Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在2016财年，我们继续研究贝塞登颗粒杆菌，这是慢性肉芽肿病（CGD）患者中的一种新兴病原体。 我们完成了对暴露于正常或CGD PMN的颗粒杆菌的转录组的比较分析，以及平行地，对暴露于颗粒杆菌后的正常和CGD PMN的转录组的比较分析（Greenberg et al.，2015年）。 本研究记录了颗粒杆菌延迟中性粒细胞凋亡的可能机制，并确定了颗粒杆菌中的应激反应基因ClpB作为PMN存活所需的毒力因子。 在细胞内生长过程中颗粒杆菌代谢基因表达的改变导致丙酮酸脱氢酶作为必需基因的鉴定，并且药理学抑制剂可用作体外对抗颗粒杆菌的抗生素。 在2016财年，我们显著推进了对NIH和西班牙和葡萄牙的国际合作者提供的单个患者颗粒杆菌属分离株（9种遗传上不同的分离株）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，并结合已建立的免疫细胞功能实验室测定（例如，嗜中性粒细胞、单核细胞和巨噬细胞的吞噬作用和杀伤作用）。 鉴于我们以前的发现，G。Bethesdensis在体外巨噬细胞中持续存在，我们检查了这种生物体的细胞内运输，以帮助解释其细胞内生长。 这项工作目前正在准备出版。 与格鲁吉亚大学复合碳水化合物研究中心的Artur Muszyski合作，正在进行脂质A颗粒杆菌的NMR研究，以确定这种材料的化学性质是否导致颗粒杆菌激活人类免疫细胞（如PMN和单核细胞）的能力相对较差。

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.