Virulence factor identification by comparative transcriptomics in Candida species

通过比较转录组学鉴定念珠菌属毒力因子

• 批准号: 8646883
• 负责人: Michael C Lorenz
• 金额: $ 19万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-12 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8646883
• 关键词: Accounting; Animal Model; Antifungal Agents; Antifungal Therapy; Bioinformatics; Bone Marrow; Candida; Candida albicans; Candida glabrata; Cells; Clinical; Coculture Techniques; Codon Nucleotides; Complex; Control Animal; Cues; Data; Dendritic Cells; Diagnosis; Disease; Disseminated candidiasis; Endothelial Cells; Epidemiology; Epithelial Cells; Event; Explosion; Frequencies; Gene Expression Profiling; Genes; Genetic; Genetic Variation; Genome; Genomics; Glyoxylates; Goals; Health; High-Throughput RNA Sequencing; Immune response; Immunity; Incidence; Infection; Interferons; Knock-out; Libraries; Life; Literature; Mammalian Cell; Maps; Mediating; Metabolic; Microbe; Molecular; Mus; Mycoses; Outcome; Pathogenesis; Pathway interactions; Pattern; Phagocytes; Phagocytosis; RNA; RNA Interference; RNA Sequences; Reading; Refractory; Relative (related person); Reporting; Resistance; Resources; Rest; Role; Saccharomyces cerevisiae; Side; Stress; Technology; Testing; Text; Transcript; Translating; Vaginitis; Variant; Virulence; Virulence Factors; Virulent; Work; base; cell type; clinically significant; comparative; fungus; genome annotation; genome sequencing; glyoxylate; immunoregulation; killings; knock-down; macrophage; mortality; mutant; neutrophil; novel; oropharyngeal thrush; pathogen; public health relevance; response; tool; trait; transcriptomics

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.

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%。这带来了重大的临床挑战，因为播散性念珠菌病难以诊断，并且抗真菌治疗通常难以治愈，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，因为它们非规范地翻译 CUG 密码子。 CTG 进化枝涵盖所有临床上重要的念珠菌属物种，但光滑念珠菌（一种常见的分离病原体，尽管它与酿酒酵母的关系比与 CTG 物种的关系更为密切）和克柔念珠菌（一种罕见的病原体）除外。在 CTG 分支内，分离物种的频率存在很大差异：研究之间的精确数字有所不同，但白色念珠菌仍然造成一半以上的传播性念珠菌病感染，而热带念珠菌和近平滑念珠菌（各约 10%）通常被分离。 C. lusitaniae 和 C. guillermondii 是罕见的病原体，而 C. famata（又名 Debaromyces hansenii）则很少见到。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固有的毒力，尽管近平滑念珠菌的毒力可能低于其发病率所暗示的毒力。白色念珠菌在念珠菌病的其他常见表现中更为占主导地位，例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提供了一种以新方式了解毒力的工具：通过转录组学剖析每个物种对与宿主细胞相互作用的反应。这是针对白色念珠菌的一种非常成功的方法，确定了应激抵抗和代谢适应的途径。它还突出显示了大量未表征的基因，其中许多是 CTG 进化枝特有的，有些仅是白色念珠菌及其近亲特有的。我们在此建议在与巨噬细胞（哺乳动物抗真菌免疫的关键组成部分）共培养期间，对七个 CTG 分支物种进行比较转录分析，这些物种的毒力范围从高（白色念珠菌）到极低（汉逊杆菌）。使用高通量 RNA 测序 (RNA-seq)，我们将定量确定这些相互作用过程中真菌和哺乳动物成分的转录本丰度。生物信息学分析将识别在毒性最强的物种（白色念珠菌和热带念珠菌）中高度诱导的基因，但在毒性较低的物种中不存在或不受调节的基因。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现有的空白（尚未报道小鼠原代细胞的转录分析），更重要的是，将确定毒性最强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，但对其如何发挥作用却知之甚少。 最后，白色念珠菌在临床上的突出地位导致绝大多数分子工作都在这个单一物种中进行。即使在考虑几个实验室生成的突变体文库之前，白色念珠菌中被敲除的基因数量比所有其他 CTG 物种的基因总和还要多得多。在非白色念珠菌物种中进行的转录谱分析研究非常少，该项目生成的数据将成为了解这些重要物种并有助于这些基因组注释的极其宝贵的资源。