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，估计在美国每年发生63,000例侵袭性念珠菌病，成本估计为20 - 40亿美元。虽然从临床样本中分离出多种致病真菌，但念珠菌属的酵母菌种类是机会性真菌感染的主要原因，白色念珠菌是流行的病原体，估计造成40%的真菌血症病例。在过去的十年里，念珠菌感染已经被有效地用卡泊真菌素治疗，卡泊真菌素是一种主要的抗真菌药物，可以干扰细胞壁的合成。正如基于该药使用增加的预测，caspofungin耐药菌株感染的报告数量已从2001年的0.5%增加到2009年的3.1%，因此caspofungin耐药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耐药的众多且已被充分了解的机制相比，对caspofungin耐药的机制知之甚少。有一种公认的caspofunins临床耐药机制，涉及编码1,3- 1亚基的FKS1 （orf19.2929）基因点突变。d -葡聚糖合成酶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。在实验室实验中，从病人身上分离出的许多其他菌株含有重塑的细胞壁，并显示出对cas- pofungin的抗性增加。这种所谓的细胞壁挽救机制与增强细胞壁的可逆改变有关，特别是几丁质的增加。我们自己的数据表明，对caspofungin的实验室抗性可以通过多种分子机制获得。当在caspofungin存在下培养白色念珠菌时，我们观察到Ch5单体耐药菌株的出现，表明抗性的负调节因子驻留在该染色体上。单体Ch5导致细胞壁葡聚糖减少，几丁质增加。克隆和鉴定参与该调控机制的ch5连锁基因将有助于阐明caspofungin耐药性的关键分子途径。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基因的双重下调，分别位于Ch1和ChR上，并需要正常合成细胞壁葡聚糖。基于我们的发现，我们假设Ch5携带细胞壁成分正常合成所需的多个基因。失去一个Ch5会导致对棘白菌素类药物的实验室耐药性增加。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，它们可能参与了单体菌株对caspofungin的抗性机制。在此，我们建议开始研究编码caspofungin抗性负调节因子的基因。我们的发现将对临床医生和研究人员研究白色念珠菌耐药现象具有重要意义。

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.