Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

National Institute Of Allergy And Infectious Diseases

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Abstract

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.