磷脂酶B对马尔尼菲青霉菌生长和致病性的作用及机制研究

Penicillium marneffi（P. marneffei, PM）is an extremely important opportunistic pathogenic fungus infecting AIDS patients in Southern China, which exhibits temperature-dependent dimorphic growth and grows in two distinct cellular morphologies, hyphae at 25 C and yeast cells at 37 C. Yeast phase is a highly pathogenic one that is capable of causing disseminated infection in human host. However, the pathogenic mechanism is still unclear. Moreover, the early diagnosis for PM infection presently remains difficult and specific antifungal drugs are still unavailable. Our team had found the PLB gene expresses significantly much more in yeast phase than in mycelial phase, meanwhile identified another full-length gene of PLB from the cDNA library of the clinically dominat strain of P. marneffei in yeast phase, which had been successfully cloned and expressed to obtain the recombination protein. Many pathogenic fungi has been proved to secrete Phospholipase B (PLB) to assist them for invasion when they infect the host. It is suggested that PLB is a potential key pathogenic factor of P. marneffei in yeast phase, which provides with a breakthrough point for elucidating the pathogenic mechanism of P. marneffei, and probably supplies a new target for developing early diagnostic method and specific treatment strategy. This project aims to investigate the mechanism and function of PLB on the growth and pathogenicity of P. marneffei from two aspect: Firstly, to generate deletion mutants of 3 putative PLB genes and explore the phenotypic variations of mutant strains in growth rate, sensitivity to several external stressors, pigment secretion, sporulation, toxicity, dimorphic phase-switching and reproductive ability, compared with P. marneffei wild-type strain. Secondly, to study effects of recombination proteins of PLB on the alveolar epithelial cells and macrophages of mice vitro and their possible molecular mechanisms, observing if they could combine with these cells by utilizing immunofluorescence; surveying their enhancing effect on the proliferation of these cells by MTT assay; detecting their influences on cell life cycle by flow cytometry; identifying the components of the membrane proteins which are conjugated with PLB by combining co-immunoprecipitation, two-dimensional electrophoresis with mass spectrogram; and studying the cell signal transduction of transmembrane proteins under the effect of PLB by real-time quantitative polymerase-chain reaction.

马尔尼菲青霉菌（PM）是艾滋病患者重要机会性致病真菌，具有温度双相性，酵母相致病力强。其致病机制仍不清楚，早期诊断困难，缺乏特效药物。本课题组前期转录组研究发现酵母相磷脂酶B（PLB）基因的表达明显高于菌丝相，并成功克隆表达了酵母相文库的PLB基因。既往研究证实许多致病真菌感染宿主时分泌PLB以促进真菌侵入，这提示PLB可能是PM酵母相重要毒力因子，是阐明其致病机制突破点之一，也可能是研发诊断治疗方案的新靶点。本项目拟：观察PLB基因缺失对PM生长速率、应激抵抗力、色素分泌、毒力及双相转变和繁殖能力表型的影响；探讨PLB重组蛋白对小鼠肺泡上皮细胞和巨噬细胞的作用：免疫荧光法观察PLB是否与细胞结合，流式细胞术检测PLB对细胞周期的影响，MTT法观察PLB对细胞的促增殖作用，免疫共沉淀、双向电泳及质谱技术鉴定与PLB结合的膜蛋白成分，实时荧光定量PCR观察PLB作用下细胞的跨膜蛋白。

磷脂酶基因可能参与马尔尼菲青霉菌（目前已更名为马尔尼菲蓝状菌）的致病过程并发挥重要作用。本项目通过生物信息学分析及基因的差异性表达锚定TmPlb1基因最可能与马尔尼菲蓝状菌的致病相关。TmPlb1基因与与黄曲霉及烟曲霉的磷脂酶序列一致性分别为67%、66%，在37℃（致病相）时表达水平上调，其所编码的TmPLB1蛋白含有特征性的丝氨酸-天冬氨酸-组氨酸三体结构和关键结构域G-X-S-X-G。本研究首次通过酵母表达系统得到具有生物学活性的TmPLB1重组蛋白，浓度可达到240.4 mg/L，磷脂酶A1和磷脂酶A2的活性分别为5.96U/mg 和 1.59 U/mg。MTT实验初步提示TmPLB1蛋白可能直接抑制巨噬细胞和肺上皮细胞的存活，但仍需要进一步证实。后续的基因抑制和表型分析实验将为揭示TmPlb1基因在马尔尼菲蓝状菌致病中的作用提供更多证据支持。