RNA Enrichment Method for Quantitative Transcriptional Analysis of Pathogens In Vivo Applied to the Fungus Candida albicans
RNA Enrichment Method for Quantitative Transcriptional Analysis of Pathogens In Vivo Applied to the Fungus Candida albicans
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DOI：
10.1128/mbio.00942-15
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发表时间：
2015-09-01
期刊：
影响因子：
6.4
通讯作者：
Sanglard, Dominique
中科院分区：
文献类型：
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作者：
Amorim-Vaz, Sara;Tran, Van Du T.;Sanglard, Dominique
微生物病原体的体内转录分析通常受到宿主器官中病原体生物量比例低的阻碍，从而阻碍了完整病原体转录组的覆盖。我们的目的是解决白念珠菌，在全身感染的免疫功能低下的患者中最常见的真菌病原体，在不同宿主的全身感染的转录组谱。我们开发了一种高分辨率定量分析C。在两种不同的宿主模型中，小鼠和昆虫大蜡螟中，白念珠菌转录组直接来自全身感染的早期和晚期。我们的研究结果表明，使用生物素化诱饵探针捕获C.白色念珠菌序列。这种富集使仅相似于3%的基因的读段计数有偏差，其可以基于先验标准被鉴定和去除。这使得C的分辨率前所未有。白念珠菌体内转录组，其基因的检测率超过86%。真菌的转录反应是令人惊讶的相似，在感染的两个主机和在两个时间点，虽然可以确定一些主机和时间点特异性基因。在感染过程中高度诱导的基因参与例如应激反应、粘附、铁获取和生物膜形成。在体内调控基因中，约有10%的基因功能尚不清楚，对它们的研究具有重要意义。真菌RNA富集方法有助于更好地鉴定C.重要性了解病原体感染宿主并在宿主中引起疾病的机制对于合理的药物开发至关重要。转录组学研究可能有助于通过确定哪些基因在感染期间特异性表达来调查这些机制。到目前为止，这项任务一直很困难，因为感染组织中微生物生物量的比例通常非常低，从而限制了测序和全面转录组分析的深度。在这里，我们采用了一种技术来捕获和富集C。白色念珠菌RNA，其接下来用于直接来自两种不同宿主生物体的感染组织的深度RNA测序。高分辨率转录组揭示了大量迄今未知的参与感染的基因，这将可能构成未来研究的重点。更重要的是，该方法可以适用于在宿主感染或定殖期间进行任何其他微生物的转录物谱分析。
In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only similar to 3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host-and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens.IMPORTANCE Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.