Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System

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.

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

“…The intracellular growth of M. marinum wild-type, ΔmbtB, or ΔmbtB-ΔirtAB Hyg was determined in D. discoideum Ax3 or in A. castellanii as described previously (Arafah et al, 2013;Kicka et al, 2014;Koliwer-Brandl et al, 2019), and these protocols were adapted to determine intracellular replication in RAW 264.7 macrophages (Koliwer-Brandl et al, 2019). Briefly, the cells were adapted to the infectious conditions by placing the RAW 264.7 macrophages in Leibovitz/FCS medium for at least 4 days or the D. discoideum amoebae in sterilefiltered HL5-C for at least 1 day, respectively.…”

“…marinum is widely used as a versatile M. tuberculosis infection model (Arafah et al, 2013;Bouz & Al Hasawi, 2018;Cardenal-Munoz, Barisch, Lefrancois, Lopez-Jimenez, & Soldati, 2017;Hagedorn, Rohde, Russell, & Soldati, 2009;Hagedorn & Soldati, 2007;Lienard & Carlsson, 2017;Prouty, Correa, Barker, Jagadeeswaran, & Klose, 2003;Shiloh & Champion, 2010;Tükenmez et al, 2019). M. marinum can be handled at biosafety Level 2 and is appreciated for its relatively rapid growth (doubling time of 4-10 hr vs. >20 hr for M. tuberculosis; Clark & Shepard, 1963), the M. tuberculosis-like behaviour during intracellular infection, such as replication within a distinct Mycobacterium-containing vacuole (MCV; Barker, George, Falkow, & Small, 1997;Pozos & Ramakrishnan, 2004;Smith et al, 2008;Tobin & Ramakrishnan, 2008;López-Jiménez et al, 2018;Koliwer-Brandl et al, 2019), as well as its wide spectrum of host cells including macrophages (Barker et al, 1997;Bouley, Ghori, Mercer, Falkow, & Ramakrishnan, 2001;Koliwer-Brandl et al, 2019;Ramakrishnan, Federspiel, & Falkow, 2000) and the amoeba Dictyostelium discoideum (Cardenal-Munoz et al, 2017;Hagedorn et al, 2009;Koliwer-Brandl et al, 2019;Solomon, Leung, & Isberg, 2003) or Acanthamoeba castellanii (Harrison et al, 2013;Kicka et al, 2014).…”

Identification of novel anti-bacterial compounds from a chemically highly diverse pathways-based library using phenotypic screens in amoebae host models

“…Each of the three assays was established and optimized separately prior to testing the 1,255 compounds (Harrison et al 2013; Kicka et al 2014; Froquet et al 2009). The three screens were run simultaneously using the same compounds batch.…”

“…Interestingly, elucidation of the mode of action of hit compounds revealed emerging concepts in the field of host-pathogen interactions, demonstrating that intracellular bacteria proliferation can be affected by targeting bacterial metabolic pathways key to their intracellular life, but also by modulating host pathways (VanderVen et al 2015; Sundaramurthy et al 2014). Alternative in cellulo screening systems have also been established to take advantage of previously underexplored amoeba host-models (Kicka et al 2014; Harrison et al 2013). For example, D. discoideum and A. castellanii were used to characterize compounds of the GSK TB-set of anti-mycobacterial compounds (Trofimov et al 2018).…”

Adrenergic antagonists restrict replication of Legionella

“…Inhibition of Legionella by adrenergics et al, 2013; Kicka et al, 2014). In brief, A. castellanii were seeded to 96-well plates at 2|10 4 cells per plate and grown overnight, following which they were infected at an m.o.i.…”