Pathogens as sensors for measuring immune defence efficiency in the on-going infection

了解免疫防御如何影响病原体生物学具有根本重要性：免疫系统的作用模式，即病原体是否被杀死，抑制其生长或空间限制，对感染的结果，免疫病理学和病原体对治疗的敏感性具有广泛的影响。然而，到目前为止还不可能确定正在进行的免疫应答的作用模式。在本项目中，我们将在细胞内寄生虫利什曼原虫中使用体内报告系统来分析病原体如何对免疫系统施加的压力作出反应。这将通过荧光蛋白构建体来实现，所述荧光蛋白构建体允许测量活寄生虫中的两个不同生物学参数：（1）应激相关蛋白酶的活性和（2）寄生虫细胞膜的完整性。使用病原体作为传感器输出免疫效应活性的策略将使我们能够根据其对病原体生理的实际影响来绘制免疫防御。通过使用活体双光子显微镜，我们将能够调查正在进行的感染过程中的保护性免疫反应的作用模式。使用抑制剂和基因敲除小鼠缺乏生产活性氧和氮中间体（ROI和RNI），我们还将确定特定的细胞防御机制的重要性，为不同模式的行动病原体遏制。通过在具有荧光标记的吞噬细胞群体的转基因小鼠中应用ROI/RNI产生抑制剂，我们将分析ROI/RNI用于防御特定细胞类型中的病原体的需求。最后，我们将使用混合骨髓嵌合体方法，将部分ROI产生缺陷小鼠与RNI产生抑制相结合，以评估ROI和RNI在其抗微生物活性中的协同作用程度。拟议的实验将阐明免疫反应诱导的细胞防御机制如何影响病原体生物学，以及这种影响如何与有效控制感染有关。理解这种关系将大大增强我们对免疫系统如何执行保护性反应的了解。

Understanding how the immune defence impacts on pathogen biology is of fundamental importance: The immune system's mode of action, i.e. whether a pathogen is killed, inhibited in its growth, or spatially confined, has broad implications for the outcome of the infection, immunopathology, and the pathogen's susceptibility to treatment. However, it has so far been impossible to determine the mode of action of an on-going immune response. In the presented project, we will use in vivo reporter systems in the intracellular parasite Leishmania major to analyse how the pathogen reacts to the stress imposed by the immune system. This will be achieved via fluorescent protein constructs that permit the measurement of two different biological parameters in the living parasite: (1) the activity of stress-related proteases and (2) the integrity of the parasite's cellular membrane. The strategy of using pathogens as sensors for the output of immune effector activity will enable us to map the immune defence according to its actual effect on pathogen physiology. By using intravital 2-photon microscopy, we will be able to survey the mode of action of a protective immune response in the course of an on-going infection. Using inhibitors and knockout mice deficient in the production of reactive oxygen and nitrogen intermediates (ROI and RNI), we will also determine the importance of specific cellular defence mechanisms for the different modes of action of pathogen containment. By applying ROI/RNI production inhibitors in transgenic mice with fluorescently labelled phagocyte populations, we will analyse the requirement of ROI/RNI for defence against pathogens in specific cell types. Finally, we will use a mixed bone marrow chimera approach, combining partially ROI production deficient mice with RNI production inhibition, in order to assess the extent of synergism between ROI and RNI in their antimicrobial activity. The proposed experiments will elucidate how cellular defence mechanisms induced by the immune response impact on pathogen biology and how this impact relates to the efficient control of infection. Understanding this relationship will greatly enhance our knowledge of how the immune system carries protective responses into execution.