Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在2016财年，我们继续对伯氏颗粒菌的研究，这是一种在慢性肉芽肿疾病(CGD)患者中出现的病原体。我们完成了对暴露于正常或CGD PMN的Granulibacter的转录本的比较分析，同时完成了暴露于Granulibacter后的正常和CGD PMN的转录本的比较分析(Greenberg等人，2015)。本研究记录了Granulibacter延缓中性粒细胞凋亡的可能机制，并确定Granulibacter的应激反应基因ClpB是PMN生存所需的毒力因子。Granulibacter代谢基因在胞内生长过程中表达的变化导致了丙酮酸脱氢酶是一个必需的基因，一种药物抑制剂可以作为体外抗Granulibacter的抗生素。 在2016财年，我们显著推进了由NIH和西班牙和葡萄牙的国际合作者提供的单个患者Granulibacter分离株(9个基因不同的分离株)的基因组和表型属性的研究。对这些分离株进行了全基因组测序，并正在结合已建立的免疫细胞功能(例如，中性粒细胞、单核细胞和巨噬细胞的吞噬和杀伤)的实验室分析进行分析。 鉴于我们之前的发现，在体外巨噬细胞中持续存在贝氏球虫，我们研究了这种生物的细胞内运输，以帮助解释它在细胞内的生长。这部作品目前正在筹备出版中。与佐治亚大学复合碳水化合物研究中心的阿图尔·穆兹斯基合作，正在对脂质A颗粒状杆菌进行核磁共振研究，以确定这种材料的化学性质是否解释了颗粒状杆菌激活人类免疫细胞(如PMN和单核细胞)的能力相对较差的原因。

During FY16, we continued our studies of Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). We completed our comparative analysis of the transcriptomes of Granulibacter exposed to normal or CGD PMN and, in parallel, the transcriptomes of normal and CGD PMN following exposure to Granulibacter (Greenberg et al., 2015). This study documented possible mechanisms by which Granulibacter delays neutrophil apoptosis, and identified ClpB, a stress response gene in Granulibacter, as a virulence factor required for survival in PMN. Alterations in Granulibacter metabolic gene expression during intracellular growth lead to the identification of pyruvate dehydrogenase as an essential gene and that a pharmacologic inhibitor could be used as an antibiotic against Granulibacter in vitro. During FY16, we have significantly advanced our study of the genomes and phenotypic attributes of individual patient Granulibacter isolates (9 genetically distinct isolates) provided by NIH and international collaborators in Spain and in Portugal. Total genome sequencing of these isolates was performed and is being analyzed in combination with established laboratory assays of immune cell function (e.g., phagocytosis and killing by neutrophils, monocytes, and macrophages). Given our previous finding that G. bethesdensis persists in macrophages in vitro, we examined the intracellular trafficking of this organism to help explain its intracellular growth. This work is currently in preparation for publication. In collaboration with Artur Muszyski of the University of Georgia Complex Carbohydrate Research Center, NMR studies of the lipid A Granulibacter bethesdensis are underway to determine whether the chemical nature of this material accounts for the relatively poor ability of Granulibacter to activate human immune cells such as PMN and monocytes.