During FY15, we continued our studies of Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). We completed our comparative analysis of the transcriptomes of Granulibacter exposed to normal or CGD PMN and, in parallel, the transcriptomes of normal and CGD PMN following exposure to Granulibacter (Greenberg et al., 2015). Several findings emerged from this study including the recognition of transcriptional and protein changes by PMN in response to Granulibacter that account, at least in part, for the unusual finding that, unlike most microbes, Granulibacter delays PMN apoptosis. Further studies will be required to determine whether or not this delay in apoptosis contributes to persistence of this pathogen in the host, for example by providing a pathway for bacteria internalized within PMN to enter into macrophages (the cells that phagocytosis PMN) thereby facilitating their long-term persistence. Another major finding was the identification of ClpB, a stress response gene in Granulibacter, as a virulence factor required for survival in PMN. We demonstrated, for the first time, that phosphorodiamidate morpholino oligomers could be used to interfere with this genes function and that inhibition of ClpB expression increased the sensitivity of Granulibacter to killing by both normal and CGD PMN. Metabolic alterations in Granulibacter gene expression during intracellular growth in PMN included upregulation of pyruvate dehydrogenase and we were able to show that pharmacologic inhibition of this enzyme could exert and antibiotic effect on Granulibacter. During FY15, our section performed functional studies of in vitro differentiated PMN produced induced pluripotent stem cells. These cells isolated from patients with CGD, were then corrected using gene-therapy. This study provided a proof-of-principle that gene-corrected iPS cells have potential as in vitro tools in the laboratory as well as potential clinical applications in the future. Our current projects include the comparison of genotypic and phenotypic attributes of individual patient isolates (8 genetically distinct isolates) provided by NIH and international collaborators. Total genome sequencing of these isolates was performed and is being analyzed in combination with evaluation of these isolates in established laboratory assays of immune cell function (e.g., phagocytosis and killing by neutrophils, monocytes, and macrophages). Given our finding in the last FY that G. bethesdensis appears to persist in macrophages for extended periods of time, we have been examining the intracellular trafficking of this organism in macrophages to identify organelles and pathways associated with intracellular growth of this pathogen. In collaboration with Artur Muszyński of the University of Georgia Complex Carbohydrate Research Center, NMR studies of the lipid A Granulibacter bethesdensis are underway to determine whether the chemical nature of this material accounts for the relatively poor ability of Granulibacter to activate human immune cells such as PMN and monocytes.
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