Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在2016财年，我们继续对慢性肉芽肿病（CGD）患者中的一种新出现的病原体——贝塞斯致密颗粒杆菌（Granulibacter bethesdensis）进行研究。我们完成了暴露于正常或CGD PMN的颗粒杆菌转录组的比较分析，同时也完成了暴露于颗粒杆菌后正常和CGD PMN转录组的比较分析（Greenberg et al., 2015）。本研究记录了颗粒杆菌延缓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是PMN存活所需的毒力因子。颗粒杆菌在细胞内生长过程中代谢基因表达的改变导致丙酮酸脱氢酶被鉴定为必需基因，并且一种药物抑制剂可以用作颗粒杆菌的体外抗生素。

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.