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

金黄色念珠菌作为一种医院内致病菌仅在十年前才出现，现已发展成为全球健康威胁，死亡率约为50%。C.Auris通常具有多药耐药性，粘在皮肤和医疗器械上，很难从高度依赖的医院单元中根除。因此，包括美国疾病控制中心(CDC)和英国公共卫生(PHA)在内的国家卫生当局已经发布了关于金黄色葡萄球菌的警报。位于阿伯丁的基因组生物和医学中心对覆盖四个主要分支的10个金黄色葡萄球菌分离株进行了全基因组测序；这为回答这些关键问题提供了主要资源。我们还获得了不受限制地获得来自Phe和CDC的暴发菌株的机会。(1)了解遗传多样性是如何在金黄色葡萄球菌中进化并驱动世界各地特定基因型的爆发的。我们最近的基因组测序和分析显示，与以前的报告相反，金黄色葡萄球菌具有一个完整的交配型基因座(MAT；真菌中的性别决定系统)。为了阐明Auris是否具有包括减数分裂在内的有性繁殖能力，我们将测试Mata株VPCI479/P/13是否与任何特征的mATX株有性相容。我们已经确定金龟子的基因组是单倍体的。使用脉冲场凝胶电泳法，我们还将确定--除了全基因组测序揭示的遗传多样性--金黄色葡萄球菌分离株是否显示出染色体数量和大小的差异。同一物种个体之间的核型差异带来了生殖障碍，但可能是遗传变异的驱动因素。这将指导我们选择要测试的菌株的交配组合，并将有助于不同的金黄色葡萄球菌分离株的完全组装的基因组。(2)表征金黄色葡萄球菌临床分离株的细胞壁表型多样性，并确定这是如何支持毒力的。为了阐明为什么该物种的表型高度可变以及为什么不同的分离物显示出显著的毒力差异，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球菌素类抗真菌药物的敏感性改变有关，而该物种通常对棘球菌素类药物具有抗药性。细胞黏附是假丝酵母菌的既定毒力因素，有趣的是，金黄色念珠菌的感染很容易在人与人之间传播，很难根除。这表明，在金黄色葡萄球菌中，粘附性和毒力有很好的相关性。这一目标对于阐明尚不清楚的金黄色葡萄球菌感染的流行病学方面具有重要的潜力。(3)研究金黄色葡萄球菌临床分离株多药耐药基因的遗传多样性及其对毒力的影响。Auris有一个编码MDR药物外排泵的基因家族，了解这些基因如何对其整体多药耐药表型做出贡献是至关重要的。在这个项目中，生物信息学分析将揭示哪些菌株中存在哪些药物外排因子。这一信息将被用来选择其药物外排因子将接受实时聚合酶链式反应分析的4个分支中的每一个分支的代表，以确定哪些在暴露于不同类别的抗真菌药物(氮唑、棘球菌素、两性抗菌素)后上调。那些在抗真菌作用下上调的基因将通过CRISPR-Cas9技术选择性地消除，我们现在对白色念珠菌有丰富的经验。本项目对阐明金黄色葡萄球菌临床分离株之间耐药性差异的来源具有很大的潜力，最终将为治疗感染金黄色葡萄球菌的患者提供指导。

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