Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在19财年，我们继续对颗粒状杆菌变性的研究，这是慢性肉芽肿病(CGD)患者的新出现的病原体。根据已发表的病例，CGD患者感染这种细菌的病死率为30%。然而，先前的研究表明，这种没有临床明显疾病的生物体的长期持久性也可能发生在一些患者身上。为了更好地了解这种细菌的发病机制，我们收集了这10例报告病例中的9例，并进行了完整的基因组测序和各种实验室研究，目的是解剖这种细菌的基因/表型特征。这些生物的基因组(现在可以在NCBI数据库中找到)显示出显著的多样性。在某些情况下，虽然16S rDNA序列99%相同，但高达11%的开放阅读框对每个分离物都是独一无二的。 在19财年，我们与佐治亚大学复合碳水化合物研究中心的拉塞尔·卡尔森和阿图尔·穆兹斯基合作，完成了对Granulibacter bethedensis的脂肪A的分析，这是一个多年的项目。我们的手稿，在审查中，记录了具有不寻常的类脂A结构的显著的耐酸性脂多糖。在这项研究的基础上，在19财年，我们提纯了致死菌株特有的一种可能的荚膜多糖，并正在对这种材料进行结构和功能研究，以确定它是否与致死菌株激活宿主细胞的能力显著低下有关 在19财年，我们开始利用我们针对Granulibacter甲醇脱氢酶的定制抗体，在实验感染动物的石蜡切片以及存档的人类临床活检中检测体内与Granulibacter相关的细胞类型，以解决这种病原体在长时间的临床沉默感染(类似于分枝杆菌感染)中的持久性。我们还完成了使用免疫金透射电子显微镜分析甲醇脱氢酶在颗粒菌中的亚细胞分布的研究。 Granulibacter bethedensis已经抵制了以前使用普通实验室质粒(例如PBR系列)进行基因操作的努力。在19财年，我们使用广泛的宿主范围的质粒成功地转化了Granulibacter，并正在开发工具来敲除或敲除特定的Granulibacter基因，以测试与某些酶在Granulibacter独特的脂质A生物合成、MDH调节以及中性粒细胞和单核细胞中的微生物复制中的重要性相关的假设。最近的一项重大技术进步是发现从转化的Granulibacter中分离出的质粒数在进一步转化Granulibacter时更有效，这表明物种特异性的修饰，例如甲基化，在努力改造这种有机体的过程中是重要的。转化的直接应用是产生一株表达dsRED的颗粒状杆菌，可用于体外定量评估中性粒细胞的吞噬能力。

During FY19, we continued our studies of Granulibacter bethesdensis, emerging pathogen in patients with chronic granulomatous disease (CGD). Based on published cases, infection of CGD patients with this organism has a case fatality rate of 30%. Previous studies have shown, however, that long-term persistence of this organism without clinically apparent disease may also occur in some patients. To better understand pathogenesis by this organism, we have collected isolates from 9 of these 10 reported cases and performed complete genome sequencing as well as a variety of laboratory studies aimed dissecting genotype/phenotype characteristics of this organism. Genomes of these organisms (now available in NCBI databases), demonstrate remarkable diversity. In some cases, while the 16S rDNA sequences are >99% identical, up to 11% of open reading frames can be unique to each isolate. During FY19 we completed our analysis of the Lipid A of Granulibacter bethesdensis, a multi-year project in collaboration with Russell Carlson and Artur Muszyski of the University of Georgia Complex Carbohydrate Research Center. Our manuscript, in review, documents the remarkably acid resistant LPS with an unusual Lipid A structure. Further to this study, during FY19 we have purified a putative capsular polysaccharide that is unique to the lethal strains and are performing structural and functional studies of this material to determine if it is responsible for the remarkably low ability of the lethal strains to activate host cells During FY19 we began to exploit our custom antibody to Granulibacter Methanol Dehydrogenase examine the cell types associated with Granulibacter in vivo in paraffin sections of experimentally infected animals as well as archived human clinical biopsies to address persistence of this pathogen over long periods of clinically silent infection (akin to Mycobacterial infections). We are also completing studies using immunogold transmission electron microscopy to analyze the subcellular distribution of methanol dehydrogenase in Granulibacter. Granulibacter bethesdensis had resisted previous efforts for genetic manipulation using common lab plasmids (e.g., pBR series). During FY19, we successfully transformed Granulibacter using broad host-range plasmids and are pursuing the development of tools to knock out or knock down specific Granulibacter genes to test hypotheses relating to the importance of certain enzymes in the unique lipid A biosynthesis in Granulibacter, MDH regulation, and microbial replication in neutrophils and monocytes. One major recent technical advance was the finding that plasmids isolated from transformed Granulibacter were several logs more effective in further transformations of Granulibacter suggesting that species specific modifications, e.g., methylation, are important in efforts to modify this organism. On immediate application of transformation has been the generation of a dsRED expressing strain of Granulibacter that could be used for quantitative assessment of phagocytosis by neutrophils in vitro.