Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在22财年，我们继续研究颗粒状杆菌变性，这是慢性肉芽肿性疾病(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22财年期间，我们发表了我们对颗粒状杆菌类脂A糖脂的分析。此前，我们已经证明，与大肠杆菌相比，贝氏革兰氏菌具有低刺激作用，即在人血中产生细胞因子或激活NOX2所需的贝氏革兰氏菌cfu/ml是大肠杆菌的10-100倍。我们分离了它的脂多糖(GbLPS)，并对其脂类A进行了鉴定。与典型的肠杆菌科脂类A不同的是，它能从典型的脂多糖中用冰醋酸进行水解，但它的类脂A分子对酸非常稳定，需要在盐酸中延长水解期。核磁共振和质谱学分析表明，糖脂的碳水化合物部分由-MANP-(14)-GlcpN3N-(16)-GlcpN-(11)-GlcpA四糖取代，取代了5个酰基链：酰胺连接的N-3‘14：0(3-OH)、N-2’16：0(3-O16：0)、N-2 18：0(3-OH)和酯连接的O-3 14：0(3-OH)和16：0。鉴定甘油-D-羟基-辛酸-2-磺酸(KO)是脂多糖核心区的第一组分，共价结合在脂质骨架的GlcpN3N上，这可能是上述耐酸性的原因。此外，Ko和只有五个酰基链的存在可能解释了GbKo-lipidA作为人类白细胞刺激剂(细胞因子诱导和NOX2激活)比大肠杆菌脂质A更弱的原因。总之，这些不同寻常的特性可能有助于颗粒状杆菌逃避免疫系统并抵抗阳离子抗菌肽。这项研究发表在《炎症》杂志(Muszyski等人)上。 在22财年，我们继续描述了一种碳水化合物(很可能是胶囊碳水化合物)，这种碳水化合物似乎与致命性分离株有关，而在非致命性临床分离株中不存在。在与复杂碳水化合物研究中心(佐治亚州雅典)的合作下，我们几乎完成了这种表面上独特的富含鼠李糖的高分子量聚合物的结构表征，预计将于明年发表。这种碳水化合物是否对致死性分离株的毒力或组织嗜性改变负责，这将是未来工作的主题。我们还试图提高这种多糖的抗体，并表征它们与细菌抗原的相互作用。 在22财年，我们完成了对NADPH氧化酶家族成员的改进分析，以便于对其活性和潜在的调节或抑制物质进行比较分析。我们进一步发展了从小鼠中分离粒细胞的方法，以评估在没有和存在各种物质的情况下小鼠NADPH氧化酶2的活性。

During FY22, 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 FY22 we published our analysis of the Lipid A-like glycolipid of Granulibacter bethesdensis. Previously, we had shown that G.bethesdensis was hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood or activation of NOX2 required 10-100 times more G. bethesdensis CFU/mL than E. coli. We isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike typical Enterobacteriaceae Lipid A that can be hydrolyzed from typical LPS with acetic acid, the Lipid A-like molecule from G. bethesdensis was remarkably acid-stabile and required extended hydrolysis in HCl. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of -Manp-(14)--GlcpN3N-(16)--GlcpN-(11)--GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3' 14:0(3-OH), N-2' 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance described above. Furthermore, the presence of Ko and only five acyl chains may explain the poor potency of GbKo-lipidA compared to E. coli lipid A as a stimulator of human leukocytes (cytokine induction and NOX2 activation). Together, these unusual properties may contribute to Granulibacters ability to evade the immune system and resist cationic antimicrobial peptides. This work was published in Inflammation (Muszyski et al.). During FY22, we continued our characterization of a carbohydrate (likely a capsular carbohydrate) that appears to be associated with lethal isolates and absent in non-lethal clinical isolates. In collaboration with the Complex Carbohydrate Research Center (Athens, Georgia) we have nearly completed the structural characterization of this apparently unique rhamnose-rich high molecular weight polymer and anticipate publication in the next year. Whether or not this carbohydrate is responsible for virulence or altered tissue tropism of lethal isolates will be the subject of future work. We have also attempted to raise antibodies to this polysaccharide and are characterizing their interaction with bacterial antigens. During FY22, we completed development of improved assays of NADPH oxidase family members to facilitate comparative analysis of their activity and of potential regulatory or inhibitory substances. We further developed methods to isolate granulocytic leukocytes from mice to evaluate activation of murine NADPH Oxidase 2 in the absence and presence of various substances.