title: High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.
abstract: The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.
authors.affiliation: Biology of Intracellular Pathogens Inserm Avenir Group, Institut Pasteur Korea, Seongbuk-gu, Seoul, Korea. pbrodin@ip-korea.org
authors.forename: Priscille
authors.initials: P
authors.lastname: Brodin
authors.forename: Yannick
authors.initials: Y
authors.lastname: Poquet
authors.forename: Florence
authors.initials: F
authors.lastname: Levillain
authors.forename: Isabelle
authors.initials: I
authors.lastname: Peguillet
authors.forename: Gerald
authors.initials: G
authors.lastname: Larrouy-Maumus
authors.forename: Martine
authors.initials: M
authors.lastname: Gilleron
authors.forename: Fanny
authors.initials: F
authors.lastname: Ewann
authors.forename: Thierry
authors.initials: T
authors.lastname: Christophe
authors.forename: Denis
authors.initials: D
authors.lastname: Fenistein
authors.forename: Jichan
authors.initials: J
authors.lastname: Jang
authors.forename: Mi-Seon
authors.initials: MS
authors.lastname: Jang
authors.forename: Sei-Jin
authors.initials: SJ
authors.lastname: Park
authors.forename: Jean
authors.initials: J
authors.lastname: Rauzier
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Carralot
authors.forename: Rachel
authors.initials: R
authors.lastname: Shrimpton
authors.forename: Auguste
authors.initials: A
authors.lastname: Genovesio
authors.forename: Jesus A
authors.initials: JA
authors.lastname: Gonzalo-Asensio
authors.forename: Germain
authors.initials: G
authors.lastname: Puzo
authors.forename: Carlos
authors.initials: C
authors.lastname: Martin
authors.forename: Roland
authors.initials: R
authors.lastname: Brosch
authors.forename: Graham R
authors.initials: GR
authors.lastname: Stewart
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gicquel
authors.forename: Olivier
authors.initials: O
authors.lastname: Neyrolles
mesh_terms: D000818:Animals; D005260:Female; D006017:Glycolipids / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000382:microbiology*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010587:Phagocytosis; D010588:Phagosomes / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014376:Tuberculosis / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology*
chemical_list: D006017:Glycolipids; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides