不规则毛霉致原发性皮肤接合菌病的易感基因和毒力基因的筛选和验证

The primary chronic skin infection caused by Mucor irregular in China in recent more than 20 years in the report, today reported so far have been about 20 cases, and Asia reported only 3 cases over the same period abroad. According to our analysis of published data, the clinical features of the infections caused by M. irregular are as follows : The vast majority of patients for immunocompetent; the deep infection of the non skin tissue caused by the fungi is very few；No obvious blood vessels invasion and tissue necrosis as in the infections caused by other Mucor species；The infection is not easily lead to death, but easy to disfigurement and disability. These clinical characteristics largely because of the host defense also be caused by the virulence factors from the fungal pathogen or caused by the host and the pathogen interaction between the two sides? On the basis of the preliminary works , we will study on two aspects, namely the susceptibility genes of the patients and the virulence genes of the M. irregular.1.the host susceptibility genes : collect the specimens of the cases,, and then using exon capture sequence and screening of the candidate genetic susceptibility, next verified to determine whether the infection susceptibility genes in healthy people and other members of the families. And by the analysis of the genes, proteins and immune cell function , clearly the role of genes in the infections development process. 2. using whole genome sequencing , the comparative genomics and bioinformatics technologies, find the virulence genes of M, irregular, such as ion metabolism and temperature control related genes and carry out functional verification. The purpose of this project is to provide a basis for clinical scientific understanding and effectively prevention and control the fungal infection.

不规则毛霉所致原发性慢性皮肤感染近20余年来在我国屡有报告，迄今累计报告已达20余例，而国外迄今仅亚洲报告3例。据我们对已发表病例资料分析，该菌所致的感染有如下临床特征：绝大多数患者为免疫健全者；极少引起非皮肤组织的深部感染；无明显其他毛霉具有的嗜血管性；虽不易致死但易致毁容或畸残。引起这些特征主要源于宿主防御因素还是菌种毒力因素抑或二者相互作用所致？拟在前期工作基础上，开展宿主和真菌两方面的致病基因研究：1、宿主易感基因：收集病例标本，采用外显子捕获测序、筛选候选易感基因、并通过家系其他成员和健康人群验证确定该感染的易感基因，并进行树突状细胞、单核细胞及动物模型不同水平的对比分析，明确该基因在疾病发生发展过程中的作用；2、真菌毒力基因：采用全基因组测序及比较基因组学和生物信息学技术，找出不规则毛霉的与致病可能相关的基因并加以功能验证。研究目的是为临床科学认识和有效防治该感染提供科学依据。

毛霉菌病作为一种侵入性真菌病，已引起全球公共卫生的广泛关注。在我国，由不规则毛霉（Mucor irregularis）所引起的皮肤毛霉病在逐渐增多。在致病过程中不规则毛霉必须面对并适应宿主皮肤内的低氧环境（～6 % O2），然而，其对低氧胁迫环境的致病性和适应机制尚不清楚。在本研究中，我们通过RNA-seq技术对不规则毛霉在正常氧含量（21 % O2）及低氧（6 % O2）这两种不同条件下的转录组进行测序，解析低氧作用下该菌的基因表达变化。结果表明发现低氧胁迫下不规则毛霉的多种代谢途径基因的表达变化都与区别于其他真菌。其主要通过干扰该菌的能量生成、低氧响应来抑制其在皮下低氧环境中的感染进程。为适应低氧环境，该菌通过增强胞内脂质代谢，并利用内吞作用吸收宿主表皮上分泌的甘油三酯等脂质成分，来为其低氧生长提供能量。该部分结果为揭示不规则毛霉所致特异性皮肤组织侵袭性感染的机制提供新思路，为进一步寻找病理条件下可能的治疗靶点提供一定的研究基础。.在真菌的有性生殖过程中，交配型（mating type, MAT）基因对真菌的性别控制和遗传进化起着决定性作用。为了了解交配型与不规则毛霉毒力之间的关系，我们首先通过tBlastN同源比对的方法获取了不规则毛霉的MAT基因位点序列，并鉴定了我国17株不规则毛霉临床株的交配类型，发现我国的不规则毛霉在交配型上存在明显的偏分离现象。此外，通过一株突变菌株，证实MAT基因位点对不规则毛霉的性别控制及交配过程起着决定性作用。随后利用小鼠动物模型来探究不规则毛霉交配型与毒力的关系，发现SexP(+)注射组小鼠的炎症及免疫反应的严重程度均显著高于SexM(-)注射组。因此我们认为，不规则毛霉菌株的交配型可能与毒力及临床病症的严重程度具有一定的相关性。该部分结果证明不规则毛霉的MAT基因参与性发育的过程，并且菌株的交配类型对皮肤感染的严重程度具有一定的影响。