Molecular mechanisms of caspofungin resistance in the pathogen Candida albicans

描述(由申请人提供)：念珠菌病是最常见的真菌感染，估计每年在美国发生6.3万例侵袭性念珠菌病，估计成本为20-40亿美元。虽然已从临床标本中分离出多种致病真菌，但念珠菌属的酵母菌是机会性真菌感染的主要原因，其中白色念珠菌是流行的病原体，估计导致40%的真菌血症。在过去的十年里，卡泊芬净有效地治疗了念珠菌感染，卡泊芬净是棘球菌素类的一种主要抗真菌药物，它干扰细胞壁的合成。随着卡泊芬净使用量的增加，卡泊芬净耐药菌株的感染报告数量已经增加，从2001年的0.5%上升到2009年的3.1%，因此卡泊芬净耐药性预计将在不久的将来成为一个主要问题。然而，与白色念珠菌对氟康唑的众多耐药机制相比，对其耐药机制(S)知之甚少。卡泊芬净临床耐药有一种公认的机制，涉及FKS1(orf19.2929)基因的点突变，该基因编码1，3-D-葡聚糖合成酶的一个亚单位，是正常合成葡聚糖所必需的，葡聚糖是细胞壁的主要成分。FKS1突变的形成总是与治疗失败有关。许多来自患者的其他分离株含有重塑的细胞壁，并在实验室实验中显示出对卡泊芬净的耐药性增加。这种所谓的细胞壁挽救机制与增强细胞壁的可逆变化有关，特别是甲壳素的增加。我们自己的数据表明，实验室对卡泊芬净的抗性可以通过多种分子机制获得。在卡泊芬净存在下培养白色念珠菌时，我们观察到了对CH5的耐药单体的出现，表明该染色体上存在耐药的负调控因子。单体CH5导致细胞壁中葡聚糖的减少和甲壳素的增加。克隆和鉴定参与该调节机制的CH5连锁基因将有助于阐明卡泊芬净耐药的关键分子途径。此外，在初步研究中，我们已经获得证据表明，CH5单体涉及FKS1(见上文)或GSL2基因的两倍下调，分别位于Ch1和ChR上，这是正常合成细胞壁葡聚糖所必需的。根据我们的发现，我们假设CH5携带正常合成细胞壁成分所需的多个基因。丢失一种CH5会导致实验室对棘球绦虫类药物的耐药性增加。为了支持这一假设，我们的初步分析揭示了CH5上的两个候选基因，它们可能参与单体菌株对卡泊芬净的耐药机制。在这里，我们建议启动对卡泊芬净耐药性负调控基因的研究。我们的发现将对临床医生和研究白念珠菌耐药现象的研究人员具有很高的意义。

DESCRIPTION (provided by applicant): Candidiasis is the most common fungal infection, with an estimated 63,000 episodes of invasive candidiasis per year occurring in the United States, with a cost estimated at $2-4 billion. Although a diverse selection of pathogenic fungi have been isolated from clinical samples, the yeast species of the genus Candida are the predominant cause of opportunistic fungal infections, with Candida albicans the prevalent pathogen, causing an estimated 40% of cases of fungemia. For the last decade, Candida infections have been effectively treated with caspofungin, a major antifungal of the echinocandin class that interferes with cell wall synthesis. As antici- pated based on increased use of this drug, the number of reports of infections with caspofungin-resistant strains has increased, from 0.5% in 2001 to 3.1% in 2009, and so caspofungin resistance is expected to be- come a major concern in the near future. However, in contrast to the numerous and well-understood mecha- nisms of C. albicans resistance to fluconazole, little is known regarding the mechanism(s) of resistance to cas- pofungin. There is one recognized mechanism of caspofungin clinical resistance that involves point mutations in the FKS1 (orf19.2929) gene encoding a subunit of 1,3-?-D-glucan synthase required for normal synthesis of glucan, a major component of the cell wall. Formation of FKS1 mutations is always associated with therapeutic failure. Many other isolates from patients contain remodeled cell wall and show increase of resistance to cas- pofungin in laboratory experiments. This co-called cell wall salvage mechanism is associated with reversible alterations that strengthen the cell wall, particularly increased chitin. Our own data show that laboratory resistance to caspofungin can be attained via multiple molecular mech- anisms. When culturing C. albicans in the presence of caspofungin, we have observed the emergence of re- sistant strains monosomic for Ch5, indicating that negative regulators of resistance are resident on this chro- mosome. Monosomic Ch5 leads to decreased glucan and increased chitin in the cell wall. Cloning and charac- terization of the Ch5-linked genes involved in this regulator mechanism will help elucidate key molecular pathways of caspofungin resistance. Moreover, in preliminary studies, we have obtained evidence that Ch5 monosomy involves two-fold downregulation of the FKS1 (see above) or GSL2 genes, residing on Ch1 and ChR, respectively, and required for normal synthesis of cell wall glucan. Based on our findings, we hypothesize that Ch5 carries multiple genes required for normal synthesis of cell wall components. Loss of one Ch5 leads to increased laboratory resistance to drugs of the echinocandin class. In support of this hypothesis, our initial analyses have revealed two candidate genes on Ch5 that are likely to be involved in the mechanism of caspofungin resistance in monosomic strains. Here, we propose to initiate the study of the genes that encode negative regulators of caspofungin resistance. Our findings will be of high significance to clinicians and researchers investigating the phenomenon of drug resistance in C. albicans.