1) To study the determinants of monocyte and macrophage host defenses against infection by the novel bacterial pathogen, Granulibacter bethesdensis Our recent publication (Zarember et al., Infection and Immunity, 2012) demonstrated that killing of G. bethesdensis by neutrophils from patients with chronic granulomatous disease (CGD) was defective and that this Gram-negative pathogen was remarkably resistant to complement and to cationic antimicrobial peptides. During FY13, Jessica Chu (Postdoctoral IRTA) completed a study demonstrating that CGD human monocytes and monocyte-derived macrophages were unable to kill G. bethesdensis normally and that this organism could persist in CGD macrophages. This paper also demonstrated a positive correlation between microbial killing by IFNgamma-treated monocytes and residual neutrophil NADPH oxidase activity. This finding has potential diagnostic implications in the use of IFN (Actimune) to treat CGD patients. 2) G. bethesdensis Lipopolysaccharide (LPS) During our studies of the interaction of Granulibacter bethesdensis with immune cells, we found that this organism is remarkably hypostimulatory of the human innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. We are collaborating with Yossi Shiloach (NIDDK) and Russell Carlson of the University of Georgia Complex Carbohydrate Research Center to complete the purification and structural characterization of the atypical lipopolysaccharide (LPS) of this organism and determine whether it acts as an anti-inflammatory inhibitory LPS. 3) G.bethesdensis Methanol Dehydrogenase In order to develop our serological testing for G.bethesdensis infection (see ZIA AI000155-36), we purified Methanol Dehydrogenase from this organism. Using ion exchange and gel filtration, highly enriched enzyme was prepared and biochemical testing is underway to identify substrate specificity, identify inhibitors, and s indicate a much broader substrate specificity that originally thought. In collaboration with Peter Steinbach (NIH Center for Molecular Modeling), we have modeled the structure of G. bethesdensis MDH.
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