白念珠菌细胞壁α-1,6-甘露糖骨架在侵染宿主过程中的作用机制研究

Invasive fungal infections are associated with significant morbidity and mortality. Candida albicans is the major opportunistic fungal pathogen of humans. N-mannan, which is located in the outermost layer of the cell wall, plays a crucial role in cell wall structure and virulence of the opportunistic pathogen. However, the molecular mechanism by which this occurs remains unclear. In our previous study, we first identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans, and determined that α-1,6-mannose backbone which is catalyzed by Mnn10 protein was crucial for the invasive infection development. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Our results support the notion of an enhanced host antifungal defense related to this backbone change. In the present project, we intend to further investigate the cellular and molecular mechanisms of cell wall α-1,6-mannose backbone of C. albicans involved in the invasive infection development, using mnn10 mutant strain as a tool. On the one hand, we will investigate the change and exposure of pathogen-associated molecular patterns (PAMPs) on the cell wall of mnn10 mutant strain, compared to parental and revertant strain. On the other hand, we will explore the cellular and molecular mechanisms of the enhanced host defense against C. albicans mnn10Δ/Δ, of which α-1,6-mannose backbone is abolished. Finally, we will explore the structure-activity relationship of Mnn10p involving in the invasive infection development using site-directed mutagenesis technique. The present project will provide theoretical and experimental basis for new effective strategy and antifungal agent development.

临床上侵袭性真菌感染发病率逐年上升，病死率居高不下，白念珠菌是主要的致病菌之一。N-甘露聚糖是白念珠菌细胞壁最外层的多糖结构，与致病力密切相关，其参与侵染宿主的机制尚不明确。本项目前期首次发现白念珠菌α-1,6-甘露糖骨架是N-甘露聚糖参与白念珠菌侵染宿主的关键结构，由Mnn10蛋白催化合成；我们发现MNN10基因敲除对白念珠菌侵袭宿主能力无影响，宿主免疫应答能力增强是MNN10基因缺失菌不能形成侵袭性感染的原因。本项目拟进一步以MNN10基因缺失菌为工具菌，一方面研究白念珠菌MNN10基因缺失后细胞壁PAMPs分子暴露以及含量的变化；另一方面研究宿主免疫细胞对MNN10基因缺失菌的免疫识别和免疫效应，关联分析α-1,6-甘露糖骨架参与白念珠菌侵染宿主的细胞与分子作用机制，并采用定点突变技术研究Mnn10蛋白的活性位点，为发现防控侵袭性白念珠菌感染的新靶标、新途径提供理论依据。

N-甘露聚糖是白念珠菌细胞壁最外层的多糖结构，与致病力密切相关，其参与侵染宿主的机制尚不明确。本项目前期首次发现白念珠菌α-1,6-甘露糖骨架是N-甘露聚糖参与白念珠菌侵染宿主的关键结构，由Mnn10蛋白催化合成。本项目进一步以MNN10基因缺失菌为工具菌，一方面研究白念珠菌MNN10基因缺失后细胞壁PAMPs分子暴露以及含量的变化；另一方面研究宿主免疫细胞对MNN10基因缺失菌的免疫识别和免疫效应，关联分析α-1,6-甘露糖骨架参与白念珠菌侵染宿主的细胞与分子作用机制。本项目研究发现白念珠菌Mnn10p可以催化细胞壁α-1,6-甘露糖骨架结构的延伸、MNN10基因的缺失会导致细胞壁甘露聚糖成分的减少、β-(1,3)-葡聚糖暴露显著的增加，对抗真菌药物的敏感性增加；抑制α-1,6-甘露糖骨架结构的延伸后细胞表面部分毒力因子蛋白的含量没有明显改变，因此并不影响菌丝形成、对宿主细胞的粘附、侵袭和损伤等能力；抑制白念珠菌α-1,6-甘露糖骨架结构合成后，宿主固有免疫细胞（中性粒细胞、巨噬细胞）对白念珠菌的免疫应答能力（NF-κB和MAPK信号通路的激活，验证细胞因子的释放等）显著增强，主要依赖于宿主Dectin-1受体的识别，与Toll样和Dectin-2等受体无关；抑制白念珠菌α-1,6-甘露糖骨架的合成会增强宿主Th1/Th17介导的适应性免疫应答，并主要依赖于α/β T细胞产生的IFN-γ和IL-17介导清除小鼠体内病原菌。本项目研究为发现防控侵袭性白念珠菌感染的新靶标、新途径提供了实验基础和理论依据。