Molecular mechanisms of caspofungin susceptibility in the pathogen Candida albicans

念珠菌病是美国最常见的真菌感染，估计有6.3万例侵袭性病例 每年发生念珠菌病，估计费用为20-40亿美元。白色念珠菌，流行的物种， 导致高达50%的血液感染。自2001年以来，白色念珠菌感染首先得到了有效的治疗 卡泊芬净(CAS)，后来与其他棘球菌素(ECN)一起使用。然而，ECN的扩大使用导致了 由于耐药性，突破性感染的数量增加。公认的白色念珠菌ECN耐药的唯一机制是编码催化亚单位的FKS1基因的特异点突变 1，3-β-D-葡聚糖合成酶复合体。有趣的是，许多没有FKS1突变的临床分离株表现出 在标准微量稀释法中增加了对CNs的最小抑菌浓度(MIC)。我们自己的数据 研究表明，在体外直接将白色念珠菌细胞暴露于CAS会产生突变株，这些突变株缺乏FKS1抗性突变，但表现出降低的CAS敏感性和重塑的细胞壁。这些突变体中有一半保持着 正常的二倍体，而其余的要么丢失5号染色体(CH5)的一个副本，要么将 将CH5克隆到具有两个右臂的染色体上(iso-Ch5R)。重要的是，54个基因在二体CH5上的表达是 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 CH5基因影响CAS易感性。我们发现，新发现的机制控制着同样的情况 基因在体外具有类似的方式，并且可以在ECN敏感性降低的临床分离株中发挥作用。此外，对上述模型突变体的dna-seq分析显示，有两条独立的突变途径，其中 突变热点数量有限。越来越多的影响ECN易感性的因素还包括：a)存在于CH5上的多个负或正调控基因；b)同源基因 FKS3，FKS1的负调控因子，在所有类型的突变体和MIC增加的分离物中下调；c)对非整倍体染色体的表观遗传调控，可能也对二体CH5起作用。在……里面 ECN治疗的患者中没有一系列连续的分离株，包括药物-幼稚分离株 和临床耐药分离株，体外机制可以降低ECN敏感性的特征 VIVE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机制。 降低临床分离株对ECN的敏感性并允许存活直到出现耐药突变的变化 发生FKS1。为了确定ECN耐药性的进化机制，我们建议1)使用模型突变体提供的信息来确定在ECN易感性降低的临床分离株中被激活的代谢和突变途径；2)确定表观遗传调控是否控制基因调控 在二体CH5上，以及3)确定体外鉴定的机制与TRUE进化的相关性 临床分离株的耐药性。我们的发现将对临床医生和研究白念珠菌耐药现象的研究人员具有很高的意义。

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.