Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

在FY22期间，我们继续对慢性肉芽肿病（CGD）患者的新发病原体——贝塞斯致密颗粒杆菌进行研究。根据已发表的病例，CGD患者感染该菌的病死率为30%。然而，先前的研究表明，在一些患者中也可能出现这种生物体长期存在而无临床明显疾病。为了更好地了解该菌的发病机制，我们收集了10例报告病例中9例的分离株，并进行了全基因组测序和各种实验室研究，旨在解剖该菌的基因型/表型特征。这些生物的基因组（现已在NCBI数据库中提供）显示出显著的多样性。在某些情况下，虽然16S rDNA序列是99%相同的，但每个分离物中高达11%的开放阅读帧是唯一的。

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.