Fluconazole-Induced Ploidy Change in Cryptococcus neoformans Results from the Uncoupling of Cell Growth and Nuclear Division.
Fluconazole-Induced Ploidy Change in Cryptococcus neoformans Results from the Uncoupling of Cell Growth and Nuclear Division.
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DOI：
10.1128/msphere.00205-17
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发表时间：
2017-05
期刊：
影响因子：
4.8
通讯作者：
Kozubowski L
中科院分区：
文献类型：
--
作者：
Altamirano S;Fang D;Simmons C;Sridhar S;Wu P;Sanyal K;Kozubowski L
唑类是抗真菌药，由于相对低的毒性和治疗成本而被广泛使用。然而，它们的缺点之一是唑类主要是细胞抑制性的，使真菌细胞能够产生耐药性。人类病原体新型隐球菌通过非整倍体的发展获得对唑类药物氟康唑（FLC）的抗性，导致关键抗性基因的表达升高，这一机制对于白色念珠菌（K. J. Kwon-Chung和Y. C. Chang，PLoS Pathog 8：e1003022，2012，https：//doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser，J Microbiol 54：192-201，2016，https：//doi.org/10.1007/s12275-016-5628-4）。然而，FLC有助于增加这些重要真菌病原体中任一种的抗性的确切方式仍不清楚。在这里，我们发现FLC处理通过多种机制导致新型隐球菌中DNA含量的增加，从而可能增加从中选择具有增加的抗性的非整倍体的细胞池的大小。这项研究证明了FLC对生长和胞质分裂的抑制作用在产生对药物敏感性降低的细胞群中的重要性。新型隐球菌是一种致病性酵母菌，可导致免疫功能低下患者的致命性隐球菌脑膜炎。治疗隐球菌病的挑战之一是对唑类抗真菌药产生耐药性。以前的研究将唑类药物耐药与非整倍体细胞中关键耐药基因拷贝数的增加联系起来。然而，在唑类药物存在下如何形成非整倍体仍不清楚。这项研究表明，治疗与抑制浓度的唑类药物，氟康唑（FLC），导致在一个显着的人口与DNA含量增加的细胞，通过以下缺陷：抑制出芽，过早有丝分裂，并抑制胞质分裂，然后在母细胞中复制。胞质分裂的抑制和/或延迟导致具有两个或更多个子细胞附着的细胞（多聚体细胞）的形成。为了研究胞质分裂的哪一部分在FLC的存在下失败，用延时显微镜分析了肌动球蛋白环（AMR），septins和Cts 1（一种参与细胞分离的蛋白质）的动力学。随着AMR的收缩，间隔蛋白组装并且在母细胞和子细胞之间形成间隔。但是，隔膜的最终降解受到影响。具有异常形态的增大细胞，包括多聚体细胞，在FLC存在下表现出增殖潜力增加。这些发现表明，FLC对生长和有丝分裂的多效性作用导致DNA含量增加，导致细胞对药物不敏感。DNA含量增加的细胞继续增殖，因此增加了形成耐药群体的机会。唑类药物是一种广泛使用的抗真菌药物，因为其毒性和治疗成本相对较低。然而，它们的缺点之一是唑类主要是细胞抑制性的，使真菌细胞能够产生耐药性。人类病原体新型隐球菌通过非整倍体的发展获得对唑类药物氟康唑（FLC）的抗性，导致关键抗性基因的表达升高，这一机制对于白色念珠菌（K. J. Kwon-Chung和Y. C. Chang，PLoS Pathog 8：e1003022，2012，https：//doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser，J Microbiol 54：192-201，2016，https：//doi.org/10.1007/s12275-016-5628-4）。然而，FLC有助于增加这些重要真菌病原体中任一种的抗性的确切方式仍不清楚。在这里，我们发现FLC处理通过多种机制导致新型隐球菌中DNA含量的增加，从而可能增加从中选择具有增加的抗性的非整倍体的细胞池的大小。这项研究证明了FLC对生长和胞质分裂的抑制作用在产生对药物敏感性降低的细胞群中的重要性。
Azoles are antifungals that are widely utilized due to relatively low toxicity and cost of treatment. One of their drawbacks, however, is that azoles are primarily cytostatic, leaving fungal cells capable of developing drug resistance. The human pathogen Cryptococcus neoformans acquires resistance to the azole drug fluconazole (FLC) through the development of aneuploidy, leading to elevated expression of key resistance genes, a mechanism that is also common for Candida albicans (K. J. Kwon-Chung and Y. C. Chang, PLoS Pathog 8:e1003022, 2012, https://doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser, J Microbiol 54:192–201, 2016, https://doi.org/10.1007/s12275-016-5628-4). However, the exact ways in which FLC contributes to increased resistance in either of these important fungal pathogens remain unclear. Here we found that FLC treatment leads to an increase in DNA content in C. neoformans through multiple mechanisms, potentially increasing the size of a pool of cells from which aneuploids with increased resistance are selected. This study demonstrated the importance of FLC’s inhibitory effects on growth and cytokinesis in the generation of cell populations with decreased sensitivity to the drug. Cryptococcus neoformans is a pathogenic yeast that causes lethal cryptococcal meningitis in immunocompromised patients. One of the challenges in treating cryptococcosis is the development of resistance to azole antifungals. Previous studies linked azole resistance to elevated numbers of copies of critical resistance genes in aneuploid cells. However, how aneuploidy is formed in the presence of azole drugs remains unclear. This study showed that treatment with inhibitory concentrations of an azole drug, fluconazole (FLC), resulted in a significant population of cells with increased DNA content, through the following defects: inhibition of budding, premature mitosis, and inhibition of cytokinesis followed by replication in the mother cell. Inhibition of and/or a delay in cytokinesis led to the formation of cells with two or more daughter cells attached (multimeric cells). To investigate which part of cytokinesis fails in the presence of FLC, the dynamics of the actomyosin ring (AMR), septins, and Cts1, a protein involved in cell separation, were analyzed with time-lapse microscopy. Following the constriction of the AMR, septins assembled and the septum was formed between the mother and daughter cells. However, final degradation of the septum was affected. Enlarged cells with aberrant morphology, including multimeric cells, exhibited an increased potential to proliferate in the presence of FLC. These findings suggest that pleiotropic effects of FLC on growth and mitotic division lead to an increase in DNA content, resulting in cells less sensitive to the drug. Cells with increased DNA content continue to proliferate and therefore increase the chance of forming resistant populations. IMPORTANCE Azoles are antifungals that are widely utilized due to relatively low toxicity and cost of treatment. One of their drawbacks, however, is that azoles are primarily cytostatic, leaving fungal cells capable of developing drug resistance. The human pathogen Cryptococcus neoformans acquires resistance to the azole drug fluconazole (FLC) through the development of aneuploidy, leading to elevated expression of key resistance genes, a mechanism that is also common for Candida albicans (K. J. Kwon-Chung and Y. C. Chang, PLoS Pathog 8:e1003022, 2012, https://doi.org/10.1371/journal.ppat.1003022; J. Morschhäuser, J Microbiol 54:192–201, 2016, https://doi.org/10.1007/s12275-016-5628-4). However, the exact ways in which FLC contributes to increased resistance in either of these important fungal pathogens remain unclear. Here we found that FLC treatment leads to an increase in DNA content in C. neoformans through multiple mechanisms, potentially increasing the size of a pool of cells from which aneuploids with increased resistance are selected. This study demonstrated the importance of FLC’s inhibitory effects on growth and cytokinesis in the generation of cell populations with decreased sensitivity to the drug.