title: A mycobacterial phosphoribosyltransferase promotes bacillary survival by inhibiting oxidative stress and autophagy pathways in macrophages and zebrafish.
abstract: Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.
authors.affiliation: From the School of Biotechnology, Campus-11, KIIT University, Bhubaneswar, Orissa-751024, India.
authors.forename: Soumitra
authors.initials: S
authors.lastname: Mohanty
authors.affiliation: From the School of Biotechnology, Campus-11, KIIT University, Bhubaneswar, Orissa-751024, India, the AU-KBC Research Center, MIT Campus, Anna University, Chromepet, Chennai, Tamil Nadu 600025, India.
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Jagannathan
authors.affiliation: From the School of Biotechnology, Campus-11, KIIT University, Bhubaneswar, Orissa-751024, India.
authors.forename: Geetanjali
authors.initials: G
authors.lastname: Ganguli
authors.affiliation: From the School of Biotechnology, Campus-11, KIIT University, Bhubaneswar, Orissa-751024, India.
authors.forename: Avinash
authors.initials: A
authors.lastname: Padhi
authors.affiliation: the Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Debasish
authors.initials: D
authors.lastname: Roy
authors.affiliation: the Department of Microbiology, Immunology, and Glycobiology, Institute of Laboratory Medicine, Lund University, 221 00 Lund, Sweden.
authors.forename: Nader
authors.initials: N
authors.lastname: Alaridah
authors.affiliation: the Bioinformatics Center, Indian Institute of Science, Bangalore, Karnataka 560012, India, and.
authors.forename: Pratip
authors.initials: P
authors.lastname: Saha
authors.affiliation: the Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Upendra
authors.initials: U
authors.lastname: Nongthomba
authors.affiliation: the Department of Microbiology, Immunology, and Glycobiology, Institute of Laboratory Medicine, Lund University, 221 00 Lund, Sweden.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Godaly
authors.affiliation: the AU-KBC Research Center, MIT Campus, Anna University, Chromepet, Chennai, Tamil Nadu 600025, India.
authors.forename: Ramesh Kumar
authors.initials: RK
authors.lastname: Gopal
authors.affiliation: the AU-KBC Research Center, MIT Campus, Anna University, Chromepet, Chennai, Tamil Nadu 600025, India, the Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Sulagna
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: From the School of Biotechnology, Campus-11, KIIT University, Bhubaneswar, Orissa-751024, India, asonawane@kiitbiotech.ac.in.
authors.forename: Avinash
authors.initials: A
authors.lastname: Sonawane
mesh_terms: D000818:Animals; D017209:Apoptosis; D001343:Autophagy; D015153:Blotting, Western; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002473:Cell Wall / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D054884:Host-Pathogen Interactions; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability; D019910:Mycobacterium marinum / Q000254:growth & development / Q000472:pathogenicity*; D009169:Mycobacterium tuberculosis / Q000254:growth & development / Q000472:pathogenicity*; D016328:NF-kappa B; D054409:Nicotinamide Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D010587:Phagocytosis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D014376:Tuberculosis / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D014774:Virulence / Q000276:immunology; D015027:Zebrafish / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology
chemical_list: D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D054409:Nicotinamide Phosphoribosyltransferase
keywords: Mycobacterium marinum; Mycobacterium smegmatis; Mycobacterium tuberculosis; autophagy; macrophage; mimG; mitogen-activated protein kinase (MAPK); oxidative stress; phosphoribosyltransferase; zebrafish
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