High-Content Imaging of Mycobacterium Tuberculosis -Infected Macrophages: An In Vitro Model for Tuberculosis Drug Discovery

Abstract

Macrophages are reservoirs for replicating mycobacterium during tuberculosis (TB) infections. In this study, small molecules to be developed as anti-tubercular treatments were investigated for their ability to kill intracellular bacteria in in vitro macrophage models. High-content imaging technologies offer a high-throughput method to quantify a drug's ability to inhibit Mycobacterium tuberculosis intracellular invasion and multiplication in host cells. Dedicated image analysis enables the automated quantification of infected macrophages, and compounds that inhibit mycobacteria proliferation can be tested using this method. Furthermore, the implementation of the assay in 384-well microtiter plates combined with automated image acquisition and analysis allows large-scale screening of compound libraries in M. tuberculosis-infected macrophages. Here we describe a high-throughput and high-content workflow and detail its utility for the development of new TB drugs.

High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis

“…This model may help bridge the gap between the in vitro evaluation of MICs and costly in vivo efficacy studies in guinea pigs. In particular, compounds that are effective in the in vitro granuloma model could be prioritized for guinea pig studies.High-content screening (HCS) technology has been used to identify anti-TB compounds (12)(13)(14). This technology has several advantages over traditional phenotypic screening approaches.…”

“…High-content screening (HCS) technology has been used to identify anti-TB compounds (12)(13)(14). This technology has several advantages over traditional phenotypic screening approaches.…”

“…This technology has several advantages over traditional phenotypic screening approaches. This technology has mostly been used in single-cell experiments, because it is adapted to identify and analyze images (12,13); however, in this paper, we focus on the use of granulomas instead of single cells. In this report, we describe the development of a novel HCS method to evaluate the activities of reference compounds against a green fluorescent protein (GFP)-expressing H37Rv strain of Mycobacterium tuberculosis (MTB-GFP) within 5 days, and we compared the MIC 50 values with those of classical liquid cultures (extracellular), intracellular growth (single-cell assay), and within granulomas, all using the same bacterial strain.…”

“…We used INH as a reference drug against MTB-GFP as previously described for a singlecell assay (12,13). Dose-response curves were calculated with concentrations ranging from 1 ϫ 10 Ϫ9 M to 1 ϫ 10 Ϫ4 M, which were the same as those used for a previously reported single-cell assay (12).…”

“…2B). In addition, the single-cell assay results have been demonstrated to show a good correlation between the fluorescence counts and CFU counts obtained for reference compounds (12,13). However, HCS screening would never replace the CFU determination method, which is currently the only technique that enables the exact determination of intracellular bacterial load, even though it gives a faster determination of the ability of a given drug to kill bacteria and enables a faster determination of the MIC of a drug.…”