Virulence factor identification by comparative transcriptomics in Candida species

描述(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属引起的。这带来了巨大的临床挑战，因为播散性念珠菌病很难诊断，而且往往对抗真菌治疗难以奏效，导致死亡率几十年来一直居高不下，约为40%。与念珠菌属重叠的是一组被称为CTG分支的物种，因为它们以非规范的方式翻译CUG密码子。CTG分支包括所有具有临床意义的念珠菌，除了光滑念珠菌和克鲁斯念珠菌，光滑念珠菌是一种常见的分离病原体，尽管与酿酒酵母的亲缘关系比CTG种更近，但克鲁斯假丝酵母菌是一种罕见的病原体。在CTG分支中，分离物种的频率有很大的差异：不同研究的确切数字不同，但白色念珠菌仍然导致一半以上的播散性念珠菌感染，而热带念珠菌和近缘念珠菌(分别约10%)通常被分离。卢氏隐孢子虫和吉氏隐孢子虫是少见的病原体，法玛塔隐孢子虫(又名汉森变形杆菌)也很少见。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固有的毒力，尽管近缘念珠菌的毒力可能比其发病率所暗示的要小。白色念珠菌甚至在其他常见的念珠菌病表现中占主导地位，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：通过转录组学剖析这些物种中的每个物种对与宿主细胞相互作用的反应。对于白色念珠菌来说，这是一种非常成功的方法，确定了抗应激和代谢适应的途径。它还强调了大量未定性的基因，其中许多是CTG分支所特有的，有些只是白念珠菌及其近亲所特有的。在这里，我们建议使用七个CTG分支物种的比较转录图谱，在与巨噬细胞共培养期间，这些物种的毒力范围从高(白念珠菌)到极低(汉森葡萄球菌)，巨噬细胞是哺乳动物抗真菌免疫的关键组成部分。使用高通量RNA测序(RNA-seq)，我们将定量测定真菌和哺乳动物在这些相互作用中的转录丰度。生物信息学分析将确定在毒力最强的物种(白念珠菌和热带念珠菌)中高度诱导的基因，但在毒力较弱的物种中要么不存在，要么不受调控。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，小鼠巨噬细胞的图谱将填补目前文献中的一个空白(尚未报道对小鼠初级细胞的转录分析)，更重要的是，将确定最具毒性的物种如何钝化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，但对它们如何发挥作用知之甚少。最后，白念珠菌在临床上的突出地位导致了绝大多数分子工作都是在这个单一物种中进行的。即使在考虑几个实验室产生的突变文库之前，白念珠菌中被敲除的基因比所有其他CTG物种的总和还要多得多。在非白念珠菌物种中进行的转录谱研究很少，该项目产生的数据将是了解这些重要物种和有助于对这些基因组进行注释的极其宝贵的资源。

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.