During FY18, 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. We are preparing two manuscripts relating to this project that we anticipate submitting in late FY18 or early FY19. During FY18 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. We are submitting a manuscript describing the remarkably acid resistant LPS with an unusual Lipid A structure. Further to this study, during FY18 we have partially purified what is apparently a capsular polysaccharide that is unique to the lethal strains. We plan on completing the purification and identification of this structure in FY19. During FY18 we created an antibody to Granulibacter methanol dehydrogenase to permit detection of Granulibacter in paraffin-embedded infected mouse and human clinical samples as well as evaluate the cells this pathogen is associated with in vivo. We have demonstrated, both by confocal microscopy and by stimulated emission depletion microscopy that some isolates demonstrate unique tissue tropism compared with others and that intact, apparently replicating microbes can be identified in some tissues. We are currently examining the cell types associated with Granulibacter in vivo to address persistence of this pathogen over long periods of clinically silent infection (perhaps akin to Mycobacterial infection). Granulibacter bethesdensis had resisted previous efforts for genetic manipulation using common lab plasmids (e.g., pBR series). During FY18, we achieved transformation of this organism using broad host-range plasmids which could be recovered from cultures of transformed Granulibacter demonstrating stable replication. During FY19 we will pursue the knockout of specific genes in Granulibacter 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.

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Myles, Ian A; Anderson, Erik D; Earland, Noah J et al. (2018) TNF overproduction impairs epithelial staphylococcal response in hyper IgE syndrome. J Clin Invest 128:3595-3604
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