During FY17, we continued our studies of Granulibacter bethesdensis, emerging pathogen in patients with chronic granulomatous disease (CGD). Based on published cases, infection 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. 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 (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 FY17, we published a manuscript (Chu et al.) describing the internalization and subcellar trafficking of G. bethesdensis in macrophages in vitro. Previous observations demonstrated that Granulibacter can persist for at least one week in primary human macrophages from CGD but not healthy controls. Although these studies strongly implicated NOX2 as a central component of intracellular killing of Granulibacter by macrophages, it was still possible that intracellular trafficking of this organism might differ between cells from normal or CGD patients leading to the distinct outcomes. To formally test this possibility, we used both fluorescence and electron microscopy to examine the association of Granulibacter with lysosomal components early during infection of macrophages in vitro. The main findings of this study were that there was no apparent difference between normal and CGD cells in the association of Granulibacter with lysosomal markers. Furthermore, under these conditions, Granulibacter appeared to remain in membrane-surrounded phagolysosomes. This study offered further evidence that, in the absence of NOX2-dependent oxidative killing mechanisms, Granulibacter is capable of resisting attack by the normally antimicrobial components of macrophage, monocyte, and neutrophil phagolysosomes such as proteases, antimicrobial peptides and proteins, and reactive nitrogen radicals. Our laboratory has shown that on a per cell basis, Granulibacter is a 10-100 times less potent activator of the neutrophil respiratory burst (NOX2-activation) as well as inducer of cytokine production in anticoagulated blood ex vivo. We hypothesized that the lipopolysaccharide (LPS) of this Gram-negative organism was different from that of typical enteric microbes such as Escherichia coli. Hypostimulatory LPS play roles in microbial pathogenesis and may have therapeutic utility in vaccine development. We therefore initiated studies aimed at determining the structure of Granulibacter LPS and Lipid A in collaboration with Artur Muszyski of the University of Georgia Complex Carbohydrate Research Center. We are currently writing a manuscript describing the presence of acid resistant LPS with an unusual Lipid A structure.
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