Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection within its human host are entry and survival in the macrophage. The macrophage mannose receptor (MR) participates in the phagocytosis of virulent strains of M.tb. Components of the M.tb cell wall serve as ligands for host cell receptors and can modulate host microbicidal and inflammatory responses. The M.tb cell envelope is heavily mannosylated containing lipoglycans such as lipoarabinomannan (LAM) which serves as a ligand for the MR. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of M.tb to interact with the MR as well as to modulate macrophage function and consequently host responses, enabling the establishment of infection. PimB was recently described as M.tb phosphatidyl myo-inositol monomannoside transferase (pimB). We have used allelic-exchange to inactivate pimB in M.tb strain Erdman. Macrophages display marked cellular adhesion following infection with wild-type M.tb. In contrast, macrophages infected with the pimB mutant display minimal cellular adhesion and a significant increase in the rate of macrophage death. We have developed an assay in which Salmonella mannose-specific binding pili agglutinate M.tb LAM coated microspheres that we will develop as a screen for alterations in M.tb surface mannosylation. We propose to further characterize the role of pimB and other selected enzymes potentially involved in mannosylation of M.tb surface molecules in the biology of the M.tb-host interaction, to develop a novel screening strategy for M.tb clones altered in surface mannosylation, and to evaluate these bacterial clones for anomalous host cell interaction.
Our specific aims are to: 1A. Determine the mechanism for reduced homotypic adhesion and increased rate of macrophage death following infection with the pimB mutant of M.tb: 1 B. Determine the biochemical nature of the pimB mutation. Analyze the structure of LAM and other mannosylated cell wall glycoconjugates from wild type, the pimB mutant, and pimB overproducing M.tb strains. 2. Perform transcription and genetic studies of genes encoding the biosynthetic enzymes of LAM and mannose glycoconjugates: 2A. Quantify the level of transcription of pimB and its homologues in M.tb grown in broth, solid medium and within human macrophages using the AbI 7700 (TaqMan) """"""""real-time"""""""" quantitative PCR system; 2B. Construct and analyze genetically defined M.tb strains with alterations in the mannose biosynthetic genes. 3). Utilize Salmonella mannose-binding (type 1) pili to screen for M.tb mutants and clones respectively altered in surface mannosylation from an M.tb transposon library and M. smegmatis library complemented with M.tb genes to characterize the effect of alterations in bacterial mannosylation on macrophage interaction. The assembled investigators will combine techniques in genetics, biochemistry, and cell biology to accomplish the goals of this proposal.
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