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，大约75%的播散性或侵袭性真菌感染是由念珠菌引起的。这构成了一个重大的临床挑战，因为弥散性念珠菌病难以诊断，而且通常对抗真菌治疗难以治愈，导致数十年来死亡率一直居高不下，约为40%。重叠念珠菌属是一组被称为CTG分支的物种，因为它们非规范地翻译CUG密码子。CTG分支包括所有临床重要的念珠菌物种，除了C. glabrata（一种常见的分离病原体，尽管它与酿酒酵母的关系比与CTG物种的关系更密切）和C. krusei（一种罕见的病原体）。在CTG分支中，该物种被分离的频率差异很大：不同研究之间的精确数字不同，但白色念珠菌仍然占播散性念珠菌感染的一半以上，而热带念珠菌和副念珠菌（各约10%）通常被分离出来。C. lusitaniae和C. guillermondii是罕见的病原体，C. famata（又名Debaromyces hansenii）很少见到。来自精心控制的动物模型的数据广泛证实，这种流行病学模式反映了这些物种的固有毒力，尽管副棘球蚴病的毒力可能低于其发病率所暗示的。白色念珠菌在念珠菌病的其他常见表现中更占优势，如阴道炎和口咽鹅口疮。这些相关物种所包含的遗传多样性为以一种新的方式理解毒力提供了一种工具：通过转录组学分析这些物种对与宿主细胞相互作用的反应。这是一种非常成功的方法，可以识别出白色念珠菌的抗逆性和代谢适应途径。它还突出了大量未表征的基因，其中许多是CTG分支所特有的，有些仅针对白色念珠菌及其近亲。我们建议在与巨噬细胞（哺乳动物抗真菌免疫的关键组成部分）共培养过程中，使用7种CTG分支物种的比较转录分析，这些物种具有从高毒力（白色念珠菌）到极低毒力（D. hansenii）的范围。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成分的转录物丰度。生物信息学分析将确定在毒性最强的物种（白色念珠菌和热带念珠菌）中高度诱导的基因，但在毒性较弱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目前文献中的空白（尚未报道小鼠原代细胞的转录分析），更重要的是，将确定最毒的物种如何削弱典型的抗真菌反应以促进生存；已经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，临床突出的白色念珠菌已经导致绝大多数的分子工作是在这个单一的物种进行。即使不考虑几个实验室生成的突变文库，在白色念珠菌中被敲除的基因数量也远远超过所有其他CTG物种的总和。目前对非白色念珠菌进行的转录本分析研究非常少，本项目所产生的数据将为了解这些重要物种和对这些基因组的注释做出贡献提供极有价值的资源。

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.