Table_3_Comparative Analysis of Clinical and Environmental Strains of Exophiala spinifera by Long-Reads Sequencing and RNAseq Reveal Adaptive Strategies.XLSX

Exophiala spinifera, a capsule-producing black yeast, is overrepresented as agent of disseminated infection in humans with inherited dysfunction of the CARD9 gene. In a review of published caspase recruitment domain-containing protein 9 (CARD9) deficiency cases, black fungi were linked to mutations other than those prevalent in yeast and dermatophyte cases, and were found to respond to a larger panel of cytokines. Here, we sequenced and annotated the genomes of BMU 08022 from a patient with CARD9 deficiency and two environmental strains, BMU 00051 and BMU 00047. We performed genomic and transcriptomic analysis for these isolates including published black yeasts genomes, using a combination of long-read (PACBIO) and short-read (Illumina) sequencing technologies with a hybrid assembly strategy. We identified the virulence factors, fitness, and the major genetic and gene expression differences between the strains with RNAseq technology. Genome assembly reached sub-chromosome level with between 12,043 and 12,130 predicted genes. The number of indels identified in the clinical strain was higher than observed in environmental strains. We identify a relatively large core genome of 9,887 genes. Moreover, substantial syntenic rearrangements of scaffolds I and III in the CARD9-related isolate were detected. Seventeen gene clusters were involved in the production of secondary metabolites. PKS-cluster 17 was consistently found to be absent in the clinical strain. Comparative transcriptome analysis demonstrated that 16 single-copy genes were significantly differentially expressed upon incubation in brain-heart infusion broth vs. Sabouraud glucose broth. Most of the single-copy genes upregulated with Brain Heart Infusion (BHI) were transporters. There were 48 unique genes differentially expressed exclusively to the clinical strain in two different media, including genes from various metabolic processes and transcriptional regulation. Up-regulated genes in the clinical strain with Gene Ontology (GO) enrichment are mainly involved in transmembrane transport, biosynthetic process and metabolic process. This study has provided novel insights into understanding of strain-differences in intrinsic virulence of the species and indicated that intraspecific variability may be related to habitat choice. This indicates that strains of E. spinifera are differentially prone to cause infection in susceptible patient populations, and provides clues for future studies exploring the mechanisms of pathogenic and adaptive strategies of black yeasts in immunodeficient patients.

棘状外瓶霉（Exophiala spinifera）是一种产荚膜黑色酵母，在携带CARD9基因遗传性功能障碍的人类播散性感染致病菌中占比偏高。在一项针对已发表的含半胱天冬酶募集结构域蛋白9（caspase recruitment domain-containing protein 9, CARD9）缺乏症病例的综述研究中，黑色真菌与酵母菌和皮肤癣菌病例中常见的突变类型存在差异，并被发现对更多种类的细胞因子产生应答。本研究对来自CARD9缺乏症患者的菌株BMU 08022以及两株环境菌株BMU 00051、BMU 00047进行了基因组测序与注释。我们结合长读长（PACBIO）与短读长（Illumina）测序技术，采用混合组装策略，对这些分离株（包含已发表的黑色酵母基因组）开展基因组与转录组分析。通过RNA测序（RNAseq）技术，我们鉴定了各菌株的毒力因子、适应度，以及主要的遗传与基因表达差异。基因组组装达到亚染色体水平，预测基因数量介于12043至12130个之间。临床菌株中鉴定到的插入缺失（indels）数量高于环境菌株。我们鉴定出一个规模相对较大的核心基因组，共包含9887个基因。此外，在与CARD9相关的分离株中，检测到支架I和III存在大量共线性重排。共有17个基因簇参与次级代谢产物的合成。聚酮合酶簇17（PKS-cluster 17）在临床菌株中始终缺失。比较转录组分析显示，在脑心浸液肉汤（brain-heart infusion broth, BHI）与沙氏葡萄糖肉汤（Sabouraud glucose broth）中培养时，有16个单拷贝基因存在显著差异表达。在脑心浸液（BHI）中上调表达的单拷贝基因大多为转运蛋白。在两种培养基中，仅临床菌株存在差异表达的独特基因共有48个，这些基因涉及多种代谢过程与转录调控。临床菌株中经基因本体（Gene Ontology, GO）富集分析得到的上调基因主要参与跨膜运输、生物合成过程与代谢过程。本研究为理解该物种内在毒力的菌株差异提供了全新视角，并表明种内变异可能与栖息地选择相关。这提示棘状外瓶霉的不同菌株在易感患者群体中引发感染的易感性存在差异，同时为未来探索免疫缺陷患者体内黑色酵母的致病与适应策略机制提供了研究线索。