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Molecular Microbiology Jan 2023Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of...
Mammalian professional phagocytic cells ingest and kill invading microorganisms and prevent the development of bacterial infections. Our understanding of the sequence of events that results in bacterial killing and permeabilization in phagosomes is still largely incomplete. In this study, we used the Dictyostelium discoideum amoeba as a model phagocyte to study the fate of the bacteria Klebsiella pneumoniae inside phagosomes. Our analysis distinguishes three consecutive phases: bacteria first lose their ability to divide (killing), then their cytosolic content is altered (permeabilization), and finally their DNA is degraded (digestion). Phagosomal acidification and production of free radicals are necessary for rapid killing, membrane-permeabilizing proteins BpiC and AlyL are required for efficient permeabilization. These results illustrate how a combination of genetic and microscopical tools can be used to finely dissect the molecular events leading to bacterial killing and permeabilization in a maturing phagosome.
Topics: Animals; Dictyostelium; Phagosomes; Klebsiella pneumoniae; Membrane Proteins; Bacteria; Mammals
PubMed: 36416195
DOI: 10.1111/mmi.15004 -
Cells Oct 2021Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including...
Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including autophagy induction. Recent studies into the medium-chain triglyceride (MCT) ketogenic diet have suggested that medium-chain fatty acids (MCFAs) provided in the diet, decanoic acid and octanoic acid, cause specific therapeutic effects independent of glucose reduction, although a role in autophagy has not been investigated. Both autophagy and MCFAs have been widely studied in , with findings providing important advances in the study of autophagy-related pathologies such as neurodegenerative diseases. Here, we utilize this model to analyze a role for MCFAs in regulating autophagy. We show that treatment with decanoic acid but not octanoic acid induces autophagosome formation and modulates autophagic flux in high glucose conditions. To investigate this effect, decanoic acid, but not octanoic acid, was found to induce the expression of autophagy-inducing proteins (Atg1 and Atg8), providing a mechanism for this effect. Finally, we demonstrate a range of related fatty acid derivatives with seizure control activity, 4BCCA, 4EOA, and Epilim (valproic acid), also function to induce autophagosome formation in this model. Thus, our data suggest that decanoic acid and related compounds may provide a less-restrictive therapeutic approach to activate autophagy.
Topics: Autophagosomes; Autophagy; Decanoic Acids; Dictyostelium; Phosphatidylinositol Phosphates; Proto-Oncogene Proteins c-akt
PubMed: 34831171
DOI: 10.3390/cells10112946 -
Frontiers in Cell and Developmental... 2021The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of...
The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism , the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in , allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in . We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in .
PubMed: 34485304
DOI: 10.3389/fcell.2021.721630 -
Frontiers in Cell and Developmental... 2021Preserving genome integrity through repair of DNA damage is critical for human health and defects in these pathways lead to a variety of pathologies, most notably... (Review)
Review
Preserving genome integrity through repair of DNA damage is critical for human health and defects in these pathways lead to a variety of pathologies, most notably cancer. The social amoeba is remarkably resistant to DNA damaging agents and genome analysis reveals it contains orthologs of several DNA repair pathway components otherwise limited to vertebrates. These include the Fanconi Anemia DNA inter-strand crosslink and DNA strand break repair pathways. Loss of function of these not only results in malignancy, but also neurodegeneration, immune-deficiencies and congenital abnormalities. Additionally, displays remarkable conservations of DNA repair factors that are targets in cancer and other therapies, including poly(ADP-ribose) polymerases that are targeted to treat breast and ovarian cancers. This, taken together with the genetic tractability of , make it an attractive model to assess the mechanistic basis of DNA repair to provide novel insights into how these pathways can be targeted to treat a variety of pathologies. Here we describe progress in understanding the mechanisms of DNA repair in , and how these impact on genome stability with implications for understanding development of malignancy.
PubMed: 34692705
DOI: 10.3389/fcell.2021.752175 -
Evolution Letters Jun 2022Symbiotic interactions change with environmental context. Measuring these context-dependent effects in hosts and symbionts is critical to determining the nature of...
Symbiotic interactions change with environmental context. Measuring these context-dependent effects in hosts and symbionts is critical to determining the nature of symbiotic interactions. We investigated context dependence in the symbiosis between social amoeba hosts and their inedible bacterial symbionts, where the context is the abundance of host food bacteria. have been shown to harm hosts dispersed to food-rich environments, but aid hosts dispersed to food-poor environments by allowing hosts to carry food bacteria. Through measuring symbiont density and host spore production, we show that this food context matters in three other ways. First, it matters for symbionts, who suffer a greater cost from competition with food bacteria in the food-rich context. Second, it matters for host-symbiont conflict, changing how symbiont density negatively impacts host spore production. Third, data-based simulations show that symbiosis often provides a long-term fitness advantage for hosts after rounds of growth and dispersal in variable food contexts, especially when conditions are harsh with little food. These results show how food context can have many consequences for the - symbiosis and that both sides can frequently benefit.
PubMed: 35784451
DOI: 10.1002/evl3.281 -
Genes Mar 2021Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good... (Comparative Study)
Comparative Study Review
Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as "proto-cell types" for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the "proto-cell type" gene regulatory networks.
Topics: Amoeba; Biological Evolution; Cold Temperature; Dictyostelium; Evolution, Molecular; Life Cycle Stages; Phylogeny; Signal Transduction; Stress, Physiological
PubMed: 33801615
DOI: 10.3390/genes12040487 -
BioTechniques Aug 2022We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This...
We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This method yields a substantial amount at microgram levels of gDNA per gel cleanup with high purity. An RNase A treatment step can be omitted. The quality of gDNA is suitable for next-generation sequencing, resulting in >10 Mb reads and high-quality read data (Phred score >28 up to 100 of 150 base reads). Furthermore, the gDNA can be kept intact in a gel slice for several days. This method has been tested for dictyostelids, bacteria and plants.
Topics: Bacteria; DNA; Electrophoresis, Agar Gel; High-Throughput Nucleotide Sequencing
PubMed: 35950336
DOI: 10.2144/btn-2022-0013 -
Frontiers in Cell and Developmental... 2021In aerobic organisms, oxygen is essential for efficient energy production, and it acts as the last acceptor of the mitochondrial electron transport chain and as...
In aerobic organisms, oxygen is essential for efficient energy production, and it acts as the last acceptor of the mitochondrial electron transport chain and as regulator of gene expression. However, excessive oxygen can lead to production of deleterious reactive oxygen species. Therefore, the directed migration of single cells or cell clumps from hypoxic areas toward a region of optimal oxygen concentration, named aerotaxis, can be considered an adaptive mechanism that plays a major role in biological and pathological processes. One relevant example is the development of O gradients when tumors grow beyond their vascular supply, leading frequently to metastasis. In higher eukaryotic organisms, aerotaxis has only recently begun to be explored, but genetically amenable model organisms suitable to dissect this process remain an unmet need. In this regard, we sought to assess whether cells, which are an established model for chemotaxis and other motility processes, could sense oxygen gradients and move directionally in their response. By assessing different physical parameters, our findings indicate that both growing and starving cells under hypoxic conditions migrate directionally toward regions of higher O concentration. This migration is characterized by a specific pattern of cell arrangement. A thickened circular front of high cell density () forms in the cell cluster and persistently moves following the oxygen gradient. Cells in the colony center, where hypoxia is more severe, are less motile and display a rounded shape. Aggregation-competent cells forming streams by chemotaxis, when confined under hypoxic conditions, undergo stream or aggregate fragmentation, giving rise to multiple small loose aggregates that coordinately move toward regions of higher O concentration. By testing a panel of mutants defective in chemotactic signaling, and a catalase-deficient strain, we found that the latter and the pkbR1 exhibited altered migration patterns. Our results suggest that in Dictyostelium, like in mammalian cells, an intracellular accumulation of hydrogen peroxide favors the migration toward optimal oxygen concentration. Furthermore, differently from chemotaxis, this oxygen-driven migration is a G protein-independent process.
PubMed: 34888305
DOI: 10.3389/fcell.2021.720623 -
Frontiers in Cell and Developmental... 2021The abnormal accumulation of the tau protein into aggregates is a hallmark in neurodegenerative diseases collectively known as tauopathies. In normal conditions, tau...
The abnormal accumulation of the tau protein into aggregates is a hallmark in neurodegenerative diseases collectively known as tauopathies. In normal conditions, tau binds off and on microtubules aiding in their assembly and stability dependent on the phosphorylation state of the protein. In disease-affected neurons, hyperphosphorylation leads to the accumulation of the tau protein into aggregates, mainly neurofibrillary tangles (NFT) which have been seen to colocalise with other protein aggregates in neurodegeneration. One such protein is α-synuclein, the main constituent of Lewy bodies (LB), a hallmark of Parkinson's disease (PD). In many neurodegenerative diseases, including PD, the colocalisation of tau and α-synuclein has been observed, suggesting possible interactions between the two proteins. To explore the cytotoxicity and interactions between these two proteins, we expressed full length human tau and α-synuclein in alone, and in combination. We show that tau is phosphorylated in and colocalises closely (within 40 nm) with tubulin throughout the cytoplasm of the cell as well as with α-synuclein at the cortex. Expressing wild type α-synuclein alone caused inhibited growth on bacterial lawns, phagocytosis and intracellular proliferation rates, but activated mitochondrial respiration and non-mitochondrial oxygen consumption. The expression of tau alone impaired multicellular morphogenesis, axenic growth and phototaxis, while enhancing intracellular proliferation. Direct respirometric assays showed that tau impairs mitochondrial ATP synthesis and increased the "proton leak," while having no impact on respiratory complex I or II function. In most cases depending on the phenotype, the coexpression of tau and α-synuclein exacerbated (phototaxis, fruiting body morphology), or reversed (phagocytosis, growth on plates, mitochondrial respiratory function, proliferation) the defects caused by either tau or α-synuclein expressed individually. Proteomics data revealed distinct patterns of dysregulation in strains ectopically expressing tau or α-synuclein or both, but down regulation of expression of cytoskeletal proteins was apparent in all three groups and most evident in the strain expressing both proteins. These results indicate that tau and α-synuclein exhibit different but overlapping patterns of intracellular localisation, that they individually exert distinct but overlapping patterns of cytotoxic effects and that they interact, probably physically in the cell cortex as well as directly or indirectly in affecting some phenotypes. The results show the efficacy of using as a model to study the interaction of proteins involved in neurodegeneration.
PubMed: 34552934
DOI: 10.3389/fcell.2021.741662 -
Methods and Protocols Jul 2020We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the...
We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system.
PubMed: 32630359
DOI: 10.3390/mps3030047