High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.
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.
Brodin
Priscille
P
Biology of Intracellular Pathogens Inserm Avenir Group, Institut Pasteur Korea, Seongbuk-gu, Seoul, Korea. pbrodin@ip-korea.org
Poquet
Yannick
Y
Levillain
Florence
F
Peguillet
Isabelle
I
Larrouy-Maumus
Gerald
G
Gilleron
Martine
M
Ewann
Fanny
F
Christophe
Thierry
T
Fenistein
Denis
D
Jang
Jichan
J
Jang
Mi-Seon
MS
Park
Sei-Jin
SJ
Rauzier
Jean
J
Carralot
Jean-Philippe
JP
Shrimpton
Rachel
R
Genovesio
Auguste
A
Gonzalo-Asensio
Jesus A
JA
Puzo
Germain
G
Martin
Carlos
C
Brosch
Roland
R
Stewart
Graham R
GR
Gicquel
Brigitte
B
Neyrolles
Olivier
O
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*
D006017:Glycolipids; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides