Role of pathogen-derived capping carbohydrates in altering immunity

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DESCRIPTION (provided by applicant): Structurally diverse carbohydrates are abundantly expressed on outer surfaces of pathogens. This project utilizes cap sugars of two surface carbohydrates, Lipophosphoglycans (LPG) and lipoarabinomannans (LAM), from Leishmania and Mycobacterium respectively. These two carbohydrate chains from virulent species delay phagosome maturation and promote a localized or systemic tolerogenic immune response that supports continuation of chronic infection. Unique cap sugars are associated with virulent Mycobacterial spp.; it is not known which specific portions of LPG/LAM cause immune alterations. Difficult carbohydrate chemistry precluded the ability to specifically test the role of this most-external portion of LAM/LPG. In this proposal, through breakthrough technology provided by chemist Dr. Nicola Pohl, this problem was overcome. Beads coated with LAM and LPG cap sugars have been produced. This proposal challenges two central hypotheses A) cap sugars are an immune- inhibitory portion of LAM and LPG and B) the in vitro and in vivo responses to four structurally different pathogen-derived cap sugars will differ in alteration of pathogen processing and destruction. Experiments outlined in this proposal are designed to 1) Confirm that synthetically-produced caps sugars have correct conformation and biologic activity 2) Determine how LAM or LPG mannose cap sugar-coated beads process in macrophages as compared to control phagocytosed beads, 3) Test the compared ability of bead-treated macrophages to kill internalized pathogens, 4) Characterize the in vivo immune response to LAM or LPG cap sugar-coated beads. We predict that beads coated with cap sugars derived from virulent Mycobacterium or Leishmania sp. will deter phagolysosome maturation and macrophage pathogen removal. In addition we predict that these cap sugars will inhibit in vivo bead clearance and promotion of a Th1 cytokine response. As pathogen-surface-expressed cap sugars partially determine pathogenic strains of bacteria and parasites, these studies will identify how changes in carbohydrate molecule cap structure lead to outcome-altering differences in immunity. PUBLIC HEALTH RELEVENCE: Understanding the specific role of cap sugars, the external surface coat for many pathogens, in altering host immune responses can lead to immediate and important discoveries in pathogen assemby that should be targeted for drug discovery. These pathogen surface- expressed cap sugars have been shown to predict differences between pathogenic and non- pathogenic strains of bacteria and parasites, therefore the proposed studies will provide a model system to identify the role of these molecules in determining virulence and how changes in cap structure leads to outcome-altering differences in immune responses. Due to the important function of carbohydrates in directing anti-pathogen immune responses, it is necessary to determine the specific role of pathogen-derived cap sugars in altering the in vivo and in vitro immune response.