Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection are entry and survival in the macrophage. The M.tb cell envelope is heavily mannosylated with the abundant lipoglycans lipoarabinomannan (ManLAM), lipomannan (LM), and phosphatidyl inositol mannosides (PIMs). These lipoglycans bind to C-type lectins expressed on macrophages and dendritic cells (DC). The mannose receptor (MR) is a C-type lectin that mediates phagocytosis of virulent strains of M.tb by human macrophages. DC-SIGN is expressed abundantly on DCs and is also expressed on alternatively activated macrophages such as alveolar macrophages. ManLAM is a major ligand for the MR and DC-SIGN. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of the bacterium to interact with C-type lectins (the MR and DC-SIGN) and thereby modulate macrophage function and host responses. The cytosolic nucleotide mannose carrier GPD-mannose is an essential donor either directly or indirectly in the biosynthesis of LAM, LM, and PIMs as well as cell wall mannoproteins. This proposal focuses on the core enzymes that are required to produce GPD-mannose in mycobacteria. Genes encoding these enzymes will be over-expressed or knocked out inM. smegmatis and M.tb and mutant bacteria with altered surface mannosylation will then be studied in human macrophages and in the mouse aerosol model of TB pathogenesis. One such gene is M.tb manB which we have found encodes a functional phosphomannomutase. Overexpression of manB in M. smegmatis leads to a significant increase in LAM, LM, and PIMs produced as well as a 13-fold increase in bacterial association with macrophages. In addition to the targeted gene approach, we will screen M.tb libraries for clones that are altered in surface mannosylation.
Specific aims :
Aim 1 : Clone, over-express and knock out a defined set of M.tb genes involved in GDP-mannose biosynthesis, Aim 2: Determine whether mycobacteria mutants are altered in surface mannosylation, Aim 3: Determine how genes involved in mannose biosynthesis impact M.tb interactions with the MR and DC-SIGN, the biology of bacteria in human macrophages, and M.tb pathogenesis in the mouse, Aim 4: Screen a M.tb transposon library and a M. smegmatis library over-expressing M.tb genes using Salmonella mannose-binding (type 1)pili and surfactant protein D to identify mutants and clones altered in surface mannosylation. The overall goal of this proposal is to combine molecular, biochemical, and cell biology techniques to identify key enzymes for GDP-mannose biosynthesis in M.tb and, by altering then- level of expression, determine their impact on 1)M.tb cell wall mannosylation and 2) the biology of bacterial behavior in human macrophages and the mouse. Information gained from these studies should enhance our knowledge of TB pathogenesis and also potentially identify new therapeutic targets since mannose metabolism has been shown to be essential for the survival of mycobacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052458-05
Application #
7750035
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Jacobs, Gail G
Project Start
2006-01-15
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2011-12-31
Support Year
5
Fiscal Year
2010
Total Cost
$351,850
Indirect Cost
Name
Ohio State University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Yang, Lanhao; Sinha, Tejas; Carlson, Tracy K et al. (2013) Changes in the major cell envelope components of Mycobacterium tuberculosis during in vitro growth. Glycobiology 23:926-34
Rajaram, Murugesan V S; Ni, Bin; Morris, Jessica D et al. (2011) Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b. Proc Natl Acad Sci U S A 108:17408-13
Keiser, Tracy L; Azad, Abul K; Guirado, Evelina et al. (2011) Comparative transcriptional study of the putative mannose donor biosynthesis genes in virulent Mycobacterium tuberculosis and attenuated Mycobacterium bovis BCG strains. Infect Immun 79:4668-73
Torrelles, Jordi B; Sieling, Peter A; Arcos, Jesús et al. (2011) Structural differences in lipomannans from pathogenic and nonpathogenic mycobacteria that impact CD1b-restricted T cell responses. J Biol Chem 286:35438-46
Sweet, Lindsay; Singh, Prachi P; Azad, Abul K et al. (2010) Mannose receptor-dependent delay in phagosome maturation by Mycobacterium avium glycopeptidolipids. Infect Immun 78:518-26
Torrelles, Jordi B; Schlesinger, Larry S (2010) Diversity in Mycobacterium tuberculosis mannosylated cell wall determinants impacts adaptation to the host. Tuberculosis (Edinb) 90:84-93
Rajaram, Murugesan V S; Brooks, Michelle N; Morris, Jessica D et al. (2010) Mycobacterium tuberculosis activates human macrophage peroxisome proliferator-activated receptor gamma linking mannose receptor recognition to regulation of immune responses. J Immunol 185:929-42
Torrelles, Jordi B; DesJardin, Lucy E; MacNeil, Jessica et al. (2009) Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death. Glycobiology 19:743-55
Valdivia-Arenas, Ma; Amer, A; Henning, Ln et al. (2007) Lung infections and innate host defense. Drug Discov Today Dis Mech 4:73-81