A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Abstract

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. Video LinkThe video component of this article can be found at

A Macrophage Infection Model to Predict Drug Efficacy AgainstMycobacterium Tuberculosis

“…More recently, this problem has been recognized and drug screening assays against nonreplicating, latent Mtb 8,9 or intracellular Mtb have been established, [10][11][12] but even these more advanced assays do not consider the penetration barriers that drugs encounter in the nonvascularized pulmonary lesions, or the necrotic foci at the site of infection. The ability to examine direct tissue penetration of drugs is only achieved using advanced experimental approaches and high-end instrumentation such as matrix-assisted laser desorption/ ionization mass spectrometry imaging.…”

Phenotypic assays for Mycobacterium tuberculosis infection

“…The adjustment of parameters for image acquisition as well as the miniaturization of infection assays has been exhaustively described in a previous study (25). The adjustment of parameters for image acquisition as well as the miniaturization of infection assays has been exhaustively described in a previous study (25).…”

Drug permeation and metabolism in Mycobacterium tuberculosis: Prioritising local exposure as essential criterion in new TB drug development

“…The use of in vitro macrophage culture to represent Mtb’s intracellular environment has shown some success with the identification of compound Q203 (86). Similar assays have been undertaken in various cell types including mouse-derived macrophages and epithelial cells, with a limited number of studies having used cell lines such as the human-derived THP-1 macrophage-like cells (60,115,116). Progress in the use of metabolomics to obtain PK/PD information from treated mycobacterial cells (117,118) has been especially useful in determining the systems-level impact of drug treatment on Mtb physiology, and has the capacity to provide key insights into the mechanisms of action of new (117,119) and even known (118,120,121) anti-TB drugs.…”