Characterization of an Anaplasma phagocytophilum protein interfering with eukaryo

描述（由申请人提供）：嗜吞噬细胞无原体是人类无原体病的病原体，是一种蜱传病原体，对一线免疫防御细胞（中性粒细胞）具有不寻常的嗜性。虽然这种革兰氏阴性细菌操纵哺乳动物宿主细胞的几个方面是已知的，但对于利用宿主细胞建立成功感染所涉及的细菌蛋白质知之甚少。这种细菌的专性细胞内性质严重限制了传统遗传操作的应用，以研究潜在的毒力机制。例如，不可能对这种细菌的基因进行靶向删除。在这种情况下，我们使用酵母作为替代宿主来鉴定嗜吞噬细胞芽孢杆菌的毒力因子。酵母中细菌产物的表达可能会改变酵母的生理机能，如果它们干扰了一个限制生长速率的真核过程。该系统正在成为一种识别细菌毒力策略的有力方法。酵母和哺乳动物之间细胞生理学的许多（或可能是大多数）基本信号机制的高度保守性，以及酵母遗传学的简单性是该系统的关键优势。我们在酵母中表达了35个A. phagocytophilum蛋白（通过生物信息学方法选择），并鉴定出一个A. phagocytophilum蛋白AptA (Anaplasma phagocytophilum toxin A)，其表达严重抑制酵母的生长。AptA定位于酵母质膜，并干扰酵母液泡的运输途径，酵母液泡是一种功能上与哺乳动物溶酶体相似的细胞器。这一观察结果可能具有更大的意义，因为在哺乳动物细胞中含有膜室的嗜吞噬胞杆菌不与溶酶体融合。我们假设AptA通过干扰一种或多种真核调节蛋白的功能来改变真核内吞/空泡运输途径，并提出利用嗜吞噬细胞假单胞菌感染的体外模型HL-60细胞进一步表征其潜在机制。具体目标是：

DESCRIPTION (provided by applicant): Anaplasma phagocytophilum, the causative agent of human Anaplasmosis, is a tick-borne pathogen with an unusual tropism for the front-line immune defense cells (neutrophils). While several aspects of the manipulation of the mammalian host cells by this gram-negative bacterium are known, relatively little is known regarding the bacterial proteins involved in exploiting the host cells to establish a successful infection. The obligate intracellular nature of this bacterium severely limits the application of conventional genetic manipulation to study the underlying virulence mechanisms. For example, it is not possible to perform targeted deletion of genes of this bacterium. In this context, we used yeast as a surrogate host to identify the virulence factors of A. phagocytophilum. Expression of bacterial products in yeast can potentially alter yeast physiology if they interfere with a eukaryotic process that is rate-limiting for growth. This system is emerging as a powerful approach to identify bacterial virulence strategies. High conservation of many (or probably most) fundamental signaling mechanisms of cell physiology between yeast and mammals, as well as the simplicity of yeast genetics are the key advantages of this system. We expressed 35 A. phagocytophilum proteins (selected by bioinformatics approach) in yeast and identified one A. phagocytophilum protein, AptA (Anaplasma phagocytophilum toxin A) whose expression severely inhibits yeast growth. AptA localizes to yeast plasma membrane, and interferes with the transport pathway involving the yeast vacuole, an organelle functionally comparable to mammalian lysosomes. This observation may have larger implications because an A. phagocytophilum containing membranous compartment does not fuse with lysosomes in mammalian cells. We hypothesize that AptA alters eukaryotic endocytotic/ vacuolar transport pathway by interfering with the function of one or more eukaryotic regulatory protein(s), and herein propose to further characterize the underlying mechanism, using the HL-60 cells, the in vitro model of A. phagocytophilum infection. The specific aims are: 1) Determine the effect of AptA on mammalian endocytic pathways. 2) Determine whether AptA is exposed (secreted or surface displayed) to the host cell. 3) Identify the eukaryotic/mammalian protein(s)/pathway(s) targeted by AptA. PUBLIC HEALTH RELEVANCE: The proposed project aims to delineate the mechanism by which a protein called AptA encoded by Anaplasma phagocytophilum, a Rickettsiales' bacterial pathogen, alters eukaryotic host physiology. Anaplasma phagocytophilum, the causative agent of human Anaplasmosis that infects neutrophils, is among the highest tick-transmitted illnesses of the USA, and hence any successful outcome of this will have significant impact on our understanding on this bacterium and potentially on the related group of Rickettsial pathogens.