Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在2022财年，我们继续对慢性肉芽肿病（CGD）患者中出现的病原体--贝氏颗粒杆菌进行研究。 根据已发表的病例，CGD患者感染该微生物的病死率为30%。 然而，以前的研究表明，这种微生物的长期持续存在而没有临床上明显的疾病也可能发生在一些患者中。 为了更好地了解这种微生物的发病机制，我们从这10例报告病例中的9例中收集了分离株，并进行了全基因组测序以及各种旨在解剖这种微生物的基因型/表型特征的实验室研究。 这些生物体的基因组（现在可在NCBI数据库中获得）表现出显着的多样性。 在某些情况下，虽然16 S rDNA序列是>99%相同的，但是高达11%的开放阅读框对于每个分离物可以是独特的。 在2022财年，我们发表了对贝塞登颗粒杆菌的脂质A样糖脂的分析。 以前，我们已经证明，与大肠杆菌相比，贝塞登革菌是低刺激性的，即，人体血液中细胞因子的产生或NOX 2的激活需要10-100倍的G。bethesdensisCFU/mL比E.杆菌我们分离了它的脂多糖（GbLPS），并对其脂质A进行了表征。与典型的肠杆菌科脂质A不同，其可以用乙酸从典型的LPS水解，来自G. Bethesdensis是非常酸稳定的，需要在HCl中延长水解。 NMR和质谱法证实分离的糖脂的碳水化合物部分由-Manp-（14）--GlcpN-（16）--GlcpN-（11）--GlcpA四糖组成，其被五个酰基链取代：酰胺连接的N-3 ′ 14：0（3-OH）、N-2 ′ 16：0（3-O 16：0）和N-2 18：0（3-OH）以及酯连接的O-3 14：0（3-OH）和16：0。将甘油-d-塔罗-辛-2-酮糖酸（Ko）鉴定为共价连接至脂质骨架的GlcpN 3 N的LPS的核心区的第一组分可以解释上述的耐酸性。 此外，Ko的存在和仅五个酰基链可以解释GbKo-lipidA与E.大肠杆菌脂质A作为人白细胞的刺激物（细胞因子诱导和NOX 2活化）。 总之，这些不寻常的特性可能有助于颗粒菌逃避免疫系统和抵抗阳离子抗菌肽的能力。 这项工作发表在Inflammation（Muszyski et al.）上。 在2022财年，我们继续对一种碳水化合物（可能是荚膜碳水化合物）进行表征，该碳水化合物似乎与致死性分离株相关，但在非致死性临床分离株中不存在。 与复杂碳水化合物研究中心（雅典，格鲁吉亚）合作，我们几乎完成了这种明显独特的富含鼠李糖的高分子量聚合物的结构表征，并预计在明年出版。 这种碳水化合物是否对致死分离株的毒力或改变的组织嗜性负责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2022财年，我们完成了对NADPH氧化酶家族成员的改进检测方法的开发，以促进对其活性和潜在调节或抑制物质的比较分析。 我们进一步开发了从小鼠中分离粒细胞白细胞的方法，以评价在不存在和存在各种物质的情况下小鼠NADPH氧化酶2的活化。

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.