1) To study the determinants of monocyte and macrophage host defenses against infection by the novel bacterial pathogen, Granulibacter bethesdensis We have published a paper (Zarember et al., 2012) that demonstrated killing of G. bethesdensis by normal and not CGD neutrophils as well as documenting a high level of resistance to serum complement and purified antimicrobial cathelicidin peptide. Normal, and especially CGD neutrophil apoptosis was delayed by G. bethesdensis suggesting that the organism might proliferate in neutrophils before dissemination to other sites. We are currently exploring the roles of human monocytes and monocyte-derived macrophages from normal subjects and CGD patients in killing this organism and anticipate submission of this work for publication before the end of the FY. 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 Russel 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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