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.

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