The Dictyostelium discoideum - Mycobacterium marinum infection model, a powerful high throughput screening platform for anti-infective compounds

Tuberculosis is among the worlds deadliest diseases, causing approximately 2 million deaths annually. The urgent need for new antitubercular drugs has been intensified by the rise of drug-resistant strains. Despite recent advancements, most hits identified through traditional target-based screening exhibit limited efficacy in vivo. Consequently, there is a growing demand for whole-cell-based approaches that directly utilize host-pathogen systems. The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this article, the phenotypic host-pathogen protocol assay is presented here which relies on monitoring M. marinum during its infection of the amoeba D. discoideum. This assay is characterized by its scalability for high-throughput screening, robustness, and ease of manipulation, making it an effective system for compound screening. This system provides not only bacterial load readout via a bioluminescent M. marinum strain, but now also host survival and growth via a fluorescent D. discoideum strain enabling further host characterization by quantifying growth inhibition and potential cytotoxicity. Finally, the system was benchmarked with selected antibiotics and anti-infectives and calculated IC50s and MICs where applicable, demonstrating its capability to differentiate between antibiotics and anti-infective compounds. ImportanceThis methods paper introduces a robust, scalable, and high-throughput phenotypic host-pathogen assay based on the well-established Dictyostelium discoideum-Mycobacterium marinum system. In contrast to conventional target-based drug screening approaches, which often struggle to translate effectively in vivo, this platform directly monitors pathogen-host interactions, providing comprehensive insights into bacterial load, host survival, and potential cytotoxicity. By employing bioluminescent M. marinum and fluorescent D. discoideum strains, we validated the system using established antibiotics and anti-infective compounds, effectively distinguishing their effects through IC50 and MIC calculations.