Genetic diversity as a major driver of phenotypic variability and virulence in local outbreak strains of the emerging pathogen Candida auris

耳念珠菌仅在十年前才作为医院病原体出现，并已发展成为全球健康威胁，死亡率约为50%。C.耳炎通常具有多重抗药性，粘附在皮肤和医疗设备上，并且难以从高度依赖性的医院病房中根除。因此，国家卫生当局，包括美国疾病控制中心（CDC）和英国公共卫生局（PHA）已经发布了关于C的警报。阿伯丁的基因组生物学和医学中心进行了10个C. auris的全基因组测序。auris分离物覆盖4个主要分支;这为回答这些关键问题提供了主要资源。我们还被赋予了不受限制地获得来自PHE和CDC的爆发菌株的权利。(1)了解遗传多样性如何在C.我们最近的基因组测序和分析表明，C. auris和驱动世界各地的特定基因型的爆发。与以前的报道相反，auris有一个完整的交配型位点（MAT;真菌的性别决定系统）。阐明C. auris能够进行有性生殖，包括减数分裂，我们将测试MATa-菌株VPCI 479/P/13是否与任何表征的MATx-菌株性相容。我们已经确定C. auris基因组为单倍体。使用脉冲场凝胶电泳，我们还将确定是否-除了通过全基因组测序揭示的遗传多样性- C。耳道分离物显示染色体数目和大小差异。同一物种的个体之间的核型差异会引入生殖障碍，但可能是遗传变异的驱动因素。这将指导我们选择待测分离株的交配组合，并将有助于完全组装不同C.耳分离物。(2)临床分离的C.为了阐明为什么这个物种在其表型上是高度可变的，以及为什么不同的分离株在其毒力上表现出显着差异，本项目将研究细胞壁组成的遗传和生理变化以及细胞壁对抗生素胁迫的反应。这与细胞的聚集特性有关，也与免疫识别和对棘白菌素抗真菌药物的敏感性改变有关，而棘白菌素抗真菌药物通常对该物种具有抗性。细胞粘附是假丝酵母菌的毒力因子，有趣的是，假丝酵母菌也是一种重要的致病菌.耳感染很容易在人与人之间传播，并且很难根除。这表明粘附和毒力在C.耳。这一目标对阐明C.目前尚不清楚的耳鼻喉感染 (3)目的：研究肺炎克雷伯菌多药耐药决定簇的遗传多样性。耳临床分离株，并确定这些如何影响耐药性。auris具有编码MDR药物外排泵的大规模扩展的基因家族，并且了解这些基因如何促成其总体多药耐药表型至关重要。在这个项目中，生物信息学分析将揭示哪些药物外排因子存在于哪些分离株中。该信息将用于选择4个进化枝中每一个的代表，其药物外排因子将进行实时PCR分析，以确定哪些因子在暴露于不同类别的抗真菌药（唑类、棘白菌素类、阿氏菌素类）后上调。那些因抗真菌暴露而上调的基因将被CRISPR-Cas9技术选择性地消除，我们现在对C.白色念珠菌。本研究对阐明临床分离株间耐药性差异的来源具有重要意义。auris，并最终将为治疗感染C.耳株

Candida auris has emerged as a nosocomial pathogen only a decade ago, and has developed into a global health threat with a mortality rate of ~50%. C. auris is often multidrug resistant, sticks to skin and medical devices, and is difficult to eradicate from high-dependency hospital units. Consequently, national health authorities, including the Centre for Disease Control in the USA (CDC) and Public Health England (PHA) have posted alerts on C. auris.The Centre for Genome Biology and Medicine at Aberdeen undertook the whole genome sequencing of ten C. auris isolates covering the 4 main clades; this provides a major resource for answering these key questions. We also have been gifted unrestricted access to outbreak strains from PHE and the CDC.(1) Understanding how genetic diversity evolves in C. auris and drives the outbreak of specific genotypes around the world Our recent genome sequencing and analysis revealed that C. auris, contrary to previous report, has a complete mating type locus (MAT; the sex determination system in fungi). To elucidate whether C. auris is capable of sexual reproduction including meiosis, we will test whether the MATa-strain VPCI479/P/13 is sexually compatible with any of the characterized MATx-strains. We have already established that the C. auris genome is haploid. Using pulsed-field gel electrophoresis we will also determine whether - in addition to the genetic diversity revealed by the whole genomic sequencing - C. auris isolates display chromosome number and size differences. Differences in karyotype between individuals of the same species introduce a reproductive barrier, but can be a driver of genetic variability. This will guide our choice of mating combinations of isolates to be tested, and will contribute to fully assembled genomes of different C. auris isolates.(2) Characterization of the cell wall's phenotypic diversity in clinical isolates of C. auris and determining how this underpins virulenceTo elucidate why this species is hypervariable in its phenotype and why various isolates show significant differences in their virulence, this project will examine the genetic and physiological variation in cell wall composition and the response of the cell wall to antibiotic stress. This is relevant to the aggregating properties of the cells, as well as to immune recognition and altered sensitivity to echinocandin antifungal drugs which this species is often resistant to. Cell adhesion is as established virulence factor of Candida species and, intriguingly, C. auris infections are passed readily from person to person and are hard to eradicate. This suggests that adherence and virulence correlate well in C. auris. This objective has significant potential for elucidating aspects of the epidemiology of C. auris infections that are not yet understood. (3) Characterization of the genetic diversity of mult-drug resistance determinants of C. auris clinical isolates and determining how these affect virulenceC. auris has a massively expanded family of genes encoding MDR drug efflux pumps, and it is crucially important to understand how these contribute to its overall multi-drug resistance phenotype. In this project bioinformatics analysis will reveal which drug efflux factors are present in which isolates. This information will be used to select representatives of each of the 4 clades whose drug efflux factors will be subjected to realtime-PCR analysis to determine which are upregulated in response to exposure to different classes of antifungals (azoles, echinocandins, amphothericin). Those genes that are upregulated in response to antifungal exposure will be selectively oblated by CRISPR-Cas9 technology, for which we now have extensive experience for C. albicans. This project has great potential to elucidate the origin of differences in drug resistance between clinical isolates of C. auris, and ultimately will provide guidance for treating patients infected with C. auris strains