分泌型磷脂酶在侵袭性阿萨希毛孢子菌感染中的毒力作用研究

Invasive trichosporosis which mainly caused by T.asahii often results in the involvement of skin and multiple systems. Among the immunocompromised patients, the incidence of disseminated trichosporosis is increasing year by year because of the mucosal colonization of asahii. We have preliminarily confirmed that asahii can secrete a variety of extracellular enzymes, including phospholipase (PL), which is closely related to the fungus invasiveness. Based on the previous findings, we designed this study to define the pathogenesis mechanism by which T.asahii adheres to and invades epithelium then causes disseminated infection with the help of secretory phospholipase. In the study, naturally occurring phospholipase high producing strains and phospholipase low/non producing strains may be screened out firstly; then the ability of adhesion to human bronchial alveolar epithelial cells and the ability of membrane degradation and destruction of different strains were evaluated by confocal scanning electron microscope and immunoassay ELASA method. Meanwhile phospholipase gene (PLB1) sequence fragments will be obtained by biological information analysis, and the gene deletion strains and recovery strains will be constructed, after that the pathogenicity of the different strains will be comparative analyzed. Finally the role of PL in the invasion of T.asahii will be further confirmed by the mouse model of invasive trichosporosis. The datas and the results of the study may provide a new scientific basis for the study of the invasive mechanism of asahii and the targets exploration of the new antifungal agents.

侵袭性毛孢子菌病主要是由阿萨希毛孢子菌（T.asahii）引起的多系统脏器感染。特别是在免疫缺陷患者中，通过黏膜定植导致播散性感染的发病率逐年上升。我们已初步证实该菌分泌多种胞外分泌酶，其中磷脂酶（PL）与其侵袭性密切相关。为进一步明确T.asahii如何利用外分泌型磷脂酶黏附和侵袭粘膜上皮并引起播散性感染的致病机制，本项目筛选出T.asahii天然高产磷脂酶菌株和低/非产磷脂酶菌株，利用共聚焦扫描电镜和免疫测定ELASA法在生物形态和细胞免疫方面观察不同菌株对人支气管肺泡上皮细胞的黏附、胞膜降解和破坏能力的差异；同时利用生物信息分析获得该菌磷脂酶基因（PLB1）序列片段，构建PLB1基因缺失株及回复株，对比分析敲除前后菌株的致病差异。并通过侵袭性感染小鼠模型进一步证实PL在T.asahii侵袭过程中的毒力作用，为研究该菌的侵袭性致病机制及新型抗真菌药物靶点提供新的科学依据。

阿萨希毛孢子菌（T.asahii）作为一种重要的条件致病性真菌，在免疫缺陷患者中，可通过气道黏膜入侵人体，导致播散性感染。该菌外分泌的磷脂酶（PL）与气道侵袭密切相关。本研究筛选出T.asahii野生高产PL菌株和野生低产PL菌株，同时利用生物信息分析获得该菌磷脂酶B1（PLB1）基因序列片段，构建PLB1基因敲降株和过表达株。通过体外和体内实验，分别从生物形态、细胞免疫及致病性等方面，观察不同菌株对人支气管肺泡上皮细胞的黏附性、细胞膜降解能力和引起机体固有免疫应答的差异，取得了以下重要成果：.（1）明确了T.asahii的两个PLB1同源基因：A1Q1_01107和A1Q1_02782..（2）利用构巢曲霉组成表达启动子，成功构建了PLB1基因过表达菌株。.（3）基于T7启动子驱动的RNAi载体构建与转化子，成功构建了PLB1基因敲降菌株。.（4）体外实验证实：野生高产酶菌株和PLB1过表达株在培养基上主要呈现孢子相生长，而野生低产酶菌株和PLB1敲降株则更易生成菌丝相。与人支气管上皮细胞共孵育后，野生高产酶株和PLB1过表达株比野生低产酶株和PLB1敲降株更容易黏附于细胞表面，并破坏细胞膜、出芽生长。共孵育的上清液中，野生高产酶组和PLB1过表达组的白三烯（cys-LT）、白三烯B4（LTB4）和前列腺素E2（PGE2）含量均较野生低产酶组和PLB1敲降组明显升高。与野生低产酶组和PLB1敲降组相比，野生高产酶组和PLB1过表达组使共孵育的上皮细胞凋亡百分比显著升高。.（5）体内实验证实：野生高产酶组和PLB1过表达组真菌感染小鼠模型的血清和支气管肺泡灌洗液中，炎症因子（髓样细胞表达触发受体-l表达、白介素6和10）和细胞表面磷脂降解产物（LTB4、PGE2）生成量均高于野生低产酶组和PLB1敲降组。支气管和肺组织标本组织病理显示：野生高产酶组和PLB1过表达组在支气管和肺组织内有更多菌丝生长，并可诱发更剧烈的巨噬细胞浸润和肺泡及间质炎性改变。.本研究结果证实T.asahii外泌性PLB1参与了对支气管肺泡上皮细胞的黏附和细胞表面磷脂成分的降解，有利于真菌的侵袭与播散，是引起微生物入侵后机体固有免疫应答的重要因素之一。抑制PLB1的分泌可能成为预防和治疗呼吸道T.asahii感染的药物新靶点。