Abstract
Tuberculosis is considered to be one of the world’s deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.
Highlights
Tuberculosis, a Serious Health Threat The negative impact that tuberculosis (TB) has on human health is hard to overestimate
A. castellanii was chosen instead of our D. discoideum model due to its ‘macrophage-like’ size that allows a higher level of bacteria uptake, detectable with our fluorescence plate reader
The percentage of infected cells at time zero and the average number of bacteria per cell is a function of the multiplicity of infection (MOI) (Figure 1A)
Summary
Tuberculosis, a Serious Health Threat The negative impact that tuberculosis (TB) has on human health is hard to overestimate. Over one third of the world population is infected by bacteria of the Mycobacterium tuberculosis (Mtb) complex. The hallmark of TB is the formation of granuloma, wellorganized multicellular structures primarily composed of mature macrophages and T-lymphocytes. Macrophages often develop into multinucleated giant cells and epitheloid cells. Granulomas contain dendritic cells, neutrophils, NK-cells, fibroblasts and Blymphocytes and are surrounded by a fibrous cuff. Epithelial cells surrounding granulomas are proposed to participate in its formation. It is generally assumed that the granuloma is a host-defensive structure that sequesters and eradicates pathogenic bacteria. Mtb mostly replicates in alveolar macrophages but can be found in dendritic cells, adipocytes and type II alveolar pneumocytes [3,4,5,6]
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