Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing.

Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or inhibit the growth of intracellular pathogens. Here, we used the soil amoeba Dictyostelium discoideum, a professional phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during phagocytosis of nonpathogenic bacteria and visualize the temporal and spatial dynamics of compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to phagosomes by fusion with 'zincosomes' of endosomal origin, and also by the action of one or more zinc transporters. We localized the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and the Golgi complex, and studied the impact of znt knockouts on zinc homeostasis. We show that zinc is delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli deficient in the zinc efflux P1B-type ATPase ZntA are killed faster than wild-type bacteria.

The ESCRT and autophagy machineries cooperate to repair ESX-1-dependent damage at the Mycobacterium-containing vacuole but have opposite impact on containing the infection.

Phagocytic cells capture and kill most invader microbes within the bactericidal phagosome, but some pathogens subvert killing by damaging the compartment and escaping to the cytosol. To prevent the leakage of pathogen virulence and host defence factors, as well as bacteria escape, host cells have to contain and repair the membrane damage, or finally eliminate the cytosolic bacteria. All eukaryotic cells engage various repair mechanisms to ensure plasma membrane integrity and proper compartmentalization of organelles, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy machineries. We show that during infection of Dictyostelium discoideum with Mycobacterium marinum, the ESCRT-I component Tsg101, the ESCRT-III protein Snf7/Chmp4/Vps32 and the AAA-ATPase Vps4 are recruited to sites of damage at the Mycobacterium-containing vacuole. Interestingly, damage separately recruits the ESCRT and the autophagy machineries. In addition, the recruitment of Vps32 and Vps4 to repair sterile membrane damage depends on Tsg101 but appears independent of Ca2+. Finally, in absence of Tsg101, M. marinum accesses prematurely the cytosol, where the autophagy machinery restricts its growth. We propose that ESCRT has an evolutionary conserved function to repair small membrane damage and to contain intracellular pathogens in intact compartments.

When Dicty Met Myco, a (Not So) Romantic Story about One Amoeba and Its Intracellular Pathogen.

In recent years, Dictyostelium discoideum has become an important model organism to study the cell biology of professional phagocytes. This amoeba not only shares many molecular features with mammalian macrophages, but most of its fundamental signal transduction pathways are conserved in humans. The broad range of existing genetic and biochemical tools, together with its suitability for cell culture and live microscopy, make D. discoideum an ideal and versatile laboratory organism. In this review, we focus on the use of D. discoideum as a phagocyte model for the study of mycobacterial infections, in particular Mycobacterium marinum. We look in detail at the intracellular cycle of M. marinum, from its uptake by D. discoideum to its active or passive egress into the extracellular medium. In addition, we describe the molecular mechanisms that both the mycobacterial invader and the amoeboid host have developed to fight against each other, and compare and contrast with those developed by mammalian phagocytes. Finally, we introduce the methods and specific tools that have been used so far to monitor the D. discoideum-M. marinum interaction.

Live imaging of Mycobacterium marinum infection in Dictyostelium discoideum.

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a recently established and powerful model system for mycobacterial infection. In this chapter, two simple protocols for live imaging of Dictyostelium discoideum infection are described. The first method is used to monitor the dynamics of recruitment of GFP-tagged Dictyostelium discoideum proteins at single time-points corresponding to the main stages of the infection (1.5-72 h post infection). The second method focuses at the early stages of the establishment of an infection (0-3 h post infection). In addition, several procedures to improve the imaging of the bacterium-containing compartment are described. Basic bacterial parameters such as bacterial growth and the recruitment of host proteins to the bacterium-containing compartment can be easily and precisely quantified using macros for ImageJ. These methods can be adapted to monitoring mycobacteria infection in other systems using mammalian cells.