Microbe Profile: Dictyostelium discoideum: model system for development, chemotaxis and biomedical research

The social amoeba Dictyostelium discoideum is a versatile organism that is unusual in alternating between single-celled and multi-celled forms. It possesses highly-developed systems for cell motility and chemotaxis, phagocytosis, and developmental pattern formation. As a soil amoeba growing on microorganisms, it is exposed to many potential pathogens; it thus provides fruitful ways of investigating host-pathogen interactions and is emerging as an influential model for biomedical research.

Ramifications of the diverse mRNA patterns in Acanthamoeba royreba

Free-living amoebae are distributed worldwide and can be found in a variety of environments. While most Acanthamoebae have been isolated from soil and water, A. royreba and A. culbertsoni were isolated from mammalian cell cultures. A. royreba’s isolation from placental and tumoral tissue, its unusual ability to grow in mammalian cell culture media at 35°C, and the presence of a primitive centriole, prompted us to investigate the potential for mammalian information in A. royreba. While some soil amoeba contain small amounts of fungal and bacterial information, presumably from the microbes they phagocytosed, the informational content of A. royreba, in some instances, was very different with > 70% of the mRNA being non-amoebic. Here we show that the proteins and mRNA content associated with A. royreba are extremely diverse and represent multiple Kingdoms, Orders, Phyla, and Genera from around the globe. The information in A. royreba, such as placental proteins from numerous mammals, the preponderance of non-amoebic mRNA, and its ability to tolerate harsh environments including Megarad irradiation, leads to a discussion regarding the possible role of this amoebae as an immunological gatekeeper protecting fetal or malignant tissue from destruction and its potential as a vehicle for panspermia.

A bleb initiation model for chemotaxis suggests a role for myosin II clusters and cortex rupture

Blebs, pressure driven protrusions of the plasma membrane, facilitate the movement of cells such as the soil amoeba Dictyostelium discoideum and other eukaryotes such as white blood cells and cancer cells. Blebs initiate or nucleate when proteins connecting the membrane to the cortex detach, either as a result of a rupture of the cortex or as a direct consequence of a build up in hydrostatic pressure. While linker detachment resulting from excess hydrostatic pressure is well understood, the mechanism by which cells rupture their cortex in locations of bleb formation is not so clear. Consequently, existing predictive models of bleb site selection do not account for it. To resolve this, we propose a model for bleb initiation which combines the geometric forces on the cell cortex/membrane complex with the underlying activity of actin binding proteins. In our model gaps, resulting from a rupture of the cortex, form at locations of high membrane energy where an accumulation of myosin II helps to weaken the cortex. We validate this model in part through a membrane energy functional which combines stresses on the cell boundary from membrane tension, curvature, membrane-cortex linker tension with hydrostatic pressure. Application of this functional to microscopy images of chemotaxing Dictyostelium discoideum cells identifies bleb nucleation sites at the highest energy locations 96.7% of the time. Sensitivity analysis of the model components points to membrane tension and hydrostatic pressure, all of which are regulated by myosin II, as critical to model predictability. Furthermore, microscopy reveals discrete clusters of myosin II along the leading edge of the cell, with blebs emerging from 80% of these sites. Together, our findings suggest a critical role for myosin II in bleb initiation through the formation of gaps and provides a predictive mathematical model for quantitative studies of blebbing.Author summaryEukaryotic cells such as white blood cells and cancer cells have been observed to move by making spherical herniation of their plasma membrane, referred to as blebs. The precise mechanism by which cells select locations around their boundary to initiate blebs is unclear. We hypothesize that blebs initiate at locations of high membrane energy where an accumulation of myosin II helps to rupture the cortex and/or detach linker proteins. We test this hypothesis by formulating a free energy functional representation of membrane energy to predict where blebs will initiate. The functional accounts for geometric forces due to membrane tension, curvature and membrane-cortex linker tension as well as hydrostatic pressure. Application of the functional to data from the soil amoeba, Dictyostelium disodium, identifies blebs at the highest energy locations over 90% of the time. Sensitivity analysis of model components points to membrane tension and hydrostatic pressure, all influenced by myosin II, as major forces driving bleb initiation. Additionally, we observe clusters of myosin II at locations of bleb initiation, further supporting its role in the process.

Dictyostelium discoideumflotillin homologues are essential for phagocytosis and participate in plasma membrane recycling and lysosome biogenesis

ABSTRACTThe metazoan flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins.Dictyostelium discoideum, a social soil amoeba, uses phagocytosis to digest, kill and feed on bacteria.D. discoideumpossesses three flotillin-like proteins, termed VacA, VacB and the recently identified VacC. All three vacuolins gradually accumulate on postlysosomes and, like flotillins, are strongly associated with membranes and partly with lipid rafts. Vacuolins are absolutely required for uptake of various particles. Their absence impairs particle recognition possibly because of defective recycling of plasma membrane or cortex-associated proteins. In addition, vacuolins are involved in phagolysosome biogenesis, although this does not impact digestion and killing of a wide range of bacteria. Furthermore, vacuolin knockout affects early recruitment of the WASH complex on phagosomes, suggesting that vacuolins may be involved in the WASH-dependent plasma membrane recycling. Altogether, these results indicate that vacuolins act as the functional homologues of flotillins inD. discoideum.

Predicting nucleation sites in chemotaxing Dictyostelium discoideum

Blebs, pressure driven protrusions of the plasma membrane, facilitate the movement of cell such as the soil amoeba Dictyostelium discoideum in a three dimensional environment. The goal of the article is to develop a means to predict nucleation sites. We accomplish this through an energy functional that includes the influence of cell membrane geometry (membrane curvature and tension), membrane-cortex linking protein lengths as well as local pressure differentials. We apply the resulting functional to the parameterized microscopy images of chemotaxing Dictyostelium cells. By restricting the functional to the cell boundary influenced by the cyclic AMP (cAMP) chemo-attractant (the cell anterior), we find that the next nucleation site ranks high in the top 10 energy values. More specifically, if we look only at the boundary segment defined by the extent of the expected bleb, then 96.8% of the highest energy sites identify the nucleation.Author summaryThis work concerns the prediction of nucleation sites in the soil amoeba-like Dictyostelium discoideum. We define a real valued functional combining input from cortex and membrane geometry such as membrane curvature and tension, cortex to membrane separation and local pressure differences. We show that the functional may be used to predict the location of bleb nucleation. In the region influenced by the cAMP gradient (the cell anterior), the next blebbing site lies in the ten highest energy functional values 70% of the time. The correctness increases to 96.8% provided we restrict attention to the segment in the general location of the next bleb. We verify these claims through the observation of microscopy images. The images are sequential at 1.66 and 0.8 seconds per image. We first identify the earliest sign of the bleb. We then use several observational factors to identify the nucleation site and estimate the corresponding location in the prior image.

Phagocyte chase behaviours: discrimination between Gram-negative and Gram-positive bacteria by amoebae

Phagocytes are cells that pursue, engulf and kill bacteria. They include macrophages and neutrophils of the mammalian immune system, as well as free-living amoebae that hunt and engulf bacteria for food. Phagocytosis can result in diverse outcomes, ranging from sustenance to infection and colonization by either pathogens or beneficial symbionts—and thus, discrimination may be necessary to seek out good bacteria while avoiding bad ones. Here we tested whether the soil amoeba Dictyostelium discoideum can discriminate among different types of bacteria using behavioural assays where amoebae were presented with paired choices of different bacteria. We observed variation in the extent to which the amoebae pursued different types of bacteria, as well as preferential migration towards Gram-negative compared with Gram-positive bacteria. Response profiles were similar for amoebae that originated from different geographical locations, suggesting that chase preference is conserved across much of the species range. While prior work has demonstrated that bacteria use chemotaxis to seek out amoebae they colonize, our work suggests that the opposite also occurs—amoebae can preferentially direct themselves to particular bacteria in the environment. Preferential sensing and response may help to explain why some amoeba–bacteria associations are more common in nature than others.

The past, present and future of Dictyostelium as a model system

The social amoeba Dictyostelium discoideum has been a preferred model organism during the last 50 years, particularly for the study of cell motility and chemotaxis, phagocytosis and macropinocytosis, intercellular adhesion, pattern formation, caspase-independent cell death and more recently autophagy and social evolution. Being a soil amoeba and professional phagocyte, thus exposed to a variety of potential pathogens, D. discoideum has also proven to be a powerful genetic and cellular model for investigating host-pathogen interactions and microbial infections. The finding that the Dictyostelium genome harbours several homologs of human genes responsible for a variety of diseases has stimulated their analysis, providing new insights into the mechanism of action of the encoded proteins and in some cases into the defect underlying the disease. Recent technological developments have covered the genetic gap between mammals and non-mammalian model organisms, challenging the modelling role of the latter. Is there a future for Dictyostelium discoideum as a model organism?

Intracellular Burkholderia Symbionts Induce Extracellular Secondary Infections; Driving Diverse Host Outcomes that Vary by Genotype and Environment

Symbiotic associations impact and are impacted by their surrounding ecosystem. The association between Burkholderia bacteria and the soil amoeba Dictyostelium discoideum is a tractable model to unravel the biology underlying symbiont-endowed phenotypes and their impacts. Several Burkholderia species stably associate with D. discoideum and typically reduce host fitness in food-rich environments while increasing fitness in food-scarce environments. Burkholderia symbionts are themselves inedible to their hosts but induce co-infections with secondary bacteria that can serve as a food source. Thus, Burkholderia hosts are “farmers” that carry food bacteria to new environments, providing a benefit when food is scarce. We examined the ability of specific Burkholderia genotypes to induce secondary co-infections and assessed host fitness under a range of co-infection conditions and environmental contexts. Although all Burkholderia symbionts intracellularly infected Dictyostelium, we found that co-infections are predominantly extracellular, suggesting that farming benefits are derived from extracellular infection of host structures. Furthermore, levels of secondary infection are linked to conditional host fitness; B. agricolaris infected hosts have the highest level of co-infection and have the highest fitness in food scarce environments. This study illuminates the phenomenon of co-infection induction across Dictyostelium associated Burkholderia species and exemplifies the contextual complexity of these associations.