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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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
Chu, Jessica; Smelkinson, Margery G; Dorward, David W et al. (2017) Early Intracellular Trafficking of Granulibacter bethesdensis in Human Macrophages. Infect Immun 85:
De Ravin, Suk See; Li, Linhong; Wu, Xiaolin et al. (2017) CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci Transl Med 9:
De Ravin, Suk See; Reik, Andreas; Liu, Pei-Qi et al. (2016) Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol 34:424-9
Kuhns, Douglas B; Fink, Danielle L; Choi, Uimook et al. (2016) Cytoskeletal abnormalities and neutrophil dysfunction in WDR1 deficiency. Blood 128:2135-2143
Feingold, Paul L; Quadri, Humair S; Steinberg, Seth M et al. (2016) Thoracic Surgery in Chronic Granulomatous Disease: a 25-Year Single-Institution Experience. J Clin Immunol 36:677-83
De Ravin, Suk See; Wu, Xiaolin; Moir, Susan et al. (2016) Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8:335ra57
Merling, Randall K; Sweeney, Colin L; Chu, Jessica et al. (2015) An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease. Mol Ther 23:147-57
Greenberg, David E; Sturdevant, Daniel E; Marshall-Batty, Kimberly R et al. (2015) Simultaneous Host-Pathogen transcriptome analysis during Granulibacter bethesdensis infection of normal and chronic granulomatous disease neutrophils. Infect Immun :
Kuhns, Douglas B; Long Priel, Debra A; Chu, Jessica et al. (2015) Isolation and Functional Analysis of Human Neutrophils. Curr Protoc Immunol 111:7.23.1-16

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