Characterization of an Anaplasma phagocytophilum protein interfering with eukaryo

描述（由申请方提供）：嗜吞噬细胞无形体是人类无形体病的病原体，是一种蜱传病原体，对前线免疫防御细胞（中性粒细胞）具有不寻常的嗜性。虽然已知通过这种革兰氏阴性细菌操纵哺乳动物宿主细胞的几个方面，但关于利用宿主细胞建立成功感染所涉及的细菌蛋白质，所知相对较少。这种细菌的专性细胞内性质严重限制了常规遗传操作的应用，以研究潜在的毒力机制。例如，不可能进行该细菌的基因的靶向缺失。在此背景下，我们使用酵母作为替代宿主来鉴定A.嗜吞噬细胞菌细菌产物在酵母中的表达如果干扰限制生长速率的真核过程，则可能改变酵母的生理学。该系统正在成为鉴定细菌毒力策略的有力方法。酵母和哺乳动物之间的许多（或可能是大多数）细胞生理学的基本信号传导机制的高度保守性，以及酵母遗传学的简单性是该系统的关键优势。我们表达了35 A。利用生物信息学方法筛选出一株A.嗜吞噬细胞无形体毒素A（Anaplasma phagocytophilum toxin A），其表达严重抑制酵母生长。AptA定位于酵母质膜，并干扰涉及酵母空泡的转运途径，酵母空泡是一种功能上与哺乳动物溶酶体相当的细胞器。这一观察可能有更大的影响，因为A。在哺乳动物细胞中，含有膜隔室的嗜吞噬细胞菌不与溶酶体融合。我们假设AptA通过干扰一个或多个真核调节蛋白的功能来改变真核细胞内吞/空泡转运途径，并在此提出使用HL-60细胞（A.嗜吞噬细胞菌感染具体目标是： 1)确定AptA对哺乳动物内吞途径的影响。 2)确定AptA是否暴露（分泌或表面展示）于宿主细胞。 3)鉴别AptA靶向的真核/哺乳动物蛋白/通路。 公共卫生相关性：该项目旨在描述由立克次氏体细菌病原体Anaplasma phagocytophilum编码的称为AptA的蛋白质改变真核宿主生理学的机制。嗜吞噬细胞无形体是感染中性粒细胞的人类无形体病的病原体，是美国最高的蜱传播疾病之一，因此任何成功的结果都将对我们对这种细菌的理解产生重大影响，并可能对立克次体病原体的相关群体产生影响。

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.