Setting Up and Monitoring an Infection of Dictyostelium discoideum with Mycobacteria

Abstract

Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. Therefore, M. marinum is used as a model for the study of mycobacterial infection in various host organisms. Recently, the Dictyostelium-M. marinum system has been shown to be a valuable model that recapitulates the main features of the intracellular fate of M. marinum including phagosome maturation arrest, as well as its particular cell-to-cell dissemination mode. We present here a "starter kit" of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantitatively the intracellular bacterial growth.

Mycobacterium marinum Degrades Both Triacylglycerols and Phospholipids from Its Dictyostelium Host to Synthesise Its Own Triacylglycerols and Generate Lipid Inclusions

“…Therefore, we tested bacteria growth and metabolic activity with the help of M . marinum expressing bacterial luciferase [32]. Interestingly, no statistical difference was observed between wild type and the dgat1&2 DKO (Fig 10A).…”

“…M . marinum expressing bacterial luciferase and the ΔRD1 mutant were generated in the Soldati laboratory [32] and were grown in the presence of 25 μg/ml kanamycin. Bacteria expressing the pCherry10 plasmid [56] were grown in medium supplemented with 100 μg/ml hygromycin.…”

“…marinum expressing bacterial luciferase as previously described [32]. Briefly, infected Dictyostelium cells (dilutions between 0.5 and 2.0 × 10 5 ) were plated on non-treated, white F96 MicroWell™ plates (Nunc) and covered with a gas permeable moisture barrier seal (Bioconcept).…”

Mycobacterium marinum antagonistically induces an autophagic response while repressing the autophagic flux in a TORC1- and ESX-1-dependent manner

“…Growth of luminescent bacteria was measured as described previously [62]. Briefly, 1.0 × 10 5 luxABCDE -expressing M .…”

Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model

“…Dictyostelium discoideum amoebae have proven a valuable non-mammalian host to study bacterial virulence and host resistance with human pathogens such as Legionella , Klebsiella , Mycobacteria [9–11], Pseudomonas (reviewed in [12, 13]), Vibrio cholera [14], and Salmonella typhimurium [15]. This system has also been used to identify compounds inhibiting bacterial infectivity [6, 8, 16].…”