Echinocandin Reduced/Increased Susceptibility: Mechanism

棘白菌素降低/增加敏感性：机制

• 批准号: 8707692
• 负责人: Santosh kumar Katiyar
• 金额: $ 32.68万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-06 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8707692
• 关键词: Antifungal Agents; Aspergillus; Aspergillus fumigatus; Aspergillus nidulans; Azoles; Binding; CRSP3 gene; Candida albicans; Candida glabrata; Caspofungin; Cell Wall; Clinical; Complement; DNA Sequence Analysis; Data; Dihydrosphingosine; Exhibits; Frequencies; Genes; Genetic Screening; Hyphae; Immunocompromised Host; Individual; Infection; Integral Membrane Protein; Laboratories; Light; Lipids; Loss of Heterozygosity; Mediating; Membrane; Membrane Proteins; Micafungin; Modeling; Moths; Mutation; Mycoses; Pathway interactions; Phenotype; Phosphorylation; Predisposition; Refractory; Regulation; Resistance; Role; Saccharomyces cerevisiae; Sequence Analysis; Side; Sphingolipids; Testing; Waxes; Yeasts; base; dihydroceramide desaturase; dimorphism; fitness; fungus; high risk; inhibitor/antagonist; mutant; novel; novel strategies; pathogen; phytosphingosine; resistance mutation

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.

描述(由申请人提供)：近年来，由于免疫缺陷个体数量的增加，侵袭性真菌感染急剧增加。同时，光滑念珠菌已经成为一种重要的病原菌，主要是因为它对广泛使用的唑类抗真菌药物治疗无效。卡泊芬净(CSP)和米卡芬净(MCF)等棘球绦菌素对光滑棘球绦虫(MIC0.06g/ml)有较强的抗菌活性，最近被提升为治疗这种酵母菌感染的一线药物。对这些脂肽合成抑制剂的罕见获得性耐药(MIC1)与棘球绦虫交叉耐药和整膜蛋白Fks1或Fks2的突变有关。我们最近描述了出现频率相对较高的新的实验室突变株，这些突变株表现出Csp降低的敏感性(CRs；MIC=0.120.5)，而Mcf增加了敏感性(MIs；MIC=0.0005至0.008)；临床分离株也具有这种表型。有趣的是，FKS测序和基因破坏证明CRS-MIS是FKS不依赖的。阐明CRS-MIS机制很重要，因为它将(1)支持并建议加强棘球蛋白治疗的新方法(例如，序贯CSP-MCF，或将MCF与模仿CRS-MIS突变的抑制剂/中间体结合)，以及(2)阐明迫切需要的Echinocandin的作用机制(即，差异性意味着可变的细胞成分直接与CSP与MCF特异的侧链结合)。为了揭示这一机制，在酿酒酵母中使用了两个不同的遗传筛选，两者都与鞘磷脂的合成有关。因此，对光滑毛囊线虫CRS-MIS突变体和临床分离株进行了脂质分析，发现二氢鞘氨醇和植鞘氨醇水平增加。基于这些数据，对特定的基因进行了测序，实际上，在SUR2、FEN1、SUR4和IFA38CRS-MIS中发现的突变并不局限于光滑念珠菌：(A)在测试的6株白色念珠菌中，有1株在高频下产生突变(8倍CRS，16倍MIS)，脂类分析/DNA测序表明鞘磷脂途径基因TSC13/TSC13丢失杂合性；以及(B)一株nidulans Basa(SUR2)突变株表现出2倍CRS，8倍MIS。为了扩展这些研究，我们提出了三个目标：(1)光叶锦鸡儿。将确定与CRS-MI相关的全部突变，评估Galeria模型中的适合性，检查通路抑制剂和中间体的影响，并评估CRS-MI和FKS突变之间的相互作用。(2)酿酒酵母。Cka2调节鞘磷脂途径的机制将被探索，鞘脂改变通过改变FKS-膜拓扑结构而导致CRS-MI的假说将得到验证。(3)白色念珠菌和致病性曲霉菌。将分析表现为CRS-MIS高危的白色念珠菌菌株的基因相关性和机制，并检查烟曲霉菌和土霉的CRS-MIS能力和机制。