog of <i>S. cerevisiae</i> was named <i>GCS2</i> as <i>GCS1</i> has already been used to designate glucosylceramide synthase in <i>C. neoformans</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033022#pone.0033022-Rittershaus1" target="_blank">[62]</a>.</p

Frequency of Chr3 disomy is increased in the strains with <i>SEY1</i> and <i>GLO3</i> translocation to Chr3.

<p>The wild type genes <i>SEY1</i> and <i>GLO3</i> were each inserted into Chr3 of the respective deletant strains <i>sey1Δ</i> and <i>glo3Δ</i>. 6–8 resistance clones grown at 3LHF were analyzed for copy number of the genes specific to each chromosome. Each bar represents the frequency of disomy formation in each chromosome.</p

CGH analysis of the <i>sey1Δglo3Δ</i> double deletants isolated from 3LHF.

<p>CGH was performed with genomic DNA extracted from double deletants that showed Type 2 duplication patterns at 3LHF in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033022#pone-0033022-g003" target="_blank">Figure 3</a>. H99 and the resistant clones of H99 grown at 3LHF were included as controls. Only the Chr1 status is shown. No additional disomy was found in any of the clones except for the clone from H99 at 3LHF (data not shown). Arrows indicate the location of <i>ERG11</i>. Each bar represents the copy number of each gene residing on Chr1 or 4 in log₂ scale.</p

Chr4 disomy is associated with survival of <i>C. neoformans</i> at high concentrations of FLC.

<p>16 resistant clones grown at 128 µg/ml FLC (3LHF) were randomly selected and the numbers of each chromosome in the genome were estimated by measuring the copy number of the genes specific to each chromosome. The histogram displays the percentage of presumed disomy for each chromosome in the 16 clones.</p

The FIB-SEM showed all mutants to have variable degrees of aberration in ER morphology.

<p>Left column shows orthoslices representing central slices through the volume. 3D reconstructions of the ER in indicated strains were generated from data collected by serial 3–10 nm cuts of each sample through the cell. ER and nuclei were psuedo-colored with green and blue color, respectively. The FIB-SEM picture of H99 was taken at different angle from the orthoslice to have a clearer view of the ER structure.</p

Disruption of <i>SEY1, GCS2 and GLO3</i> causes ER elongation.

<p><b>A</b>) A GFP fusion of the ER protein Sec61βp was expressed in each indicated strain. Cells from fresh overnight culture grown on YPD agar at 30°C were visualized under a florescent microscope. White arrow head = ER; grey arrow head = nucleus (determined by co-localized Hoechst 3342 dye staining; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033022#pone.0033022.s004" target="_blank">Figure S4</a>). <b>B</b>) Transmission Electron Microscopy (TEM) of each deletant and the wild type cells. Cells were grown overnight on a shaker at 30°C in YPD broth to mid log phase and cells were prepared for and examined by TEM.</p

Analysis of the <i>ERG11</i> chromosomal location in the Type2 <i>sey1Δglo3Δ</i> clones.

<p><b>A</b>) CHEF gel analysis. Chromosomes of the indicated strains were separated by CHEF and stained with ethidium bromide. <b>B</b>) Southern blot analysis. DNA of the CHEF gel was transferred to membrane and hybridized with <i>ERG11</i> (Chr1) and <i>CNAG_06646</i> (Chr7) probes. Star indicates the non-Chr1 located <i>ERG11</i>. <b>C</b>) Copy-number of <i>ERG11</i>. White bar indicates the copy number of <i>ERG11</i> from the CHEF quantified by phosphor-imager and the black bar represents the copy number of <i>ERG11</i> analyzed by qPCR. Gray bar = non-Chr1 <i>ERG11</i> indicates the copy number of <i>ERG11</i> from the CHEF quantified by phosphor-imager of the band marked with a star from B). Error bar = 2 SD.</p