High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

PLoS Pathogens

Carlos Martin¹  Priscille Brodin¹  Auguste Genovesio¹  Jesus A. Gonzalo-Asensio¹  Sei-Jin Park¹  Rachel Shrimpton¹  Fanny Ewann¹  Florence Levillain²  Thierry Christophe²  Gerald Larrouy-Maumus²  Olivier Neyrolles²  Jichan Jang²  Brigitte Gicquel³  Roland Brosch⁴  Graham R. Stewart⁵  Jean-Philippe Carralot⁵  Jean Rauzier⁶  Yannick Poquet⁷  Germain Puzo⁸  Denis Fenistein⁹  Isabelle Peguillet⁹  Mi-Seon Jang⁹  Martine Gilleron⁹ 

[1] Biology of Intracellular Pathogens Inserm Avenir Group, Institut Pasteur Korea, Seongbuk-gu, Seoul, Korea;Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France;Department of Microbiology and Public Health, Faculty of Medicine, University of Zaragoza, Zaragoza, Spain;Division of Microbial Science, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom;Image Mining, Institut Pasteur Korea, Seongbuk-gu, Seoul, Korea;Screening Technologies and Pharmacology, Institut Pasteur Korea, Seongbuk-gu, Seoul, Korea;UP Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France;Unit of Mycobacterial Genetics, Institut Pasteur, Paris, France;Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France

关键词: Mycobacterium tuberculosis;    Phagosomes;    Macrophages;    Lipids;    Biosynthesis;    Genetic loci;    Transposable elements;    Vacuoles;   

DOI  :  10.1371/journal.ppat.1001100

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY