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

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.新型隐球菌（Cryptococcus neoformans）的异质性耐药是其对唑类药物的内在适应性耐药，而异质性耐药表型与染色体二体性密切相关。此前已有研究报道，1号染色体（Chr1）上的两个基因——氟康唑靶标基因ERG11与药物转运蛋白编码基因AFR1，是1号染色体二体性出现的核心驱动因素。本研究证实，在高浓度氟康唑（FLC）胁迫环境中，4号染色体（Chr4）二体性是第二常见的染色体变异类型，并对该染色体上参与二体性形成的内源基因的重要性进行了系统表征。我们针对9个假定参与麦角固醇生物合成、膜组成/完整性调控或药物转运过程（上述过程均可影响氟康唑胁迫下4号染色体二体性形成）的4号染色体基因开展了基因敲除实验。在这9个敲除菌株中，3个与酿酒酵母Sey1（一种GTP酶）、Glo3和Gcs2（ADP核糖基化因子GTP酶激活蛋白）同源的基因被敲除后，异质性耐药克隆中4号染色体二体性的发生频率显著降低。进一步研究发现，源自sey1Δglo3Δ双敲除菌株的氟康唑耐药克隆，既未出现4号染色体二体性，也未检测到1号染色体二体性，反而使1号染色体上ERG11位点邻近基因的拷贝数显著增加。由于这3个基因在酿酒酵母中对于维持内质网（endoplasmic reticulum, ER）的完整性至关重要，我们以Sec61ß-GFP融合蛋白作为标记物，对突变菌株的内质网结构进行了观察。荧光显微镜结果显示，sey1Δ突变株的细胞质内质网发生了明显伸长，但gcs2Δ和glo3Δ突变株的内质网未出现可识别的形态改变。不过，通过透射电子显微镜可清晰观察到这3个突变菌株均存在异常的内质网形态。利用聚焦离子束扫描电子显微镜（Focused Ion Beam Scanning Electron Microscopy, FIB-SEM）进行的三维重建结果显示，glo3Δ和gcs2Δ菌株的内质网网状结构显著减少。与野生型菌株相比，sey1Δ突变株的内质网网状结构几乎无法被检测到，且膜潴泡发生了广泛扩张。上述实验数据表明，在唑类药物胁迫下，维持内质网完整性所需的基因，对新型隐球菌染色体二体性的形成具有关键调控作用。