Molecular mechanisms of caspofungin susceptibility in the pathogen Candida albicans

白色念珠菌卡泊芬净敏感性的分子机制

基本信息

批准号：
9926826
负责人：
ELENA RUSTCHENKO
金额：
$ 38.5万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-07 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9926826
关键词：
ATAC-seq Acetylation Affect Aneuploidy Architecture Azoles Bioinformatics Biological Assay Biological Models Blood Candida Candida albicans Candidiasis Caspofungin Catalytic Domain Cell Wall Cell physiology Cells Cessation of life ChIP-seq Chromatin Chromosome 5 Chromosomes Clinical Complement Complex DNA Sequence Alteration DNA sequencing Data Development Diploidy Drug resistance Epigenetic Process Evolution Exhibits Frequencies Gene Activation Gene Expression Gene Expression Regulation Genes Genetic Genome Histones Hot Spot Human Immunocompromised Host In Vitro Incidence Individual Infection Knock-out Laboratories Lead Metabolic Metabolic Pathway Methods Methylation Minimum Inhibitory Concentration measurement Modality Modeling Molecular Multiple Fungal Drug Resistance Mutate Mutation Mycoses Orthologous Gene Pathway interactions Patients Pharmaceutical Preparations Ploidies Point Mutation Post-Translational Protein Processing Predisposition Prophylactic treatment Regulator Genes Research Personnel Resistance Resistance development Role Sepsis Series Tern Testing Tissue-Specific Gene Expression Toxic effect Ursidae Family Yeasts arm base beta-Glucans clinical development cost estimate echinocandin resistance epigenetic regulation experimental study fungus gene repression genome-wide glucan synthase in vitro Model in vivo mutant new therapeutic target novel overexpression pathogen polyglucosan resistance mutation transcriptome transcriptome sequencing

项目摘要

Candidiasis is the most common fungal infection in the US, with an estimated 63,000 episodes of invasive candidiasis occurring per year and an estimated cost of $2-4 billion. Candida albicans, the prevalent species, causes up to 50% of blood infections. Since 2001, C. albicans infections have been effectively treated first with caspofungin (CAS) and later with other echinocandins (ECNs). However, expanding use of ECNs has led to an increase in the number of breakthrough infections due to resistance. The only generally recognized mecha- nism of C. albicans ECN resistance are specific point mutations in the FKS1 gene encoding a catalytic subunit of 1,3-β-D-glucan synthase complex. Intriguingly, many clinical isolates that are free of FKS1 mutations exhibit increased minimum inhibitory concentrations (MICs) for ECNs in standard microdilution assays. Our own data show that direct exposure of C. albicans cells to CAS in vitro generates mutants that lack FKS1 resistance mu- tations but exhibit decreased CAS susceptibilities and remodeled cell walls. A half of these mutants remain as normal diploids, whereas the remainder either lose one copy of chromosome 5 (Ch5) or convert one copy of Ch5 into a chromosome with two right arms (iso-Ch5R). Importantly, expression of 54 genes on disomic Ch5 is downregulated twofold or more both in the mutants that are normal diploids, suggesting that some of these Ch5 genes affect CAS susceptibility. We are finding that the newly-discovered mechanisms control same genes in a similar fashion in vitro and can operate in clinical isolates with decreased ECN susceptibility. Fur- thermore, DNA-seq analysis of the above model mutants, revealed 2 independent mutational pathways with a limited number of mutational hot spots. A growing body of factors influencing ECN susceptibilities also in- cludes: a) multiple genes for either negative or positive regulators residing on Ch5; b) the orthologous gene FKS3, a negative regulator of FKS1 that is downregulated in all types of mutants and in an isolate with in- creased MICs; c) epigenetic regulation on aneuploid chromosomes that might also operate on disomic Ch5. In the absence of series of multiple consecutive isolates from ECN-treated patients including drug-naïve isolate and clinically resistant isolate, characterization of in vitro mechanisms that can decrease ECN susceptibility in vivo is of a special importance. We propose that the genetic mechanisms discovered in vitro are adaptive initial changes that decrease ECN susceptibility in clinical isolates and allow survival until a resistance mutation in FKS1 occurs. To define mechanisms of evolution of ECN resistance we propose to 1) use the information pro- vided by the model mutants to determine metabolic and mutational pathways that are activated in clinical iso- lates with decreased ECN susceptibility; 2) determine whether epigenetic regulation governs gene regulation on the disomic Ch5, as well as 3) determine the relevance of mechanisms identified in vitro to evolution of true resistance in clinical isolates. Our findings will be of high significance to clinicians and researchers investigat- ing the phenomenon of drug resistance in C. albicans.

念珠菌病是美国最常见的真菌感染，估计有 63,000 起侵袭性真菌感染念珠菌病每年发生一次，估计造成 2-40 亿美元的损失。自 2001 年以来，白色念珠菌感染首先得到有效治疗。卡泊芬净 (CAS) 以及后来与其他棘白菌素 (ECN) 一起使用然而，ECN 的使用范围扩大导致了由于耐药性而导致突破性感染数量的增加。白色念珠菌 ECN 抗性的本质是编码催化亚基的 FKS1 基因中的特定点突变有趣的是，许多临床分离株没有 FKS1 突变。我们自己的数据提高了标准微量稀释测定中 ECN 的最低抑制浓度 (MIC)。结果表明，在体外将白色念珠菌细胞直接暴露于 CAS 会产生缺乏 FKS1 抗性的突变体 mu- 但这些突变体中有一半仍表现出 CAS 敏感性降低和细胞壁重塑。正常的二倍体，而其余的要么丢失 5 号染色体 (Ch5) 的一份拷贝，要么转化 5 号染色体的一份拷贝。 Ch5 进入具有两个右臂的染色体 (iso-Ch5R) 重要的是，二体 Ch5 上的 54 个基因的表达是。在正常二倍体的突变体中均下调两倍或更多，这表明其中一些 Ch5 基因影响 CAS 易感性，我们发现新发现的机制也控制着 CAS 易感性。基因在体外以类似的方式进行，并且可以在 ECN 敏感性降低的临床分离株中发挥作用。 thermore 对上述模型突变体进行 DNA-seq 分析，揭示了 2 条独立的突变途径，其中数量有限的突变热点也影响 ECN 敏感性。包括：a) Ch5 上的负调节子或正调节子的多个基因 b) 直系同源基因； FKS3，FKS1 的负调节因子，在所有类型的突变体和具有 in- 的分离株中下调增加的 MIC；c) 对非整倍体染色体的表观遗传调控也可能对二体 Ch5 起作用。不存在来自 ECN 治疗患者的多个连续分离株，包括未接触过药物的分离株和临床耐药分离株，可降低 ECN 敏感性的体外机制特征我们认为体外发现的遗传机制是适应性初始的。降低临床分离株对 ECN 敏感性的变化，并允许存活直至出现耐药突变为了定义 ECN 抵抗的演变机制，我们建议 1）使用亲信息。由模型突变体拍摄视频，以确定临床异构体中激活的代谢和突变途径 2）确定表观遗传调控是否支配基因调控对二体 Ch5 进行研究，以及 3) 确定体外鉴定的机制与真实进化的相关性我们的研究结果对于追随者和研究人员的研究具有重要意义。白色念珠菌的耐药现象。