Echinocandin Reduced/Increased Susceptibility: Mechanism

描述（由申请人提供）：近年来，由于免疫功能低下个体数量的增加，侵袭性真菌感染急剧增加。与此同时，光滑念珠菌已成为一种重要的病原体，主要是因为它是难治性的治疗与广泛使用的唑类抗真菌药。包括卡泊芬净（CSP）和米卡芬净（MCF）在内的棘白菌素类药物对C.光叶甘草素（MIC 0.06？g/ml），并且最近被提升为治疗该酵母感染的一线药剂。对这些细胞壁合成脂肽抑制剂的罕见获得性耐药性（MIC 1）与棘白菌素交叉耐药性和整合膜蛋白Fks 1或Fks 2的突变有关。我们最近描述了以相对高的频率出现的新型实验室突变体，其表现出CSP降低的敏感性（CRS; MIC = 0.12至0.5），但MCF增加的敏感性（MIS; MIC = 0.0005至0.008）;还鉴定了具有这种表型的临床分离株。有趣的是，FKS测序和基因破坏表明CRS-MIS是FKS非依赖性的。阐明CRS-MIS机制是重要的，因为它将（1）支持和建议增强棘白菌素治疗的新方法（例如，连续CSP-MCF，或将MCF与模拟CRS-MIS突变的抑制剂/中间体组合）和（2）揭示了棘白菌素作用机制急需的光（即，差异亲和性意味着改变的细胞组分直接结合CSP与MCF特异性侧链）。为了揭示这一机制，在酿酒酵母中采用了两种不同的遗传筛选，都涉及鞘脂合成。因此，C.对glabrata CRS-MIS突变体和临床分离株进行了研究，揭示了增加的二氢鞘氨醇和植物鞘氨醇水平。基于这些数据，对特定的基因进行测序，实际上，在SUR 2、FEN 1、SUR 4和IFA 38中鉴定的突变CRS-MIS不限于C。光滑：（a）6种白色念珠菌菌株中有1种以高频率产生突变体（8倍CRS，16倍MIS），脂质分析/DNA测序表明鞘脂途径基因TSC 13/tsc 13杂合性缺失;（B）构巢曲霉basA（Sur 2）突变体显示2倍CRS，8倍MIS。为了扩展这些研究，我们提出了三个目标：（1）C.光滑的将鉴定赋予CRS-MIS的全部突变，在Galleria模型中评估适应性，检查途径抑制剂和中间体的影响，并评价CRS-MIS和Fks突变之间的相互作用。(2)S.啤酒。将探讨Cka 2调节鞘脂途径的机制，并检验鞘脂变化通过改变Fks-膜拓扑结构赋予CRS-MIS的假设。(3)C.白色念珠菌和致病性曲霉菌。C.将分析显示高风险CRS-MIS的白色念珠菌菌株的基因型相关性和机制，以及A. fumigatus和A.对terreus进行了CRS-MIS能力和机制的审查。

DESCRIPTION (provided by applicant): Invasive fungal infections have increased dramatically in recent years due to increased numbers of immunocompromised individuals. In parallel, Candida glabrata has emerged as an important pathogen, largely because it is refractory to treatment with widely used azole antifungals. Echinocandins including caspofungin (CSP) and micafungin (MCF) have potent activity versus C. glabrata (MIC 0.06 ?g/ml) and were recently elevated to first-line agents for treating infections with this yeast. Rare acquired resistance (MIC 1) to these lipopeptide inhibitors of cell wall synthesis is associated with echinocandin cross-resistance and mutations in the integral membrane proteins Fks1 or Fks2. We recently described novel laboratory mutants arising at relatively high frequency that exhibit CSP reduced susceptibility (CRS; MIC = 0.12 to 0.5) but MCF increased susceptibility (MIS; MIC = 0.0005 to 0.008); clinical isolates with this phenotype were also identified. Intriguingly, FKS sequencing and gene disruption demonstrate that CRS-MIS is Fks-independent. Elucidating the CRS-MIS mechanism is important since it will (1) support and suggest new approaches to enhancing echinocandin therapy (e.g., sequential CSP-MCF, or combining MCF with an inhibitor/intermediate that mimics the CRS-MIS mutation) and (2) shed much needed light on echinocandin mechanism of action (i.e., differential susceptibilities imply that the alterd cellular component directly binds to a CSP versus MCF-specific side chain). To uncover this mechanism, two distinct genetic screens in Saccharomyces cerevisiae were employed, both implicating sphingolipid synthesis. Consequently, lipid analysis of C. glabrata CRS-MIS mutants and clinical isolates was pursued, revealing increased levels of dihydrosphingosine and phytosphingosine. Based on these data specific genes were sequenced and, indeed, mutations identified in SUR2, FEN1, SUR4, and IFA38 CRS-MIS is not limited to C. glabrata: (a) 1 of 6 Candida albicans strains tested yielded mutants (8-fold CRS, 16-fold MIS) at high frequency, and lipid analysis/DNA sequencing implicated sphingolipid pathway gene TSC13/tsc13 loss of heterozygosity; and (b) an Aspergillus nidulans basA(Sur2) mutant exhibited 2-fold CRS, 8-fold MIS. To extend these studies, we propose three Aims focusing on: (1) C. glabrata. The full complement of mutations conferring CRS-MIS will be identified, fitness assessed in the Galleria model, effects of pathways inhibitors and intermediates examined, and interaction between CRS-MIS and Fks mutations evaluated. (2) S. cerevisiae. The mechanism of sphingolipid pathway regulation by Cka2 will be explored, and the hypothesis that sphingolipid changes confer CRS-MIS through altered Fks-membrane topology will be tested. (3) C. albicans and pathogenic aspergilli. C. albicans strains exhibiting high risk CRS-MIS will be analyzed for genotypic relatedness and mechanism, and A. fumigatus and A. terreus examined for CRS-MIS capacity and mechanism.