A microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening.
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells.
Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille.
Christophe J
CJ
Queval
Ok-Ryul
OR
Song
Vincent
V
Delorme
Raffaella
R
Iantomasi
Romain
R
Veyron-Churlet
Nathalie
N
Deboosère
Valérie
V
Landry
Alain
A
Baulard
Priscille
P
Brodin
D056809:Alveolar Epithelial Cells / Q000382:microbiology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010641:Phenotype; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics; D014376:Tuberculosis / Q000382:microbiology
D034741:RNA, Small Interfering