Characterization of an Anaplasma phagocytophilum protein interfering with eukaryo

干扰真核生物的无形体吞噬细胞蛋白的表征

• 批准号: 7879356
• 负责人: SUKANYA NARASIMHAN
• 金额: $ 8.19万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7879356
• 关键词: Anaplasma phagocytophilum; Bacteria; Bacterial Proteins; Bioinformatics; Bovine Anaplasmosis; Cell membrane; Cell physiology; Cells; Gene Deletion; Gram-Negative Bacteria; Growth; HL-60 Cells; Human; Immune; Infection; Lysosomes; Mammalian Cell; Mammals; Nature; Organelles; Outcome; Pathway interactions; Physiology; Process; Proteins; Rickettsiales; Signal Transduction; Surface; System; Ticks; Toxin; Tropism; Vacuole; Virulence; Virulence Factors; Yeasts; factor A; genetic manipulation; genetic regulatory protein; in vitro Model; neutrophil; pathogen; public health relevance; yeast genetics

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.

描述（由申请人提供）：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）具有不寻常的趋向性。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，但关于利用宿主细胞建立成功感染所涉及的细菌蛋白知之甚少。这种细菌的专性细胞内性质严重限制了常规遗传操作在研究潜在毒力机制方面的应用。例如，不可能对该细菌的基因进行定向删除。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放线菌的毒力因子。如果细菌产物在酵母中的表达干扰了限制生长速率的真核过程，则可能会改变酵母的生理学。该系统正在成为识别细菌毒力策略的强大方法。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主要优势。我们在酵母中表达了 35 种嗜吞噬细胞曲霉蛋白（通过生物信息学方法选择），并鉴定了一种嗜吞噬细胞曲霉蛋白 AptA（嗜吞噬细胞无形体毒素 A），其表达严重抑制酵母生长。 AptA 定位于酵母质膜，并干扰涉及酵母液泡的运输途径，酵母液泡是一种功能与哺乳动物溶酶体相当的细胞器。这一观察结果可能具有更大的意义，因为含有膜区室的嗜吞噬细胞菌不与哺乳动物细胞中的溶酶体融合。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/液泡转运途径，并在此建议使用 HL-60 细胞（AptA 的体外模型）进一步表征潜在机制。 . 嗜吞噬细胞感染。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) 鉴定 AptA 靶向的真核/哺乳动物蛋白/通路。 公共健康相关性：该项目旨在阐明由嗜吞噬细胞无形体（一种立克次氏体细菌病原体）编码的 AptA 蛋白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，是美国最严重的蜱传播疾病之一，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，并可能对相关的立克次氏体群体产生重大影响病原体。