title: Mycobacterium tuberculosis Infection Manipulates the Glycosylation Machinery and the N-Glycoproteome of Human Macrophages and Their Microparticles.
abstract: Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of frontline alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases, e.g., β-galactosidase, β-hexosaminidases and α-/β-mannosidases, was observed. Relative weak infection-driven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation and concomitant down-regulation of paucimannosylation upon infection. Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique N-glycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated α-glucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.
authors.affiliation: Tuberculosis Research Program, Centenary Institute, Discipline of Medicine, Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney , Newtown, NSW 2042, Australia.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hare
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University , Sydney, NSW 2109, Australia.
authors.forename: Ling Y
authors.initials: LY
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University , Sydney, NSW 2109, Australia.
authors.forename: Ian
authors.initials: I
authors.lastname: Loke
authors.affiliation: Tuberculosis Research Program, Centenary Institute, Discipline of Medicine, Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney , Newtown, NSW 2042, Australia.
authors.forename: Warwick J
authors.initials: WJ
authors.lastname: Britton
authors.affiliation: Tuberculosis Research Program, Centenary Institute, Discipline of Medicine, Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney , Newtown, NSW 2042, Australia.|School of Life Sciences, University of Technology Sydney , Sydney, NSW 2007, Australia.
authors.forename: Bernadette M
authors.initials: BM
authors.lastname: Saunders
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University , Sydney, NSW 2109, Australia.
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
mesh_terms: D002240:Carbohydrate Sequence; D002460:Cell Line; D055252:Cell-Derived Microparticles / Q000737:chemistry / Q000378:metabolism* / Q000382:microbiology; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D037102:Lectins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism* / Q000382:microbiology; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000254:growth & development* / Q000472:pathogenicity; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D006023:Glycoproteins; D037102:Lectins; D020543:Proteome; D012794:Sialic Acids; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D008358:Mannose
keywords: LC−MS/MS; Mycobacterium tuberculosis; N-glycosylation; exoglycosidase; glycomics; glycoprofiling; glycoproteome; macrophage; microparticle; proteomics; tuberculosis