The soil-dwelling social amoeba Dictyostelium discoideum feeds on bacteria. Each meal is a potential infection because some bacteria have evolved mechanisms to resist predation. To survive such a hostile environment, D. discoideum has in turn evolved efficient antimicrobial responses that are intertwined with phagocytosis and autophagy, its nutrient acquisition pathways. The core machinery and antimicrobial functions of these pathways are conserved in the mononuclear phagocytes of mammals, which mediate the initial, innate-immune response to infection. In this review, we discuss the advantages and relevance of D. discoideum as a model phagocyte to study cell-autonomous defenses. We cover the antimicrobial functions of phagocytosis and autophagy and describe the processes that create a microbicidal phagosome: acidification and delivery of lytic enzymes, generation of reactive oxygen species, and the regulation of Zn²⁺, Cu²⁺, and Fe²⁺ availability. High concentrations of metals poison microbes while metal sequestration inhibits their metabolic activity. We also describe microbial interference with these defenses and highlight observations made first in D. discoideum. Finally, we discuss galectins, TNF receptor-associated factors, tripartite motif-containing proteins, and signal transducers and activators of transcription, microbial restriction factors initially characterized in mammalian phagocytes that have either homologs or functional analogs in D. discoideum.

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. Upon uptake by a host cell, intracellular pathogens reside in a membranous compartment called phagosome. Within the phagosome, microbes are protected from the extracellular and cytosolic immune defences, whilst access to nutrients is limited. Some microbes gain access to the host cytosol by damaging the membrane of the phagosome, a step preceding egress and dissemination. Autophagy, a major catabolic pathway in eukaryotes, has been previously proposed to contribute to autonomous cell defence and to repair the membrane damage induced by intracellular pathogens. Here, we provide evidence that, in Dictyostelium discoideum, autophagy does not work alone in the containment of vacuolar mycobacteria, but it operates together with the Endosomal Sorting Complex Required for Transport (ESCRT), a protein machinery recently shown to repair endolysosomal damage. We demonstrate that the membrane perforations induced by the ESX-1 secretion system of Mycobacterium marinum are targeted by both ESCRT and autophagy, which seal the damaged vacuole. We propose that ESCRT might mend small membrane pores, whilst autophagy patches larger cumulative wounds. Interestingly, and contrary to what has been described in mammalian cells for ESCRT-dependent endolysosomal repair, in D. discoideum, repair of sterile membrane damage appears not to require Ca²⁺. The evolutionary conservation of the function of ESCRT in membrane repair suggests that this machinery plays an ancestral and widespread role to contain a broad range of intracellular pathogens.

During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for Mycobacterium tuberculosis. Mycobacteria can be found both inside a vacuole and the cytosol, but how this impacts their access to lipids is not well appreciated. Lipid droplets (LDs) store FAs in form of triacylglycerols (TAGs) and are energy reservoirs of prokaryotes and eukaryotes. Using the Dictyostelium discoideum/Mycobacterium marinum infection model we showed that M. marinum accesses host LDs to build up its own intracytosolic lipid inclusions (ILIs). Here, we show that host LDs aggregate at regions of the bacteria that become exposed to the cytosol, and appear to coalesce on their hydrophobic surface leading to a transfer of diacylglycerol O-acyltransferase 2 (Dgat2)-GFP onto the bacteria. Dictyostelium knockout mutants for both Dgat enzymes are unable to generate LDs. Instead, the excess of exogenous FAs is esterified predominantly into phospholipids, inducing uncontrolled proliferation of the endoplasmic reticulum (ER). Strikingly, in absence of host LDs, M. marinum alternatively exploits these phospholipids, resulting in rapid reversal of ER-proliferation. In addition, the bacteria are unable to restrict their acquisition of lipids from the dgat1&2 double knockout leading to vast accumulation of ILIs. Recent data indicate that the presence of ILIs is one of the characteristics of dormant mycobacteria. During Dictyostelium infection, ILI formation in M. marinum is not accompanied by a significant change in intracellular growth and a reduction in metabolic activity, thus providing evidence that storage of neutral lipids does not necessarily induce dormancy. Mycobacterium tuberculosis (Mtb) survives the human immune defence mechanisms leading to latent tuberculosis in one third of the world population. The ability to persist latently in human macrophages is due to a remarkable physiological change that is accompanied by a slowdown in replication, low metabolism, and phenotypic tolerance to antibiotics. It was proposed that fatty acids released from bacterial intracytosolic lipid inclusions (ILIs), a characteristic of dormant Mtb, serve as carbon source during reactivation from dormancy. We show here that bacteria accumulate ILIs even in a Dictyostelium mutant that is deficient in triacylglycerol synthesis and therefore incapable to build up lipid droplets. In addition, the accumulation of ILIs is not sufficient to induce a dormancy-like phenotype in M. marinum inside its host Dictyostelium. Moreover, we propose an alternative lipid transfer route from the host to the pathogen via degradation and recycling of host phospholipids.

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.