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

Abstract

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.

Keywords: Dictyostelium discoideum; Mycobacterium marinum; host-pathogen interactions; infection; methods; model organisms; phagocytosis.

Figure 1

M. marinum infection course in D. discoideum. M. marinum is phagocytosed by D. discoideum (1) and rapidly manipulates its phagocytic pathway to reside within a replicative niche (2). The ESX-1 secretion system of M. marinum perforates the MCV (3), which induces the recruitment of phagophores for membrane repair (4). M. marinum proliferates within its MCV (5), which finally breaks (6) and release mycobacteria to D. discoideum cytosol (7). Bacteria continue growing in the host cytosol (8) prior to egress by ejection (9.1), lytic death (9.2), or exocytosis (9.3). Phagophores are also recruited to the site of ejection for plasma membrane repair. Recapture into lysosome-like compartments may precede late exocytosis. However, this has not been shown yet in D. discoideum. Early exocytosis (9.3) can be induced upon starvation. Intercellular dissemination occurs after M. marinum release from the amoeba (10).

Figure 2

Establishment of the MCV during the first 12 h of infection. M. marinum is engulfed by an F-actin-positive phagocytic cup, resulting in an early phagosome that likely fuses sequentially with early and late endosomes. Rab5a is rapidly recycled from the MCV, which transiently acquires characteristics of a late endosome (Rab7a⁺, vATPase⁺, active TORC1⁺) but does not accumulate hydrolases. The undetectable level of hydrolases might result from defects in delivery, efficient recycling or leakage out of the MCV. In the case lysosomes do fuse with the MCV and deliver hydrolases, these might be retrieved by a mechanism dependent on WASH and Arp2/3 complexes. These complexes induce actin polymerization at the MCV, contributing to the retrieval of the vATPase and possibly hydrolases. In addition, M. marinum secretes ESAT-6 that damages the membrane, which renders the MCV leaky for ions and possibly lysosomal enzymes. Moreover, present evidence does not exclude the possibility that lysosomal fusion with the MCV is blocked. The membrane perforations induce an autophagic response in D. discoideum: TORC1 is inactivated and induces transcription of autophagy genes and formation of phagophores. The phagophores are recruited to the damaged MCV to repair the membrane and possibly deliver nutrients. M. marinum consequently survives and proliferates within a repaired and permissive MCV.

Figure 3

Lipid distribution and re-arrangement during infection. (1) Soon after bacteria uptake (10 min post-infection), host lipid droplets (LDs) are clustered around the MCV (Barisch et al., 2015b). (1b) Indicates the possibility of LDs capture and translocation via lipophagy (Barisch and Soldati, 2017a); (2) LDs might be imported into the MCV by a mechanism similar to phagosomal fusion; (3) Neutral lipids (and sterols) accumulate within the MCV at late infection stages; (4) Host phospholipids are transferred to the MCV by membrane trafficking (Barisch and Soldati, 2017b); (5) Host triacylglycerols (TAGs) and phospholipids serve as fatty acid (FA) source for bacterial intracytosolic lipid inclusion (ILI) formation (Barisch et al., 2015b); (6) Dgat2-positive LDs aggregate at bacteria poles as soon as the MCV breaks, leading to the coalescence of LDs onto the M. marinum cell wall and the complete surrounding of the bacteria by Dgat2 (Barisch and Soldati, 2017a); (7) The D. discoideum homolog of perilipin reaches M. marinum from the cytosol and targets the bacterial cell wall with the help of amphipathic and hydrophobic domains (Barisch et al., ; Barisch and Soldati, 2017b).