Dictyostelium Discoideum Research Articles

Yellow polyketide pigment suppresses premature hatching in social amoeba

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

The AraC-type transcription factor TagK is a new player in the signaling cascade that induces the anti-eukaryotic T6SS of Xanthomonas citri.

Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfKT51E ) to identify the EcfK regulon, which includes a previously uncharacterized transcription factor of the AraC-family (TagK), in addition to T6SS genes and genes encoding protein homeostasis factors. Functional studies demonstrated that TagK acts downstream of EcfK, binding directly to T6SS gene promoters and inducing T6SS expression in response to contact with amoeba cells. TagK controls a small regulon, consisting of the complete T6SS, its accessory genes and additional genes encoded within the T6SS cluster. We conclude that a singular regulatory circuit consisting of a transmembrane kinase (PknS), an alternative sigma factor (EcfK) and an AraC-type transcriptional regulator (TagK) promotes expression of the X. citri T6SS in response to a protozoan predator.

Spindly is a nucleocytosolic O-fucosyltransferase in Dictyostelium and related proteins are widespread in protists and bacteria.

O-GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O-GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii, and both enzymes are now recognized as O-fucosyltransferases (OFTs). Here we investigate the evolution of spy-like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium-a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma, the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation.

Understanding the Cell's Response to Chemical Signals: Utilisation of Microfluidic Technology in Studies of Cellular and Dictyostelium discoideum Chemotaxis.

Cellular chemotaxis has been the subject of a variety of studies due to its relevance in physiological processes, disease pathogenesis, and systems biology, among others. The migration of cells towards a chemical source remains a closely studied topic, with the Boyden chamber being one of the earlier techniques that has successfully studied cell chemotaxis. Despite its success, diffusion chambers such as these presented a number of problems, such as the quantification of many aspects of cell behaviour, the reproducibility of procedures, and measurement accuracy. The advent of microfluidic technology prompted more advanced studies of cell chemotaxis, usually involving the social amoeba Dictyostelium discoideum (D. discoideum) as a model organism because of its tendency to aggregate towards chemotactic agents and its similarities to higher eukaryotes. Microfluidic technology has made it possible for studies to look at chemotactic properties that would have been difficult to observe using classic diffusion chambers. Its flexibility and its ability to generate consistent concentration gradients remain some of its defining aspects, which will surely lead to an even better understanding of cell migratory behaviour and therefore many of its related biological processes. This paper first dives into a brief introduction of D. discoideum as a social organism and classical chemotaxis studies. It then moves to discuss early microfluidic devices, before diving into more recent and advanced microfluidic devices and their use with D. discoideum. The paper then closes with brief opinions about research progress in the field and where it will possibly lead in the future.

Deciphering the cytochrome P450 genes in the microbiome of a chronically polluted soil with history of agricultural activities

BackgroundCytochrome P450 monooxygenases (CYPs) are exciting biocatalysts that catalyzes diverse regio- and stereoselective reactions of a broad range of substrates. The cytochrome P450 genes (CYPomes) of a chronically polluted soil (3S) with history of agricultural activities were deciphered via functional annotation of putative ORFs (open reading frames) using KEGG KofamKOALA, PHMMER, the Cytochrome P450 Engineering Database CYPED v6.0, and the NCBI Batch Web CD-Search tool.ResultsAnnotation revealed the detection of seventy-seven CYP families and eight standalone CYPs cutting across the three domains of life. The prokaryote CYPome has 72 CYP families, 93 subfamilies, and seven standalone CYPs. The phylum Proteobacteria and the genera Streptomyces, Mycobacterium, and Bacillus with 17, 16, 24, and 5 CYP families were predominant, while the domain Archaea was represented by CYP119A1. The phylum Cyanobacteria has two families, while 23 actinobacterial CYPs (other than Streptomyces and Mycobacterium) were also detected. The detected prokaryote CYPs are responsible for biodegradation of camphor, hydroxylation of monoterpene alcohols, biosynthesis of secondary metabolites, and hydroxylation of fatty acids and steroidal compounds. The eukaryote CYPome was represented by seven fungal CYPs (CYP505A3, CYP505B1, CYP51A, CYP51C, CYP55A1, CYP55A2, and CYP55A3) from Acremonium egyptiacum, Fusarium oxysporum, Aspergillus oryzae, Gibberella moniliformis, Aspergillus flavus, and Fusarium lichenicola, respectively, and CYP524A1 from the slime mold, Dictyostelium discoideum. The fungi CYPs were involved in biosynthesis of secondary metabolites, hydroxylation of fatty acids, and nitrate reduction and denitrification.ConclusionsThis study has established the diverse roles played by CYPs in soil, its implication for soil health and resilience, and its potentials for industrial application.

Spontaneous transitions between amoeboid and keratocyte-like modes of migration.

The motility of adherent eukaryotic cells is driven by the dynamics of the actin cytoskeleton. Despite the common force-generating actin machinery, different cell types often show diverse modes of locomotion that differ in their shape dynamics, speed, and persistence of motion. Recently, experiments in Dictyostelium discoideum have revealed that different motility modes can be induced in this model organism, depending on genetic modifications, developmental conditions, and synthetic changes of intracellular signaling. Here, we report experimental evidence that in a mutated D. discoideum cell line with increased Ras activity, switches between two distinct migratory modes, the amoeboid and fan-shaped type of locomotion, can even spontaneously occur within the same cell. We observed and characterized repeated and reversible switchings between the two modes of locomotion, suggesting that they are distinct behavioral traits that coexist within the same cell. We adapted an established phenomenological motility model that combines a reaction-diffusion system for the intracellular dynamics with a dynamic phase field to account for our experimental findings.

ДВА КОНКУРИРУЮЩИХ МЕХАНИЗМА ПРИ АГРЕГАЦИИ DICTYOSTELIUM DISCOIDEUM

In the present work, the hybrid cellular automaton model of the initial stages of aggregation of the cellular slime mold Dictyostelium discoideum, proposed by the authors earlier, is modified by taking into account the dependence of myxamoebae movement on the intracellular Ca2+ concentration. The cellular automaton is a three-dimensional grid the cells of which are either empty or contain one amoeba, the state of the automaton being determined by the distribution of amoebae over the grid. At each time step, the state of the automaton changes according to fixed rules which are determined by solutions to systems of equations that describe the distribution of aggregation factors in the medium and their concentrations inside cells. Movements of amoebae can be either directed along the concentration gradient of cyclic adenosine monophosphate, or random walk. The calculation results show that when the aggregate reaches a certain size its growth stops and the number of cells in it fluctuates around the average value. Such behavior suggests that aggregation is determined by two competing mechanisms: one promotes the growth of the aggregate while the other promotes detachment of cells or small clusters from it.

The lectin Discoidin I acts in the cytoplasm to help assemble the contractile machinery.

Cellular functions, such as division and migration, require cells to undergo robust shape changes. Through their contractility machinery, cells also sense, respond, and adapt to their physical surroundings. In the cytoplasm, the contractility machinery organizes into higher order assemblies termed contractility kits (CKs). Using Dictyostelium discoideum, we previously identified Discoidin I (DscI), a classic secreted lectin, as a CK component through its physical interactions with the actin crosslinker Cortexillin I (CortI) and the scaffolding protein IQGAP2. Here, we find that DscI ensures robust cytokinesis through regulating intracellular components of the contractile machinery. Specifically, DscI is necessary for normal cytokinesis, cortical tension, membrane-cortex connections, and cortical distribution and mechanoresponsiveness of CortI. The dscI deletion mutants also have complex genetic epistatic relationships with CK components, acting as a genetic suppressor of cortI and iqgap1, but as an enhancer of iqgap2. This work underscores the fact that proteins like DiscI contribute in diverse ways to the activities necessary for optimal cell function.

ATP levels influence cell movement during the mound phase in Dictyostelium discoideum as revealed by ATP visualization and simulation.

Cell migration plays an important role in multicellular organism development. The cellular slime mold Dictyostelium discoideum is a useful model organism for the study of cell migration during development. Although cellular ATP levels are known to determine cell fate during development, the underlying mechanism remains unclear. Here, we report that ATP-rich cells efficiently move to the central tip region of the mound against rotational movement during the mound phase. A simulation analysis based on an agent-based model reproduces the movement of ATP-rich cells observed in the experiments. These findings indicate that ATP-rich cells have the ability to move against the bulk flow of cells, suggesting a mechanism by which high ATP levels determine the cell fate of differentiation.

Generating polyketide diversity in Dictyostelium: a Steely hybrid polyketide synthase produces alternate products at different developmental stages.

The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social amoeba Dictyostelium discoideum has a high genomic potential for producing polyketides to mediate its ecological interactions, including the unique ‘Steely’ enzymes, consisting of a fusion between a fatty acid synthase and a chalcone synthase. We report here that D. discoideum further increases its polyketide potential by using the StlB Steely enzyme, and a downstream chlorinating enzyme, to make both a chlorinated signal molecule, DIF-1, during its multi-cellular development, and a set of abundant polyketides in terminally differentiated stalk cells. We identify one of these as a chlorinated dibenzofuran with potent anti-bacterial activity. To do this, StlB switches expression from prespore to stalk cells in late development and is cleaved to release the chalcone synthase domain. Expression of this domain alone in StlB null cells allows synthesis of the stalk-associated, chlorinated polyketides. Thus, by altered expression and processing of StlB, cells make first a signal molecule, and then abundant secondary metabolites, which we speculate help to protect the mature spores from bacterial infection.

Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper.

First person – Ramesh Rijal

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Ramesh Rijal is first author on ‘ Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source’, published in JCS. Ramesh is a postdoc in the lab of Richard H. Gomer at the Department of Biology, Texas A&M University, College Station, TX, investigating polyphosphate biology at the host–microbe interface.

Reduced and Nonreduced Genomes in Paraburkholderia Symbionts of Social Amoebas.

ABSTRACTThe social amoeba Dictyostelium discoideum is a predatory soil protist frequently used for studying host-pathogen interactions. A subset of D. discoideum strains isolated from soil persistently carry symbiotic Paraburkholderia, recently formally described as P. agricolaris, P. bonniea, and P. hayleyella. The three facultative symbiont species of D. discoideum present a unique opportunity to study a naturally occurring symbiosis in a laboratory model protist. There is a large difference in genome size between P. agricolaris (8.7 million base pairs [Mbp]) versus P. hayleyella and P. bonniea (4.1 Mbp). We took a comparative genomics approach and compared the three genomes of D. discoideum symbionts to 12 additional Paraburkholderia genomes to test for genome evolution patterns that frequently accompany host adaptation. Overall, P. agricolaris is difficult to distinguish from other Paraburkholderia based on its genome size and content, but the reduced genomes of P. bonniea and P. hayleyella display characteristics indicative of genome streamlining rather than deterioration during adaptation to their protist hosts. In addition, D. discoideum-symbiont genomes have increased secretion system and motility genes that may mediate interactions with their host. Specifically, adjacent BurBor-like type 3 and T6SS-5-like type 6 secretion system operons shared among all three D. discoideum-symbiont genomes may be important for host interaction. Horizontal transfer of these secretion system operons within the amoeba host environment may have contributed to the unique ability of these symbionts to establish and maintain a symbiotic relationship with D. discoideum.IMPORTANCE Protists are a diverse group of typically single cell eukaryotes. Bacteria and archaea that form long-term symbiotic relationships with protists may evolve in additional ways than those in relationships with multicellular eukaryotes such as plants, animals, or fungi. Social amoebas are a predatory soil protist sometimes found with symbiotic bacteria living inside their cells. They present a unique opportunity to explore a naturally occurring symbiosis in a protist frequently used for studying host-pathogen interactions. We show that one amoeba-symbiont species is similar to other related bacteria in genome size and content, while the two reduced-genome-symbiont species show characteristics of genome streamlining rather than deterioration during adaptation to their host. We also identify sets of genes present in all three amoeba-symbiont genomes that are potentially used for host-symbiont interactions. Because the amoeba symbionts are distantly related, the amoeba host environment may be where these genes were shared among symbionts.

Submicron 3-D mass spectrometry imaging reveals an asymmetric molecular distribution on chemotaxing cells.

Background: Dictyostelium discoideum is a ~10 µm diameter unicellular eukaryote that lives on soil surfaces. When starved, D. discoideum cells aggregate into streams of cells in a process called chemotaxis. In this report, we studied D. discoideum cells during chemotaxis using 3D - mass spectrometry imaging (3D-MSI). Methods: The 3D-MSI consisted of the sequential generation of 2D molecular maps using burst alignment coupled to delayed extraction time-of flight secondary ion mass spectrometry (TOF-SIMS) combined with a soft sputtering beam to access the different layers. Results: Molecular maps with sub-cellular high spatial resolution (~300 nm) indicated the presence of ions at m/z = 221 and 236 at the front and sides, but reduced levels at the back, of cells moving toward of aggregation streams. The 3D-MSI also detected an ion at m/z = 240 at the edges and back, but reduced levels at the front, of aggregating cells. Other ions showed an even distribution across the cells. Conclusions: Together, these results demonstrate the utility of sub-micron MSI to study eukaryotic chemotaxis.

A novel gene, Le-Dd10, is involved in fruiting body formation of Lentinula edodes

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with an 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

The dawning of deiodinases: an outer ring deiodinase activity in the social amoeba dictyostelium discoideum with high affinity for reverse t3

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Autophagy of the somatic stalk cells nurses the propagating spores of Dictyostelid social amoebas

Background: Autophagy (self-feeding) assists survival of starving cells by partial self-digestion, while dormancy as cysts, spores or seeds enables long-term survival. Starving Dictyostelium amoebas construct multicellular fruiting bodies with spores and stalk cells, with many Dictyostelia still able to encyst individually like their single-celled ancestors. While autophagy mostly occurs in the somatic stalk cells, autophagy gene knock-outs in Dictyostelium discoideum (D. discoideum) formed no spores and lacked cAMP induction of prespore gene expression. Methods: To investigate whether autophagy also prevents encystation, we knocked-out autophagy genes atg5 and atg7 in the dictyostelid Polysphondylium pallidum, which forms both spores and cysts. We measured spore and cyst differentiation and viability in the knock-out as well as stalk and spore gene expression and its regulation by cAMP. We tested a hypothesis that spores require materials derived from autophagy in stalk cells. Sporulation requires secreted cAMP acting on receptors and intracellular cAMP acting on PKA. We compared the morphology and viability of spores developed in fruiting bodies with spores induced from single cells by stimulation with cAMP and 8Br-cAMP, a membrane-permeant PKA agonist. Results: Loss of autophagy in P. pallidum reduced but did not prevent encystation. However, spore, but not stalk differentiation, and cAMP-induced prespore gene expression were lost. Spores induced in vitro by cAMP and 8Br-cAMP were smaller and rounder than spores formed multicellularly and while they were not lysed by detergent they did not germinate, unlike multicellular spores. Conclusions: The stringent requirement of sporulation on both multicellularity and autophagy, which occurs mostly in stalk cells, suggests that stalk cells nurse the spores through autophagy. This highlights autophagy as a major cause for somatic cell evolution in early multicellularity.

Evolving social behavior through selection of single-cell adhesion in Dictyostelium discoideum

SummaryThe social amoeba Dictyostelium discoideum commonly forms chimeric fruiting bodies. Genetic variants that produce a higher proportion of spores are predicted to undercut multicellular organization unless cooperators assort positively. Cell adhesion is considered a primary factor driving such assortment, but evolution of adhesion has not been experimentally connected to changes in social performance. We modified by experimental evolution the efficiency of individual cells in attaching to a surface. Surprisingly, evolution appears to have produced social cooperators irrespective of whether stronger or weaker adhesion was selected. Quantification of reproductive success, cell-cell adhesion, and developmental patterns, however, revealed two distinct social behaviors, as captured when the classical metric for social success is generalized by considering clonal spore production. Our work shows that cell mechanical interactions can constrain the evolution of development and sociality in chimeras and that elucidation of proximate mechanisms is necessary to understand the ultimate emergence of multicellular organization.

Erratum: From single to collective motion of social amoebae: A computational study of interacting cells

Moreno E, Großmann R, Beta C and Alonso S (2022) From Single to Collective Motion of Social Amoebae: A Computational Study of Interacting Cells. Front. Phys. 9:750187. doi: 10.3389/fphy.2021.750187Due to a production error, the funder CONACYT to CB was erroneously omitted. The funder Deutsche Forschungsgemeinschaft (DFG), Project-ID 318763901-SFB1294 project B02, to CB and RG was erroneously omitted.The updated funding statement can be found below: SA and EM thank grant PGC2018-095456-B-I00 funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe", by the European Union. EM also acknowledges financial support from CONACYT. CB and RG acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG)-Project-ID 318763901-SFB1294, project B02.The publisher apologizes for this mistake.The original version of this article has been updated.

The polyketide synthase StlA is involved in inducing aggregation in Polysphondylium violaceum.

In the social amoeba Dictyostelium discoideum, the polyketide MPBD (4-methyl-5-pentylbenzene-1,3-diol) regulates the gene expressions of cAMP signaling to make cells aggregation-competent and also induces spore maturation. The polyketide synthase StlA is responsible for MPBD biosynthesis in D. discoideum and appears to be conserved throughout the major groups of the social amoeba (Dictyostelia). In this study, we analyzed the function of StlA in Polysphondylium violaceum by identifying the gene sequence and creating the knockout mutants. We found that Pv-stlA- mutants had defects only in cell aggregation but not in spore maturation, indicating that the function of StlA in inducing spore maturation is species-specific. We also found that MPBD could rescue the aggregation defect in Pv-stlA- mutants whereas the mutants normally exhibited chemotaxis to their chemoattractant, glorin. Our data suggest that StlA is involved in inducing aggregation in P. violaceum by acting on signaling pathways other than chemotaxis in P. violaceum.