Hog1-MAPK信号通路调控马尔尼菲青霉潜伏感染的机制研究

The dimorphic fungus Penicillium marneffei can cause a life-threatening disseminated mycosis in AIDS patients in southeast Aisa and southern part of China. As an intracellular pathogen, P.marneffei could survive and persist inside macrophage, the common antifungal administration couldn't kill the yeast cell of P.marneffei throughly. Comprehensive understanding the mechanism that lead to P.marneffei escape or survival attack from phagocyte may shed light on the way to control the pathogen infection..In previous study, we have observed P. marneffei had the ability to sense and response to the envirnment stresses. In further study, we had identified HOG1 gene, the core gene in Hog1-MAPK pathway, had important role not only in regulation the stress response but also in regulation the synthesis of α-( 1,3 )-glucan, which is a definite fungal virulence factor. Base on the fruitful results of our previous studies, we speculate that the Hog1-MAPK pathway may have a key role in P.marneffei survive in host and contribute to its pathogenicity. In this application, we are going to investigate the genes function that involved in Hog1-MAPK pathway by construction mutant strain using deletion and overexpression strategies, compare the gene expression level under different stress conditions by real time PCR, detect the phosphorylation or non-phosphorylation of Pbsp and hog1p in the mutant strains based on western blot, in order to elucidate the regulation mechanism of Hog1-MAPK pathway in P.marneffei. Further, we will study the interaction between P.marneffei and the innate immune system by co-culture the different mutant strans with macrophage and CD4+ T cell, and by the infection mouse model. .The results should highlight the Hog1-MAPK signal pathway in P.marneffei latent infection and may contribute to even greater therapeutic strategies benefits to P.marneffei infection treatment.

双相真菌马尔尼菲青霉是我国南方艾滋病患者特征性的机会性感染因素，常规治疗难以彻底杀灭患者体内潜伏感染的马尔尼菲青霉。探讨其潜伏感染的机制，有助于找到有效遏制该菌播散的方法。本课题组前期研究发现马尔尼菲青霉对环境应激有很强的反应能力，而Hog-MAPK信号通路的核心基因HOG1不仅参与应激反应的调控，同时还调控毒力因子α- ( 1,3 )-葡聚糖合成。基于已获得的重要发现，我们首先提出Hog1-MAPK 信号通路在马尔尼菲青霉潜伏感染中发挥重要作用。拟通过基因敲除、过表达、实时 PCR 和蛋白磷酸化分析等手段，研究该通路多个重要基因的功能，探讨该通路信号调控的确切机制；进而建立基因缺陷株与巨噬细胞、CD4+T细胞相互作用体系，以及感染动物模型，探讨该通路对免疫细胞活性和功能的影响，以及在马尔尼菲青霉致病中的作用，为阐明马尔尼菲青霉体内潜伏感染的机制和发现抗真菌治疗新靶位奠定基础。

首先本课题组对Hog1-MAPK信号通路基因 HOG1和PBS2 的基因功能做了研究，比较∆hog1、∆pbs2敲除株与FRR在不同培养基中生长特性。结果显示∆hog1、∆pbs2在培养基中生长缓慢，产孢量锐减，菌落呈薄膜状，菌丝弯曲不规则，揭示基因 HOG1和PBS2 对马尔尼菲青霉菌的生长速度、生长形态、产孢能力起重要的调控作用。同时本课题组发现∆hog1、∆pbs2在含不同浓度盐离子、山梨醇、过氧化氢的培养基中生长受抑制，这提示HOG1和PBS2 对马尔尼菲青霉热应激、盐应答、渗透压、氧化应激起着关键的作用。其次本课题组与澳大利亚墨尔本大学的Alex Andrianopoulos 教授合作，对 SskA 与 Hog1-MAPK 信号通路的信号传导机制进行了探讨，SskA基因为激活Hog1-MAPK信号通路所必需，并在Hog1基因上游起着重要调控作用。再次本课题组将马尔尼菲青霉菌（PM）敲除株(∆hog1)、野生株FRR与巨噬细胞共培养，测定共培养后巨噬细胞内ROS平均荧光强度。与FRR相比，∆hog1更易被巨噬细胞杀死，并且巨噬细胞吞噬∆hog1后释放的ROS明显多于吞噬FRR，∆hog1对巨噬细胞杀灭病原微生物产生的活性氧簇具有高度敏感性，提示HOG1基因参与了PM抵抗巨噬细胞吞噬及氧化应激杀伤作用，是PM在病人细胞中潜伏感染的重要机制。通过流式细胞仪检测发现，巨噬细胞表面模式识别受体TLR-2、TLR-4、Dectin-1表达在∆hog1中表达高于FRR，进一步证实HOG1参与巨噬细胞对PM的识别和免疫应答，Hog1-MAPK信号通路在马尔尼菲青霉巨噬细胞潜伏感染中起重要作用。最后本课题组建立了侵袭性马尔尼菲青霉病小鼠模型，分别用∆hog1、野生株感染免疫受抑制的小鼠，比较小鼠存活量和存活时间以及致病率。结果显示，野生株感染后的小鼠在14天内的死亡率为100%，而∆Hog1 14天内死亡率仅33.3%。致病率试验中，∆hog1组绝大部分未培养出马尔尼菲青霉菌，与野生株组相比有明显差异。∆hog1敲除株毒力明显减弱，提示该基因在马尔尼菲青霉侵袭感染中的重要作用