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

• 批准号: 10395938
• 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
• 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/10395938
• 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; Research Personnel; Resistance; Resistance development; Role; Sepsis; Series; Testing; Tissue-Specific Gene Expression; Toxic effect; Ursidae Family; Yeasts; arm; base; beta-Glucans; breakthrough infection; clinical development; cost estimate; echinocandin resistance; epigenetic regulation; experimental study; fungus; gene regulatory network; gene repression; genome-wide; glucan synthase; in vitro Model; in vivo; mutant; new therapeutic target; novel; opportunistic pathogen; overexpression; pathogen; polyglucosan; resistance mutation; transcriptome; transcriptome sequencing

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 mechanism 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 mutations 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. Furthermore, 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 includes: 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 increased 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 provided by the model mutants to determine metabolic and mutational pathways that are activated in clinical isolates 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 investigating 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例侵袭性感染。 念珠菌病每年发生，估计费用为20 - 40亿美元。白色念珠菌是最常见的菌种， 导致高达50%的血液感染自2001年以来，C.白色念珠菌感染首先得到有效治疗， 卡泊芬净（CAS），后来与其他棘白菌素（ECN）。然而，ECN的使用不断扩大， 由于耐药性而导致的突破性感染数量增加。C.白念珠菌ECN耐药是编码催化亚单位的FKS 1基因中的特异性点突变 1，3-β-D-葡聚糖合成酶复合物。有趣的是，许多没有FKS 1突变的临床分离株表现出 在标准微量稀释试验中，ECN的最低抑制浓度（MIC）增加。我们自己的数据 表明C.白念珠菌细胞在体外对CAS的耐受性产生缺乏FKS 1抗性突变但表现出降低的CAS亲和性和重塑的细胞壁的突变体。一半的变种人 正常的二倍体，而其余的要么失去一个拷贝的染色体5（Ch 5）或转换一个拷贝的 Ch 5插入一条有两条右臂的染色体（iso-Ch 5 R）。重要的是，在二体Ch 5上的54个基因的表达是 在正常二倍体的突变体中下调了两倍或更多，这表明其中一些 Ch 5基因影响CAS易感性。我们发现，新发现的机制控制相同的 基因在体外以类似的方式表达，并且可以在ECN易感性降低的临床分离株中起作用。此外，上述模型突变体的DNA-seq分析揭示了2个独立的突变途径， 有限数量的突变热点。越来越多的影响ECN亲和性的因素还包括：a）位于Ch 5上的负或正调节因子的多个基因; B）正向调节基因 FKS 3，FKS 1的负调节因子，在所有类型的突变体和MIC增加的分离株中下调; c）对非整倍体染色体的表观遗传调节，也可能对二体Ch 5起作用。在 不存在来自ECN治疗患者的一系列多个连续分离株，包括药物初治分离株 和临床耐药分离株，表征可以降低ECN敏感性的体外机制， vivo具有特殊的重要性。我们认为，在体外发现的遗传机制是适应性的初始 这些变化降低了临床分离株的ECN敏感性，并使其存活至耐药突变。 FKS 1发生。为了确定ECN抗性的进化机制，我们建议1）使用模型突变体提供的信息来确定ECN易感性降低的临床分离株中激活的代谢和突变途径; 2）确定表观遗传调节是否支配基因调节 对二体Ch 5，以及3）确定在体外鉴定的机制的相关性，以真正的进化 临床分离株的耐药性。我们的研究结果将对临床医生和研究人员调查耐药现象的C。白色念珠菌