Virulence factor identification by comparative transcriptomics in Candida species

描述（由申请人提供）：总的来说，念珠菌属是发达国家真菌感染的最重要原因，约占播散性或侵袭性真菌感染的75%。这构成了一个重大的临床挑战，因为播散性念珠菌病很难诊断，并且通常对抗真菌治疗无效，导致数十年来死亡率一直居高不下，约为40%。与念珠菌属重叠的是一组被称为CTG进化枝的物种，因为它们非规范地翻译CUG密码子。CTG进化枝包括除C. glabrata是一种常见的分离病原体，尽管与酿酒酵母的关系比CTG物种更密切，而C.克柔丝菌一种罕见的病原体在CTG分支中，物种被隔离的频率有很大的变化：精确的数字在不同的研究中不同，但是C。白色念珠菌仍然负责一半以上的播散性念珠菌感染，而C。tropicalis和C.近平滑（约10%）通常是孤立的。C. lusitaniae和C. guillermondii是罕见的病原体，C. famata（又名汉逊德巴利酵母）是罕见的。来自仔细控制的动物模型的数据广泛地证实了这种流行病学模式反映了这些物种的固有毒力，尽管C。近平滑病的致命性可能比其发病率所暗示的要低。C.白色念珠菌在其他常见的念珠菌病表现中，如阴道炎和口咽鹅口疮中甚至更占优势。 这些相关物种所包含的遗传多样性提供了一种以新的方式了解毒力的工具：通过转录组学分析这些物种中的每一个对与宿主细胞相互作用的反应。这是一个非常成功的方法与C。白念珠菌，确定途径的压力抵抗和代谢适应。它还强调了大量的未表征的基因，其中许多是特异性的CTG分支和一些特定的C。白念珠菌及其近亲我们建议在这里使用比较转录谱的七个CTG进化枝物种，从高（C。albicans）至极低（D. Hansenii），在与巨噬细胞共培养期间，巨噬细胞是哺乳动物抗真菌免疫的关键组分。使用高通量RNA测序（RNA-seq），我们将定量确定在这些相互作用过程中真菌和哺乳动物组分的转录丰度。生物信息学分析将确定在最毒的物种中高度诱导的基因（C.白色念珠菌和C. tropicalis），但在毒性较低的物种中不存在或不受调节。然后将使用分子方法分析这些基因的优先子集，以了解它们在毒力中的作用。同时，小鼠巨噬细胞的概况将填补文献中的空白（尚未报道小鼠原代细胞的转录分析），更重要的是，将确定最毒力的物种如何减弱典型的抗真菌反应以促进存活;已提出了几种免疫调节机制，但对它们如何起作用知之甚少。 最后，C.白色念珠菌的研究导致绝大多数的分子工作都是在这个单一的物种中进行的。在C中有更多的基因被敲除。甚至在考虑由几个实验室产生的突变体文库之前，白色念珠菌的数量比所有其他CTG物种的总和还要多。在非白念珠菌物种中进行了很少的转录谱研究，该项目产生的数据将是了解这些重要物种并有助于注释这些基因组的极其宝贵的资源。

DESCRIPTION (provided by applicant): Collectively, the genus Candida is the most important cause of fungal infections in the developed world, responsible for roughly 75% of disseminated or invasive fungal infections. This poses a significant clinical challenge as disseminated candidiasis is difficult to diagnose and is often refractory to antifungal therapy, leading to a mortality rate that has remained stubbornly high, at around 40%, for decades. Overlapping the Candida genus is a group of species referred to as the CTG clade, because they translate the CUG codon non-canonically. The CTG clade encompasses all of the clinically significant Candida species with the exception of C. glabrata, a commonly isolated pathogen despite being much more closely related to Saccharomyces cerevisiae than to CTG species, and C. krusei, a rare pathogen. Within the CTG clade there is a wide variation in the frequency with which the species are isolated: precise numbers differ between studies, but C. albicans remains responsible for more than half of disseminated candidiasis infections, while C. tropicalis and C. parapsilosis (~10% each) are commonly isolated. C. lusitaniae and C. guillermondii are infrequent pathogens and C. famata (aka Debaromyces hansenii) is rarely seen. Data from carefully controlled animal models broadly confirm that this epidemiological pattern reflects the inherent virulence of these species, though C. parapsilosis is probably less virulent than its incidence rate would imply. C. albicans is even more dominant in other common manifestations of candiosis, such as vaginitis and oropharyngeal thrush. The genetic diversity encompassed by these related species offers a tool with which to understand virulence in a novel way: by dissecting the response of each of these species to interactions with host cell through transcriptomics. This has been a very successful approach with C. albicans, identifying pathways of both stress resistance and metabolic adaptations. It has also highlighted large numbers of uncharacterized genes, many of which are specific to the CTG clade and some specific only to C. albicans and its closest relatives. We propose here to use comparative transcriptional profiling of seven CTG clade species, with a range of virulence from high (C. albicans) to very low (D. hansenii), during co-cultures with macrophages, a key component of mammalian antifungal immunity. Using high-throughput RNA sequencing (RNA-seq), we will quantitatively determine transcript abundance from both the fungal and mammalian component during these interactions. Bioinformatic analysis will identify genes that are highly induced in th most virulent species (C. albicans and C. tropicalis) but either do not exist or are not regulated in the less virulent species. A prioritized subset of these genes will then be analyzed using molecular approaches to understand their role in virulence. Simultaneously, the profiles of the murine macrophages will both fill a present hole in the literature (no transcriptional analyses of murine primary cells have been reported) and, more importantly, will identify how the most virulent species may blunt the typical antifungal response to promote survival; several mechanisms of immunomodulation have been proposed but very little is known about how they might work. Finally, the clinical prominence of C. albicans has led to the vast majority of molecular work being performed in this single species. Far more genes have been knocked out in C. albicans than in all the other CTG species combined, even before considering mutant libraries generated by several labs. Very few transcript profiling studies have been performed in non-albicans species, and the data generated by this project will be an extremely valuable resource to understand these important species and to contribute to annotation of these genomes.