Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence.
Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence.
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DOI：
10.1371/journal.ppat.1000753
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发表时间：
2010-02-05
期刊：
影响因子：
6.7
通讯作者：
Whiteway M
中科院分区：
文献类型：
--
作者：
Epp E;Vanier G;Harcus D;Lee AY;Jansen G;Hallett M;Sheppard DC;Thomas DY;Munro CA;Mullick A;Whiteway M
白色念珠菌是人类的主要真菌病原体，在免疫功能低下的个体中引起危及生命的感染。由于可用的治疗选择有限，这可能经常导致治疗失败和出现耐药性。为了改善目前的治疗策略，我们结合了酿酒酵母中的综合化学基因组筛选和C。目的是鉴定可以与真菌抑制药物氟康唑结合使其具有杀真菌作用的化合物。在酵母筛选中鉴定的基因中，我们发现只有编码ADP核糖基化因子GT3激活效应蛋白的AGE 3消除了C.白色念珠菌age 3突变体对其他固醇和细胞壁抑制剂（包括卡泊芬净）更敏感。AGE 3在耐药临床分离株和组成型活性钙调神经磷酸酶信号转导突变体中的缺失恢复了氟康唑的敏感性。我们通过在野生型C中显示氟康唑和布雷菲德菌素A（ADP核糖基化因子的鸟嘌呤核苷酸交换因子的抑制剂）之间的有效杀真菌协同作用，证实了AGE 3依赖性药物敏感性。白色念珠菌以及耐药临床分离株。在氟康唑/布雷菲德菌素A组合中加入钙调磷酸酶抑制剂仅在最初提高了病原体杀灭。布雷菲德菌素A与不同药物在非白色念珠菌属以及烟曲霉中具有协同作用。微阵列研究表明，核心转录反应，两种不同的药物类别没有显着改变年龄3突变体。通过体内研究证实了抑制ARF活性的治疗潜力，这些研究表明，年龄3的突变体在野生型小鼠中是无毒的，在免疫功能低下的小鼠中毒力减弱，并且当ARF信号传导受到遗传损害时，氟康唑治疗显著更有效。这项工作描述了一种新的，广泛保守的，广谱的机制，涉及真菌的耐药性和毒力，并提供了一个潜在的途径，单一或改进的联合治疗。 白色念珠菌是一种真菌，通常作为人体肠道微生物区系的一部分。念珠菌属可引起健康人群的浅表感染（如鹅口疮）和免疫功能低下患者的危及生命的侵入性感染。真菌感染通常用唑类药物治疗，但由于这些药物的抑真菌性质，C。白色念珠菌可产生耐药性，导致治疗失败。为了改善唑类药物的作用并将其转化为杀真菌药物，我们首先系统地分析了对唑类药物氟康唑耐受性的遗传要求。我们表明，无论是遗传学还是生物学，ARF循环机制的组成部分在介导C.白色念珠菌以及其他几种致病性念珠菌和致病性烟曲霉。我们强调了ARF循环在耐药性中的重要性，表明ARF功能的遗传妥协压倒了临床样本和其他唑类/棘白菌素耐受性关键调节因子中常见的耐药机制。我们在两种小鼠模型中验证了ARF循环的治疗潜力，并提供了当ARF活动受到遗传损害时药物治疗更有效的证据。我们的研究证明了一种新的机制，涉及人类真菌病原体生物学的两个重要方面，并为改善抗真菌治疗提供了一种潜在的途径。
Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies. Candida albicans is a fungus that normally resides as part of the microflora in the human gut. Candida species can cause superficial infections like thrush in the healthy human population and life-threatening invasive infections in immunocompromised patients. Fungal infections are often treated with azole drugs, but due to the fungistatic nature of these agents, C. albicans can develop drug resistance, leading to therapy failure. To improve the action of azoles and convert them into fungicidal drugs, we first systematically analyzed the genetic requirements for tolerance to one such azole drug, fluconazole. We show, both genetically and pharmacologically, that components of the ARF cycling machinery are critical in mediating both azole and echinocandin tolerance in C. albicans as well as several other pathogenic Candida species and in the pathogenic mold Aspergillus fumigatus. We highlight the importance of ARF cycling in drug resistance by showing that genetic compromise of ARF functions overrides common drug resistance mechanisms in clinical samples and other key regulators of azole/echinocandin tolerance. We validated the therapeutic potential of ARF cycling in two mouse models and provide evidence that drug treatment is more efficacious when ARF activities are genetically compromised. Our study demonstrates a new mechanism involved in two important aspects of the biology of human fungal pathogens and provides a potential route for improved antifungal therapies.
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影响因子：
4.3
作者：
Dean, N;Poster, JB
通讯作者：
Poster, JB
影响因子：
--
作者：
Dunkel, Nico;Liu, Teresa T.;Rogers, P. David
通讯作者：
Rogers, P. David
影响因子：
6.7
作者：
Askew C;Sellam A;Epp E;Hogues H;Mullick A;Nantel A;Whiteway M
通讯作者：
Whiteway M
影响因子：
3.1
作者：
Blankenship, JR;Heitman, J
通讯作者：
Heitman, J
影响因子：
3.1
作者：
Bader, T;Bodendorfer, B;Morschhäuser, J
通讯作者：
Morschhäuser, J