Amoeba Dictyostelium Discoideum Research Articles

Exploring the Properties of Unilamellar Vesicle Bilayers Formed by Ionic Liquid Surfactants for Future Applications in Nanomedicine.

Two ionic liquids (ILs) with amphiphilic properties composed of 1-butyl-3-methylimidazolium dioctylsulfosuccinate (bmim-AOT) and 1-hexyl-3-methylimidazolium dioctylsulfosuccinate (hmim-AOT) form unilamellar vesicles spontaneously simply by dissolving the IL-like surfactant in water. These novel vesicles were characterized using two different and highly sensitive fluorescent probes: 6-propionyl-2-(dimethylaminonaphthalene) (PRODAN) and trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (HC). These fluorescent probes provide information about the physicochemical properties of the bilayer, such as micropolarity, microviscosity, and electron-donor capacity. In addition, the biocompatibility of these vesicles with the blood medium was evaluated, and their toxicity was determined using Dictyostelium discoideum amoebas. First, using PRODAN and HC, it was found that the bilayer composition and the chemical structure of the ions at the interface produced differences between both amphiphiles, making the vesicles different. Thus, the bilayer of hmim-AOT vesicles is less polar, more rigid, and has a lower electron-donor capacity than those made by bmim-AOT. Finally, the results obtained from the hemolysis studies and the growth behavior of unicellular amoebas, particularly utilizing the D. discoideum assay, showed that both vesicular systems do not produce toxic effects up to a concentration of 0.02 mg/mL. This elegant assay, devoid of animal usage, highlights the potential of these newly organized systems for the delivery of drugs and bioactive molecules of different polarities.

Switch-2 determines Mg2+ADP-release kinetics and fine-tunes the duty ratio of Dictyostelium class-1 myosins.

Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act as nucleotide sensors, can substantially determine the kinetic signature of a myosin, i.e., to either perform fast movement or enable long-range transport and tension generation. Switch-2 essentially contributes to the ATP hydrolysis reaction and determines product release. With few exceptions, class-1 myosin harbor a tyrosine in the switch-2 consensus sequence DIYGFE, at a position where class-2 myosins and a selection of myosins from other classes have a substitution. Here, we addressed the role of the tyrosine in switch-2 of class-1 myosins as potential determinant of the duty ratio. We generated constitutively active motor domain constructs of two class-1 myosins from the social amoeba Dictyostelium discoideum, namely, Myo1E, a high duty ratio myosin and Myo1B, a low duty ratio myosin. In Myo1E we introduced mutation Y388F and in Myo1B mutation F387Y. The detailed functional characterization by steady-state and transient kinetic experiments, combined with in vitro motility and landing assays revealed an almost reciprocal relationship of a number of critical kinetic parameters and equilibrium constants between wild-type and mutants that dictate the lifetime of the strongly actin-attached states of myosin. The Y-to-F mutation increased the duty ratio of Moy1B by almost one order of magnitude, while the introduction of the phenylalanine in switch-2 of Myo1E transformed the myosin into a low duty ratio motor. These data together with structural considerations propose a role of switch-2 in fine-tuning ADP release through a mechanism, where the class-specific tyrosine together with surrounding residues contributes to the coordination of Mg2+ and ADP. Our results highlight the importance of conserved switch-2 residues in class-1 myosins for efficient chemo-mechanical coupling, revealing that switch-2 is important to adjust the duty ratio of the amoeboid class-1 myosins for performing movement, transport or gating functions.

Dictyostelium discoideum -assisted pharmacognosy of plant resources for discovering antivirulence molecules targeting Klebsiella pneumoniae

The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to confront this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is a potent model organism in this effort. Employing this invertebrate model, we introduce a novel perspective to investigate natural plant extracts in search of molecules with potential antivirulence activity. Our work established an easy-scalable developmental assay targeting a virulent strain of Klebsiella pneumoniae, with Helenium aromaticum as the representative plant. The main objective was to identify tentative compounds from the Helenium aromaticum extract that attenuate the virulence of K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum fulfilled these prerequisites compared to the dichloromethane extract. Using UHPLC Q/Orbitrap/ESI/MS/MS, 63 compounds were tentatively identified in both extracts, 47 in the methanolic and 29 in the dichloromethane, with 13 compounds in common. This research underscores the potential of employing D. discoideum-assisted pharmacognosy to discover new antivirulence agents against multidrug-resistant pathogens.

Prestalk-like positioning of de-differentiated cells in the social amoeba Dictyostelium discoideum

The social amoeba Dictyostelium discoideum switches between solitary growth and social fruitification depending on nutrient availability. Under starvation, cells aggregate and form fruiting bodies consisting of spores and altruistic stalk cells. Once cells socially committed, they complete fruitification, even if a new source of nutrients becomes available. This social commitment is puzzling because it hinders individual cells from resuming solitary growth quickly. One idea posits that traits that facilitate premature de-commitment are hindered from being selected. We studied outcomes of the premature de-commitment through forced refeeding. Our results show that when refed cells interacted with non-refed cells, some of them became solitary, whereas a fraction was redirected to the altruistic stalk, regardless of their original fate. The refed cells exhibited reduced cohesiveness and were sorted out during morphogenesis. Our findings provide an insight into a division of labor of the social amoeba, in which less cohesive individuals become altruists.

In the social amoeba Dictyostelium discoideum, shortened stalks may limit obligate cheater success even when exploitable partners are available.

Cooperation is widespread across life, but its existence can be threatened by exploitation. The rise of obligate social cheaters that are incapable of contributing to a necessary cooperative function can lead to the loss of that function. In the social amoeba Dictyostelium discoideum, obligate social cheaters cannot form dead stalk cells and in chimeras instead form living spore cells. This gives them a competitive advantage within chimeras. However, obligate cheaters of this kind have thus far not been found in nature, probably because they are often enough in clonal populations that they need to retain the ability to produce stalks. In this study we discovered an additional cost to obligate cheaters. Even when there are wild-type cells to parasitize, the chimeric fruiting bodies that result have shorter stalks and these are disadvantaged in spore dispersal. The inability of obligate cheaters to form fruiting bodies when they are on their own combined with the lower functionality of fruiting bodies when they are not represent limits on obligate social cheating as a strategy.

Cell scientist to watch – Dan Dickinson

ABSTRACT Dan Dickinson is an Assistant Professor in Molecular Biosciences at the University of Texas at Austin (UT Austin), USA. He completed his doctoral studies at Stanford University in California, co-mentored by Prof. Bill Weis and Prof. James Nelson, where he became fascinated with cell polarity in the amoeba Dictyostelium discoideum. He next moved to Prof. Bob Goldstein's lab at the University of North Carolina at Chapel Hill for his postdoctoral research, where he developed a single-cell method to probe protein interactions in Caenorhabditis elegans zygotes. In 2017, he started his own lab at UT Austin. Intrigued by capacity of proteins to generate complex patterns across scales, his group aims to close the gap between biochemistry and cell biology using single-cell methods to investigate cell polarity in diverse contexts. For our Special Issue on Cell and Tissue Polarity, we spoke with Dan over Zoom about his goals as a researcher and educator and his dedication to teaching students the joy of scientific inquiry.

Temporal genome-wide fitness analysis of Mycobacterium marinum during infection reveals the genetic requirement for virulence and survival in amoebae and microglial cells.

Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A key feature of pathogenesis, conserved between the human pathogen Mycobacterium tuberculosis and the model pathogen Mycobacterium marinum, is the metabolic switch to lipid catabolism and altered expression of virulence genes at different stages of infection. This study aims to identify genes involved in sustaining viable intracellular infection. We applied transposon sequencing (Tn-Seq) to M. marinum, an unbiased genome-wide strategy combining saturation insertional mutagenesis and high-throughput sequencing. This approach allowed us to identify the localization and relative abundance of insertions in pools of transposon mutants. Gene essentiality and fitness cost of mutations were quantitatively compared between in vitro growth and different stages of infection in two evolutionary distinct phagocytes, the amoeba Dictyostelium discoideum and the murine BV2 microglial cells. In the M. marinum genome, 57% of TA sites were disrupted and 568 genes (10.2%) were essential, which is comparable to previous Tn-Seq studies on M. tuberculosis and M. bovis. Major pathways involved in the survival of M. marinum during infection of D. discoideum are related to DNA damage repair, lipid and vitamin metabolism, the type VII secretion system (T7SS) ESX-1, and the Mce1 lipid transport system. These pathways, except Mce1 and some glycolytic enzymes, were similarly affected in BV2 cells. These differences suggest subtly distinct nutrient availability or requirement in different host cells despite the known predominant use of lipids in both amoeba and microglial cells.IMPORTANCEThe emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system Dictyostelium discoideum/Mycobacterium marinum to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for M. marinum to identify conservation of virulence pathways between hosts.

Inorganic Polyphosphate Affects Biofilm Assembly, Capsule Formation, and Virulence of Hypervirulent ST23 Klebsiella pneumoniae.

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to high mortality rates and propensity to cause severe community-acquired infections in healthy individuals. The ability to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Polyphosphate kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a proteomic analysis to gain further insights into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins involved in capsule synthesis (Wzi-Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB-LpxC-PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hvKP. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.

Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.

Discovery and Quantitative Dissection of Cytokinesis Mechanisms Using Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.

Inflicting, Monitoring, Visualizing, and Quantitating Various Sterile Membrane Damages and the Repair Response in Dictyostelium discoideum.

Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.

Reduced social function in experimentally evolved Dictyostelium discoideum implies selection for social conflict in nature.

Many microbes interact with one another, but the difficulty of directly observing these interactions in nature makes interpreting their adaptive value complicated. The social amoeba Dictyostelium discoideum forms aggregates wherein some cells are sacrificed for the benefit of others. Within chimaeric aggregates containing multiple unrelated lineages, cheaters can gain an advantage by undercontributing, but the extent to which wild D. discoideum has adapted to cheat is not fully clear. In this study, we experimentally evolved D. discoideum in an environment where there were no selective pressures to cheat or resist cheating in chimaeras. Dictyostelium discoideum lines grown in this environment evolved reduced competitiveness within chimaeric aggregates and reduced ability to migrate during the slug stage. By contrast, we did not observe a reduction in cell number, a trait for which selection was not relaxed. The observed loss of traits that our laboratory conditions had made irrelevant suggests that these traits were adaptations driven and maintained by selective pressures D. discoideum faces in its natural environment. Our results suggest that D. discoideum faces social conflict in nature, and illustrate a general approach that could be applied to searching for social or non-social adaptations in other microbes.

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix.

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time is developed. As a proof of concept, real-time imaging of inter-cellular cyclic adenosine 3',5'-monophosphate (cAMP) signals in Dictyostelium discoideum amoeba cells is performed. A set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals is engineered. By combining multiple switches with different dynamic ranges, measure cAMP concentrations spanning three orders of magnitude in a single experiment can be measured. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements are achieved. Using different aptamer switches, this approach can be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

Extracellular and intracellular destruction of Pseudomonas aeruginosa by Dictyostelium discoideum phagocytes mobilize different antibacterial mechanisms.

Ingestion and killing of bacteria by phagocytic cells are critical processes to protect the human body from bacterial infections. In addition, some immune cells (neutrophils, NK cells) can release microbicidal molecules in the extracellular medium to eliminate non-ingested microorganism. Molecular mechanisms involved in the resulting intracellular and extracellular killing are still poorly understood. In this study, we used the amoeba Dictyostelium discoideum as a model phagocyte to investigate the mechanisms allowing intracellular and extracellular killing of Pseudomonas aeruginosa. When a D. discoideum cell establishes a close contact with a P. aeruginosa bacterium, it can either ingest it and kill it in phagosomes, or kill it extracellularly, allowing a direct side-by-side comparison of these two killing modalities. Efficient intracellular destruction of P. aeruginosa requires the presence of the Kil2 pump in the phagosomal membrane. On the contrary, extracellular lysis is independent on Kil2 but requires the expression of the superoxide-producing protein NoxA, and the extracellular release of the AplA bacteriolytic protein. These results shed new light on the molecular mechanisms allowing elimination of P. aeruginosa bacteria by phagocytic cells.

Adaptive pathfinding by nucleokinesis during amoeboid migration.

Motile cells encounter microenvironments with locally heterogeneous mechanochemical composition. Individual compositional parameters, such as chemokines and extracellular matrix pore sizes, are well known to provide guidance cues for pathfinding. However, motile cells face diverse cues at the same time, raising the question of how they respond to multiple and potentially competing signals on their paths. Here, we reveal that amoeboid cells require nuclear repositioning, termed nucleokinesis, for adaptive pathfinding in heterogeneous mechanochemical micro-environments. Using mammalian immune cells and the amoeba Dictyostelium discoideum, we discover that frequent, rapid and long-distance nucleokinesis is a basic component of amoeboid pathfinding, enabling cells to reorientate quickly between locally competing cues. Amoeboid nucleokinesis comprises a two-step polarity switch and is driven by myosin-II forces that readjust the nuclear to the cellular path. Impaired nucleokinesis distorts path adaptions and causes cellular arrest in the microenvironment. Our findings establish that nucleokinesis is required for amoeboid cell navigation. Given that many immune cells, amoebae, and some cancer cells utilize an amoeboid migration strategy, these results suggest that nucleokinesis underlies cellular navigation during unicellular biology, immunity, and disease.

Predation-resistant Pseudomonas bacteria engage in symbiont-like behavior with the social amoeba Dictyostelium discoideum

The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from transiently infected wild D. discoideum for ability to escape predation and infect D. discoideum fruiting bodies. Three predation-resistant strains frequently caused extracellular infections of fruiting bodies but were not found within spores. Furthermore, infection by one of these species induces secondary infections and suppresses predation of otherwise edible bacteria. Another strain can persist inside of amoebae after being phagocytosed but is rarely taken up. We sequenced isolate genomes and discovered that predation-resistant isolates are not monophyletic. Many Pseudomonas isolates encode secretion systems and toxins known to improve resistance to phagocytosis in other species, as well as diverse secondary metabolite biosynthetic gene clusters that may contribute to predation resistance. However, the distribution of these genes alone cannot explain why some strains are edible and others are not. Each lineage may employ a unique mechanism for resistance.

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E.coli OMVs trigger luminescence of the V.cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D.discoideum migration. L.pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L.pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L.pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.

Parallel evolution of the G protein-coupled receptor GrlG and the loss of fruiting body formation in the social amoeba Dictyostelium discoideum evolved under low relatedness.

Aggregative multicellularity relies on cooperation among formerly independent cells to form a multicellular body. Previous work with Dictyostelium discoideum showed that experimental evolution under low relatedness profoundly decreased cooperation, as indicated by the loss of fruiting body formation in many clones and an increase of cheaters that contribute proportionally more to spores than to the dead stalk. Using whole-genome sequencing and variant analysis of these lines, we identified 38 single nucleotide polymorphisms in 29 genes. Each gene had 1 variant except for grlG (encoding a G protein-coupled receptor), which had 10 unique SNPs and 5 structural variants. Variants in the 5' half of grlG-the region encoding the signal peptide and the extracellular binding domain-were significantly associated with the loss of fruiting body formation; the association was not significant in the 3' half of the gene. These results suggest that the loss of grlG was adaptive under low relatedness and that at least the 5' half of the gene is important for cooperation and multicellular development. This is surprising given some previous evidence that grlG encodes a folate receptor involved in predation, which occurs only during the single-celled stage. However, non-fruiting mutants showed little increase in a parallel evolution experiment where the multicellular stage was prevented from happening. This shows that non-fruiting mutants are not generally selected by any predation advantage but rather by something-likely cheating-during the multicellular stage.

Soil physical structure drives N-glycan mediated trophic interactions in soil amoebae: Mechanisms and environmental implications

Soil protozoa are an essential part of the terrestrial ecosystem, playing a vital role in the global element cycling and energy flow. However, one research gap is what are the key factors driving their diversity and environmental fates. In this study, we hypothesized that soil texture could affect soil protozoa's predation and their interactions with environmental pollutants, and we tested it by using a soil amoeba Dictyostelium discoideum as a model system. We found that soil texture affected amoeba's growth and development. In addition, environmental factors cannot explain the variation of amoeba's fitness in different soil textures. Soil sandy particles and water content rather than particle size contribute to amoeba's fitness. Furthermore, different soil textures induced distinct transcriptional responses to amoebae, especially N-glycan-related and multiple signaling pathways and the expression of key genes (e.g., Ras superfamily, cxgE, trap1). The expression of N-glycan-related pathways, which is positively correlated with amoeba predation, was inhibited in sand soil, decreasing amoeba's fitness. Finally, the results showed that soil texture also affects amoeba's interaction with environmental pollutants. In conclusion, this study shows that soil physical structures affect amoeba's interactions with bacteria and environmental pollutants. SynopsisSoil texture affects soil protozoa's growth and development and their interactions with environmental pollutants.

The Dictyostelium discoideum FimA protein, unlike yeast and plant fimbrins, is regulated by calcium similar to mammalian plastins

Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory ‘switch helix’ that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.