Mechanism of cryptococcal fitness, innate defense subversion, and the adaptive immune skewing in lungs

新生隐球菌(C.neo)是世界范围内最重要的真菌病原体之一。 然而，关于它如何与宿主相互作用以逃避和颠覆免疫反应，仍有许多未知之处。 在这里，我们建议研究截获海藻糖生物合成途径的生物学效应，该途径 我们的数据表明，影响隐球菌与多条宿主防御线的相互作用，可能是一种 真菌的“阿喀琉斯之踵”。海藻糖-6-磷酸合成酶(TPS1)催化的第一步是 产生海藻糖的生物合成途径，海藻糖是一种典型的保护C.neo(和其他 微生物)抵抗压力，而压力不是哺乳动物细胞合成或需要的。已经描述了TPS1 在多种动物感染模型中作为新城疫杆菌的毒力因子，但其机制基础是 未定义。我们建议从机械上揭示TPS1缺失如何改变真菌与宿主的相互作用，从而驱动 这增加了对真菌的控制。 我们的初步数据显示，在小鼠肺部感染模型中，缺乏海藻糖的C.neo (TPS1Δ)由于免疫保护的改善，清除非常迅速。因此，我们假设隐球菌 TPS1需要通过(1)干扰来保护真菌免受所有3条肺宿主防御线路的侵袭 当地居民防御；(2)招募/激活先天免疫细胞以进行早期真菌控制， 以及(3)自适应记忆响应的调制。我们建议需要海藻糖来保护C.neo 对抗肺泡腔中的先天因素，如表面活性物质。我们的初步数据支持 海藻糖缺乏的C.neo对照中的表面活性蛋白A和D(目标1)。我们还建议，损失 海藻糖促进常驻先天免疫细胞，特别是中性粒细胞快速清除真菌(AIM2)。 此外，我们的初步数据表明，隐球菌失去Tps1功能会导致 支持保护性偏斜和适应性免疫记忆发展的免疫刺激 回应(目标3)。这提高了免疫保护水平，支持靶向TPS途径的免疫保护 可直接辅助清除真菌，可诱导长期保护性免疫记忆作用。最终， 了解海藻糖如何改变隐球菌与宿主防御的相互作用可以支持 未来一类广泛的抗真菌抑制剂的开发，可帮助治疗多种类型的 真菌感染。总之，这项工作将为C.neo如何避免肺脏提供新的背景 宿主防御以及如何利用这些相互作用。 上述建议的研究构成了退伍军人事务部CDA-2计划的科学基础，该计划将为 Kristie Goughenour博士成为一名独立的退伍军人事务部调查员的培训和职业发展。Dr。 Goughenour完成了她在多学科T32研究培训中的博士后培训的第三年 肺病方面的计划。她的主要研究重点是病原真菌的寄主-病原体相互作用。 肺部感染。她所受的训练为她提供了技术能力和理论框架 剖析人类宿主因素和微生物毒力机制，它们相互作用，以确定 感染的后果。这一建议建立在她在真菌致病的分子机制方面的技能之上。 和宿主对真菌病原体的反应结合成在肺部宿主免疫控制方面的独特专业知识 真菌病原体和真菌免疫逃避的新机制。高格努尔博士将增强她的力量 在免疫过程的不同方面拥有专业知识，并学习几项新技术。她的指导团队 精心培养了各自领域的顶尖专家，为他们提供专业知识和职业支持 在她过渡到独立并为拟议的研究提供援助的同时。

Cryptococcus neoformans (C. neo), is one of the most significant fungal pathogens worldwide. However, much is still unknown on how it interacts with the host to evade and subvert the immune response. Here we propose to study the biological effects of intercepting the trehalose sugar biosynthetic pathway, which our data suggest affects interactions of cryptococcus with multiple lines of host defenses and could serve as an “Achilles heel” for the fungus. Trehalose-6-phosphate synthase (TPS1) catalyzes the first step in the biosynthetic pathway to generate trehalose, a disaccharide that canonically protects C. neo (and other microbes) against stress, which is not synthesized or required by mammalian cells. TPS1 has been described as a virulence factor for C. neo in multiple animal infection models, but the mechanistic underpinnings are undefined. We propose to mechanistically uncover how TPS1-deletion alters fungal-host interactions to drive this increased fungal control. Our preliminary data show that in murine models of pulmonary infection, trehalose-deficient C. neo (tps1Δ) is very rapidly cleared due to improved immune protection. As such, we hypothesize that cryptococcal TPS1 is required to protect the fungus against all 3 lines of pulmonary host defenses via (1) interference with the local resident defenses; (2) the recruitment/activation of the innate immune cells for early fungal control, and (3) modulation of the adaptive memory responses. We propose that trehalose is required to protect C. neo against innate factors in the alveolar space, such as surfactants. Our preliminary data support a role for the surfactant proteins A and D in trehalose-deficient C. neo control (Aim 1). We also propose that the loss of trehalose facilitates rapid fungal clearance by resident innate immune cells, specifically neutrophils (Aim2). Further, our preliminary data suggest that the loss of Tps1-function by Cryptococcus results in an immunostimulation that supports the development of protective skewing and an adaptive immune memory response (Aim 3). This improved the level of immunoprotection supports that targeting the TPS pathway, apart from directly aiding the fungal removal, can induce long-term protective immune memory effects. Eventually, understanding how trehalose alters cryptococcal interactions with host defenses could support the development of a future class of broad antifungal inhibitors that could aid the treatment of multiple types of fungal infections. In summary, this work will provide novel context for how C. neo avoids pulmonary host defenses and how these interactions can be exploited. The studies proposed above make up the scientific basis for the VA CDA-2 program that will provide training and career development for Dr. Kristie Goughenour into an independent VA investigator. Dr. Goughenour completes the third year of her postdoctoral training in the Multidisciplinary T32 Research Training Program in Lung Disease. Her primary research focus is on host-pathogen interactions of pathogenic fungi in pulmonary infections. Her training has provided her with both the technical ability and theoretical framework to dissect both the human host factors and the microbial virulence mechanisms that interact to determine the outcomes of infections. This proposal builds upon her skills in molecular mechanisms of fungal pathogenesis and host responses to fungal pathogens to combine into unique expertise in host immune control of pulmonary fungal pathogens and novel mechanisms of fungal immune evasion. Dr. Goughenour will strengthen her expertise in different aspects of immune processes and learn several new technologies. Her mentoring team of 5 top experts in their respective fields has been carefully developed to provide expertise and career support while she transitions towards independence and to provide aid on the proposed studies.