难治性慢性播散性毛孢子菌病宿主致病基因定位与功能验证

Disseminated Trichosporonosis is a kind of invasive infection with multi-organ involvement caused by Trichosporon spp.. It occurs in a variety of immunocompromised hosts, and shows high mortality without efficient diagnosis and treatment. We found one case of disseminated trichosporonosis in an immunocompetent patient whose parents were consanguineous, presented with onset in young age, multi-organ involvement, prolonged recurrent process, refractory to many antifungal compounds. In our series of clinical and experimental studies conducted in the past more than 10 years around the case and clinical isolate, there was not specificity being found not only pathogen virulence, but also resistance mechanisms, which reminded that host immunological defence instead of clinical isolate was possible responsible for this special clinical feature. Recently, a variety of molecular genetic studies focus on persistent refractory recurrent fungal infections showed innate immune activation associated with Th17 cells upstream pattern recognition receptors Dectin-1 and activation pathways CARD9 and other related genetic defects are the main cause. But most of researches focused on ascomycetes instead of basidiomycetes so far. This project intends to find the host pathogenic gene of refractory chronic disseminated trichosporonosis by exome capture sequencing and bioinformatics analysis, compared with pedigree members, unrelated patients and healthy people. At the same time, to verify the functions of the target gene at different levels, including genomic, transcription, protein, immune cell function, disseminated trichosporonosis model of genomic deficient mice model. It would be helpful to make clear the role of defective gene in host immunity of Trichosporon infection, thoroughly understand the host immune mechanisms of trichosporonosis.

播散性毛孢子菌病是毛孢子菌引起的多脏器受累的侵袭性感染，多见于免疫缺陷宿主，死亡率较高。我们发现1例幼年起病父母近亲的毛孢子菌慢性播散性感染，迁延反复多年，对各种治疗抵抗。已完成的临床与实验研究显示其致病菌在毒力、耐药机制等方面均无特殊，临床特殊性可能源自宿主本身。近年围绕多种难治迁延性真菌感染的分子遗传研究显示天然免疫模式识别受体Dectin-1和激活通路CARD9等基因缺陷是其主要病因，但仅集中于子囊真菌。作为担子菌的毛孢子菌，其细胞壁组分及天然免疫激活机制与子囊菌存在明显差异，提示其难治性感染可能亦存在独特的基因缺陷。本课题拟通过外显子捕获测序和生物信息分析，家系成员、非关联患者和健康人比对，寻找宿主致病基因。并在转录、蛋白、免疫细胞功能、DCs体外实验研究等不同层面进行体内外缺陷基因功能验证，明确其在毛孢子菌感染免疫中的作用，深化毛孢子菌病免疫学机制研究，为寻找新的治疗靶点提供思路

在本课题的系列研究中，我们发现并明确了一例罕见的难治性慢性毛孢子菌病患者存在的天然免疫缺陷R70W错义点突变位点，位于真菌天然免疫识别过程中的关键基因CARD9基因。在此基础上，我们围绕CARD9基因进行了系列功能验证，结果显示在阿萨希毛孢子菌感染过程中，CARD9基因缺陷可影响DC细胞的共刺激因子激活、天然免疫相关细胞因子的分泌减少、导致DC细胞对阿萨希毛孢子菌的吞噬率下降、阿萨希毛孢子菌感染小鼠的致死率增高，提示CARD9基因在阿萨希毛孢子菌感染中发挥重要的防御作用。