Aggregation Of Amoebae Research Articles

Amoeba-inspired magnetic microgel assembly assisted by engineered dextran-binding protein for vaccination against life-threatening systemic infection.

Vaccination is critical for population protection from pathogenic infections. However, its efficiency is frequently compromised by a failure of antigen retention and presentation. Herein, we designed a dextran-binding protein DexBP, which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A, together with the sequence of the fluorescent protein mCherry. DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles. The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol) framework and a reactive oxygen species-responsive linker, obtaining magnetic polymeric microgels for carrying pathogen antigen. Similar to amoeba aggregation, the microgels self-assembled to form aggregates and further induced dendritic cell aggregation. This step-by-step assembly retained antigens at lymph nodes, promoted antigen presentation, stimulated humoral immunity, and protected the mice from life-threatening systemic infections. This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections.Electronic Supplementary MaterialSupplementary material (AFM image and zeta potential of MG; TEM, FT-IR, DLS, and zeta potential of MNP−DexBP; zeta potential of MG+CaAg and MG+MNP−DexBP+CaAg; antigen release profile of MG+CaAg and MG+MNP−DexBP+CaAg; aggregation and dispersion of dendritic cells induced by MG+MNP−DexBP+CaAg; uptake of FITC-labeled CaAg (fCaAg) and intracellular distribution of fCaAg in the dendritic cells; antigen retention and dendritic cell activation in lymph nodes; and serum anti-CaAg antibody levels on day 3 after C. albicans infection in the mice pre-immunized by PBS (control), CaAg, MG+CaAg, and MG+MNP−DexBP+CaAg) is available in the online version of this article at 10.1007/s12274-022-4809-1.

Novel Single-Cell and High-Throughput Microscopy Techniques to Monitor Dictyostelium discoideum-Mycobacterium marinum Infection Dynamics.

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.

Glimpses of Dictyostelid research in India.

Simple organisms are preferred for understanding the molecular and cellular function(s) of complex processes. Dictyostelium discoideum is a lower eukaryote, a protist and a cellular slime mould, which has been in recent times used for various studies such as cell differentiation, development, cell death, stress responses etc. It is a soil amoeba (unicellular) that undertakes a remarkable, facultative shift to multicellularity when exposed to starvation and requires signal pathways that result in alteration of gene expression and finally show cell differentiation. The amoebae aggregate, differentiate and form fruiting bodies with two terminally differentiated cells: the dead stalk (non-viable) and dormant spores (viable). In India, starting from the isolation of Dictyostelium species to morphogenesis, cell signalling and social evolution has been studied with many more new research additions. Advances in molecular genetics make Dictyostelium an attractive model system to study cell biology, biochemistry, signal transduction and many more.

Slime Mold Inspired Swarm Robot System for Underwater Wireless Data Communication

Swarm robotics is a collection of mobile robots that displays swarm behavior. This paper presents a simulator of slime mold amoeba inspired swarm robot for underwater wireless communication system. The slime mold inspired robotic swarm is used to overcome the challenges of transmitting data in a large underwater environment. Underwater communication systems today are primarily acoustic technology and characterized by limited and distance dependent bandwidth, presence of multipath, and low speed of sound propagation. The robots navigate and seek the shortest path creating a virtual connection between the data transmitter and receiver similar to the foraging behavior of swarms. Each individual robot going back and forth from the transmitter to the receiver and vice-versa acts as a “physical” carrier of the data. Swarm robots navigate using swarm level intelligence based on the signal propagation technique used by slime mold amoeba aggregation using acoustics communication. The robot swarm system is developed, simulated and tested using the coded simulator. Using the slime mold inspired swarm robot system; the simulation successfully performed the data “foraging” scenario and showed the ability of the swarm to provide a virtual link in an underwater wireless communication network.

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation.

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co-occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co-incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.

Dictyostelium Cultivation, Transfection, Microscopy and Fractionation.

The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient heterohexamer membrane trafficking complex (Hirst et al., 2013). TSPOON is the product of the tstD gene in Dictyostelium and is not required for growth or the developmental cycle in this organism. Dictyostelium amoebae will exist in a vegetative phase where growth is sustained by the phagocytosis of bacteria. When this food source is spent they enter a developmental phase where the amoebae aggregate, via chemotaxis to extracellular waves of cAMP, into multicellular structures that subsequently form a fruiting body containing viable spores (Muller-Taubenberger et al., 2013). In the laboratory this cycle takes less than 24 h to complete and as a further aid to manipulation the requirement for a bacterial food source has been circumvented by the derivatisation of the wild type and isolation of axenic strains that can also grow in a nutrient rich broth. Axenic strains like Ax2 are the mainstay of laboratory research using Dictyostelium (Muller-Taubenberger et al., 2013). A description of Dictyostelium cell cultivation, the generation of cell lines that overexpress TSPOON-GFP and TSPOON null cells, and subsequent analysis (Muller-Taubenberger and Ishikawa-Ankerhold, 2013) is detailed below.

Developmental and Spatial Expression of sir2 Genes in the Cellular Slime Mold Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum grows as unicellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multicellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.

Developmental timing in Dictyostelium is regulated by the Set1 histone methyltransferase

Histone-modifying enzymes have enormous potential as regulators of the large-scale changes in gene expression occurring during differentiation. It is unclear how different combinations of histone modification coordinate regimes of transcription during development. We show that different methylation states of lysine 4 of histone H3 (H3K4) mark distinct developmental phases of the simple eukaryote, Dictyostelium. We demonstrate that the enzyme responsible for all mono, di and tri-methylation of H3K4 is the Dictyostelium homolog of the Set1 histone methyltransferase. In the absence of Set1, cells display unusually rapid development, characterized by precocious aggregation of amoebae into multicellular aggregates. Early differentiation markers are abundantly expressed in growing set1 cells, indicating the differentiation program is ectopically activated during growth. This phenotype is caused specifically by the loss of Set1 catalytic activity. Set1 mutants induce premature differentiation in wild-type cells, indicating Set1 regulates production of an extra-cellular factor required for the correct perception of growth conditions. Microarray analysis of the set1 mutants reveals genomic clustering of mis-expressed genes, suggesting a requirement for Set1 in the regulation of chromatin-mediated events at gene clusters.

Robust numerical methods for taxis–diffusion–reaction systems: Applications to biomedical problems

In this paper we consider the numerical solution of taxis–diffusion–reaction models. Such nonlinear partial differential equation models appear often in mathematical biology and we consider examples from tumour growth and invasion, and the aggregation of amoebae. These examples are characterised by a taxis term which is large in magnitude compared with the diffusion term and this can cause numerical problems. The numerical technique presented here follows the method of lines. Special attention is paid to the discretization of the taxis term in space to avoid oscillations and negative solution values. We employ splitting techniques in the time discretization to deal with the complex structure of the model and to reduce the amount of computational linear algebra. These techniques are based on explicit Runge–Kutta and linearly implicit Runge–Kutta–Rosenbrock methods. A series of numerical experiments demonstrates the good performance of the algorithm and gives rise to some implications for future modelling.

The singular limit of a chemotaxis-growth system with general initial data

We study the singular limit of a system of partial differential equations which is a model for an aggregation of amoebae subjected to three effects: diffusion, growth and chemotaxis. The limit problem involves motion by mean curvature together with a nonlocal drift term. We consider rather general initial data. We prove a generation of interface property and study the motion of interfaces. We also obtain an optimal estimate of the thickness and the location of the transition layer that develops.

Multiple Splice Variants Encode a Novel Adenylyl Cyclase of Possible Plastid Origin Expressed in the Sexual Stage of the Malaria Parasite Plasmodium falciparum

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.

A Role for a Galactose Lectin and Its Ligands During Encystment of Entamoeba1

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.

Generic modeling of chemotactic based self-wiring of neural networks

The proper functioning of the nervous system depends critically on the intricate network of synaptic connections that are generated during the system development. During the network formation, the growth cones migrate through the embryonic environment to their targets using chemical communication. A major obstacle in the elucidation of fundamental principles underlying this self-wiring is the complexity of the system being analyzed. Hence much effort is devoted to in vitro experiments of simpler (two-dimensional) 2D model systems. In these experiments neurons are placed on Poly- l-Lysine (PLL) surfaces, so it is easier to monitor their self-wiring. We developed a model to reproduce the salient features of the 2D systems, inspired by the study of the growth of bacterial colonies and the aggregation of amoebae. We represent the neurons (each composed of cell's soma, neurites and growth cones) by active elements that capture the generic features of the real neurons. The model also incorporates stationary units representing the cells' soma and communicating walkers representing the growth cones. The stationary units send neurites one at a time, and respond to chemical signaling. The walkers migrate in response to chemotaxis substances emitted by the soma and communicate with each other and with the soma by means of chemotactic “feedback”. The interplay between the chemo-repulsive and chemo-attractive responses is determined by the dynamics of the walker's internal energy which is controlled by the soma. These features enable the neurons to perform the complex task of self-wiring. We present numerical experiments of the model to demonstrate its ability to form fine structures in simple networks of few neurons. Our results raise two fundamental issues: (1) one needs to develop characterization methods (beyond number of connections per neuron) to distinguish the various possible networks; (2) what are the relations between the network organization and its computational properties and efficiency?

Crithidia fasciculata Induces Encystation of Entamoeba invadens in a Galactose-Dependent Manner

The ability of the flagellate Crithidia fasciculata to induce encystation of the reptile pathogen, Entamoeba invadens, was studied in vitro. A specific ratio of flagellate to amoeba was required; both live and heat-killed C. fasciculata induced amoebic encystation. The interaction between the Crithidia and Entamoeba cells was found to be galactose-mediated because the addition of galactose to the culture medium, or the pretreatment of the flagellate with galactosidase, eliminated its ability to induce encystation. Galactose was also found to prevent the amoeba amoeba aggregation that normally occurs in axenic cultures of encystation-induced E. invadens. Both galactose and glcNAc completely inhibited cyst formation of these induced cultures, although the latter sugar did not prevent cell aggregation. These results indicate that a galactose-mediated interaction between E. invadens cells is an early step in the in vitro encystation pathway.

Calcium, cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca2+ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca2+ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca2+, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca2+ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca2+) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.

Cell Density-sensing in Dictyostelium by Means of the Accumulation Rate, Diffusion Coefficient and Activity Threshold of a Protein Secreted by Starved Cells

The simple eukaryote Dictyostelium discoideum grows as an amoeba on leaf and soil surfaces. When starved, the amoebae aggregate and differentiate. The amoebae can also be induced to differentiate as isolated cells submerged in buffer, if the buffer contains a sufficiently high concentration of a protein (CMF) secreted by starved cells. CMF is also necessary for aggregation and differentiation on surfaces. This indicated that CMF has either an autocrine function or is part of a density-sensing system. To distinguish between these two possible functions, we first examined the rate at which CMF is accumulated and the activity threshold of cells for CMF, since both parameters will affect whether a cell can provide enough CMF to self-stimulate. We find that CMF potentiates its own accumulation, and that otherwise the accumulation rate and activity threshold are affected very little by a variety of physiological conditions. We then use diffusion calculations to show that even after many hours of continuous secretion, the CMF concentration adjacent to an isolated starved cell on a leaf or soil surface will be too low to allow differentiation, whereas an extracellular concentration of CMF sufficiently high to allow differentiation will occur when starved cells are at high densities. We find a close match between the predicted and experimentally observed density necessary for differentiation. The theoretical and observed behavior of cells at different cell densities suggests that due to its accumulation rate, diffusion coefficient, and activation threshold, CMF can function as part of a cell density-sensing system which allows Dictyostelium cells in the wild to co-ordinate their development.

Comparison of the hydrolysis of phosphatidylethanolamine and phosphatidyl ( N-acyl) ethanolamine in Dictyostelium discoideum amoebae

In Dictyostelium discoideum, ( N-acyl)ethanolamine glycerophospholipids disappear as the amoebae aggregate, whereas the amount of ethanolamine glycerophospholipids remains relatively constant, suggesting that each type of ethanolamine-containing phospholipid might have a separate metabolic pathway. To study their metabolism, phosphatidylethanolamine and phosphatidyI( N-acyl)ethanolamine containing either [ 14C]ethanolamine or a 14C-labeled sn-2 fatty acyl group were incubated with D. discoideum homogenates, and the conversion of the substrates into radioactive products was monitored. At pH values 3.8 and 4.5, phosphatidyl( N-acyl)ethanolamine was hydrolyzed by a phospholipase A 1 to form the sn-2 acyl form of the lipid. Only minor hydrolysis occurred at pH values of 5.2 or higher. ( N-acyl)Ethanolamine was also released by a phospholipase D type activity at 0.1 the rate of the lysophospholipid formation. Phosphatidyl ( N-acyl)ethanolamine was not hydrolyzed to form phosphatidylethanolamine or water soluble components. At pH 7.2 and at the low pH range of 3.8–4.5, phosphatidylethanolamine was hydrolyzed to lysophosphatidylethanolamine, which was then further degraded to water-soluble components. At pH 7.2, a phospholipase A 2 initially hydrolyzed the phosphatidylethanolamine, whereas at the low pH range a phospholipase A 1 was the most active enzyme. Although both types of ethanolamine-containing phospholipid were hydrolyzed by a phospholipase A 1 at the low pH range, phosphatidylethanolamine hydrolysis was more sensitive to inhibition by Trition X-100, and phosphatidylethanolamine was hydrolyzed to water-soluble components, whereas phosphatidyl( N-acyl)ethanolamine was not. At pH 7.2, phosphatidylethanolamine was hydrolyzed, but phosphatidyl( N-acyl)ethanolamine was not hydrolyzed at all. These results indicate that there are separate routes of degradation for the two types of ethanolamine-containing phospholipid in D. discoideum.

Localization and levels of cyclic AMP during development of Dictyostelium discoideum

Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass.

The Role of Light and an Aggregation Stimulating Factor During Aggregation of Polysphondylium violaceum

SUMMARY: Light has been shown to affect aggregation and founder cell development in the cellular slime mould Polysphondylium violaceum. An aggregation-stimulating factor, D factor, also affects aggregation of P. violaceum wild-type and aggregation-defective mutants in the complementation group aggA. Both light and D factor cause premature aggregation of amoebae and increase the number of aggregate centres formed. In addition, D factor is able to stimulate aggregation of wild-type amoebae at densities where aggregation would not normally occur. Several experiments show that amoebae are more sensitive to D factor in the presence of light, with optimum aggregation occurring in the presence of both light and D factor. Development of founder cells has been observed in the aggA mutants in the absence of D factor. However, these founder cells are inactive and do not produce aggregates until D factor is added. This class of inactive founder cells can be detected in wild-type amoebae. Production of D factor is also dependent on light with a 20- to 60-fold decrease in production over that occurring in darkness. A complex relationship between D factor, light and founder cell differentiation is established by this study.

On a mathematical model describing the aggregation of amoebae

On a mathematical model describing the aggregation of amoebae