Echinocandin Reduced/Increased Susceptibility: Mechanism

描述（由申请人提供）：近年来，由于免疫功能低下的个体数量增加，侵袭性真菌感染急剧增加。与此同时，光秃念珠菌已成为一种重要的病原体，主要是因为它难以用广泛使用的唑类抗真菌药物治疗。棘白菌素包括caspofungin （CSP）和micafungin （MCF）对光斑棘白菌(C. glabrata, MIC 0.06 ？G /ml)，最近被提升为治疗这种酵母菌感染的一线药物。对这些细胞壁合成脂肽抑制剂的罕见获得性耐药（MIC 1）与棘白菌素交叉耐药和整体膜蛋白Fks1或Fks2突变有关。我们最近描述了以相对较高的频率出现的新型实验室突变体，其表现为CSP降低了易感性（CRS, MIC = 0.12至0.5），但MCF增加了易感性（MIS, MIC = 0.0005至0.008）；具有这种表型的临床分离株也被鉴定出来。有趣的是，FKS测序和基因破坏表明CRS-MIS与FKS无关。阐明CRS-MIS机制很重要，因为它将(1)支持并提出加强棘白菌素治疗的新方法（例如，顺序CSP-MCF，或将MCF与模拟CRS-MIS突变的抑制剂/中间体结合）；(2)揭示棘白菌素的作用机制（即，不同的敏感性意味着改变的细胞成分直接结合CSP与MCF特异性侧链）。为了揭示这一机制，研究人员利用了酿酒酵母中两种不同的基因筛选，它们都与鞘脂合成有关。因此，脂质分析显示，二氢鞘氨醇和植物鞘氨醇水平升高。基于这些数据，对特定基因进行了测序，确实，在SUR2、FEN1、SUR4和IFA38中鉴定出的CRS-MIS突变并不局限于光毛念珠菌：(a) 6株白色念珠菌中有1株在高频率下产生突变（8倍CRS， 16倍MIS），脂质分析/DNA测序涉及鞘脂通路基因TSC13/ TSC13杂合性缺失；(b)细粒曲霉basA（Sur2）突变体表现为2倍CRS， 8倍MIS。为了进一步扩展这些研究，我们提出了三个目标，重点是：(1)C. glabrata。将鉴定授予CRS-MIS的全部突变，评估Galleria模型中的适应度，检查通路抑制剂和中间体的作用，并评估CRS-MIS和Fks突变之间的相互作用。(2)酿酒葡萄球菌。我们将探索Cka2调控鞘脂通路的机制，并验证鞘脂改变通过改变fks -膜拓扑结构赋予CRS-MIS的假设。(3)白色念珠菌和致病性曲霉。分析白色念珠菌发生CRS-MIS的高危菌株的基因型相关性和机制，研究烟曲霉和地芽孢杆菌发生CRS-MIS的能力和机制。

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.