Dictyostelium a Professional Phagocyte at the Border of Multicellularity

Dictyostelium cells are so-called "social amoebae" which live in the forest soil, feed on bacteria by phagocytosis and proliferate by binary fission [6]. As the food supply diminishes, the haploid cells stop growing and enter the social phase oftheir life cycle by assembling into multicellular aggregates. In a complex developmental process, approximately 105 cells form a migrating slug and finally a fruiting body, consisting of dead stalk cells and spores.

The phagocytic potential of Dictyostelium cells gradually declines when cells enter the multicellular stage. However, starving preaggregating or aggregating cells fully revert to the phagocytic stage when exposed to bacteria [7]. At the migrating slug stage "sentinel cells" may represent a reservoir of immune-like cells that engulf bacteria and sequester toxins while circulating within the slug [8]. This apparent innate immune function active during multicellular development is further substantiated by the occurrence of potential homologs to signaling proteins involved in the innate immunity ofmulticellular animals or plants, including Toll-like interleukin receptor (TIR) domain-containing proteins, WRKY transcription factors and potential leucine-rich repeat (LRR) domain receptors [8].

Dictyostelium cells are considered "professional phagocytes," as they ingest a large variety of bacteria, yeast, apoptotic cells, as well as inert particles. The phagocytosis capacity of Dictyostelium in the single cell growth phase exceeds that of neutrophils several fold, each cell being able to digest about 300 bacteria per hour. Both fluid-phase endocytosis and phagocytosis in Dictyostelium cells have been reviewed in detail [9-11].

In Dictyostelium, the first step in phagosome biogenesis is initiated by binding of the particle to cell surface receptors, which leads to rearrangement of the actin cortex, progressive surrounding of the particle by the plasma membrane and ingestion of the newly produced vesicle. Particle binding and phagocytic cup formation are inhibited in the Phg2-null mutant defective in a serine/threonine kinase that is constitutively associated to the plasma membrane [12].

Actin assembly and reorganization are known to be controlled by signals originating at the site of particle attachment and transmitted to the cell interior by hetero-trimeric and monomeric G proteins of the Ras and Rac families, membrane lipids generated by the activity of phospholipases, phosphatidylinositol kinases and phosphatases, and calcium ions [13]. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) appears to be important in both activation and localization ofactin nucleation factors and actin-binding proteins, such as profilin, coronin, gelsolin, DAPI1 or CAP. Concomitant with the disassembly of the actin coat from the internalized phagosome, a decrease of PI(4,5)P2 has been observed.

Phagocytosis in D. discoideum is efficiently inhibited by phospholipase C (PLC) inhibitors and intracellular calcium chelators [14, 15]. It has been proposed that the endoplasmic reticulum (ER), which has been shown to come into close association with phagosomes during uptake, may be required for local release of calcium. This calcium increase in turn may stimulate PLC activity and activate proteins involved in phagosomal membrane tethering and fusion [16, 17].

During matuarion, the phagosome sequentially fuses with acidic vesicles of the endocytic pathway. Early phagocytic steps are characterized by recruitment of the V-H + -ATPase to the phagosome, shedding of the actin coat and delivery of proteins regulating vesicle fusion, such as the small G protein Rab7, the soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) Vti1, syntaxin 7, syntaxin 8 and the lysosomal marker LmpB. A second step in the maturation of phagosomes, starting between 3 and 15 min post uptake, is characterized by recruitment of lysosomal enzymes, such as cathepsin D and cysteine proteinases CP-p34, together with lysosomal membrane markers, such as the CD36/LIMP family members LmpA and LmpC, and the SNARE VAMP7. This is followed by the acquisition of lysosomal glycosidases, such as a-mannosidase and b-glucosidase. The post-lysosomal marker protein vacuolin B appears 60-90 min post internalization and indicates the late phase of phagosome maturation [18].

The cycle of phagosome biogenesis is considered complete when undigested material is excreted, though retrieval and recycling of several membrane components has been shown to occur from the very early steps [19]. Since post-lysosomal vesicles are recoated with actin before excretion, a close link between heterotrimeric G protein and actin reorganization at different steps of the phagocytic process is suggested [20].

The sequence of events presented here is consistent with a half-life of 45 min for degradation of bacteria [21]. The killing and digestion of the internalized bacteria is achieved by production of toxic oxygen radicals (respiratory burst), acidification of the phagosome lumen, depletion of essential divalent metals, degradation ofthe bacterial cell wall and digestion of cytoplasmic compounds by hydrolytic enzymes [22, 23]. Since the Dictyostelium genome contains several genes encoding for lysozyme homologs and a large family of pore-forming peptides, homologous to amoebapores and naegleriapores, further bactericidal effectors may be active in these amoebae.

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