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

Priscille Brodin,Fanny Ewann,Roland Brosch,Jean-Philippe Carralot,Jean Rauzier,Brigitte Gicquel,Martine Gilleron,Graham R Stewart,Thierry Christophe,Denis Fenistein,Mi-Seon Jang,Germain Puzo,Rachel Shrimpton,Jichan Jang,Olivier Neyrolles,Auguste Genovesio,Florence Levillain,Yannick Poquet,Jesús Gonzalo-Asensio ,Gerald Larrouy‐Maumus ,Isabelle Péguillet ,Seijin Park ,Carlos Martı́n

doi:10.1371/journal.ppat.1001100

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.

Highlights

Upon engulfment by host macrophages, M. tuberculosis, the main etiological agent of tuberculosis in humans, localizes in vacuoles or phagosomes that fail to fuse with host cell lysosomes and to acidify [1,2]
One of the major virulence mechanisms of the tuberculosis bacillus, Mycobacterium tuberculosis, is its ability to resist killing by phagocytic cells of the host immune system, namely the macrophages
To identify mycobacterial genes involved in phagosome maturation arrest, we developed a novel high-throughput technology based on automated confocal microscopy

Summary

Introduction

Upon engulfment by host macrophages, M. tuberculosis, the main etiological agent of tuberculosis in humans, localizes in vacuoles or phagosomes that fail to fuse with host cell lysosomes and to acidify [1,2]. This process, called phagosome maturation arrest, may contribute to mycobacterial pathogenicity and to mycobacterial evasion from host immune surveillance. Apart from focused, candidate gene-based studies [8,9], large scale approaches have been performed using mutant libraries to obtain a comprehensive picture of the process. By use of flow cytometry, Stewart et al

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Conclusion