Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum.
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ABSTRACT:
SUBMITTER: Livermore TM
PROVIDER: S-EPMC4743807 | biostudies-other | 2016 Jan
REPOSITORIES: biostudies-other
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Project description:Dictyostelium discoideum, a social slime mold that forms fruiting bodies with spores, depends on inorganic polyphosphate (poly P) for its cycles of development and for nutritional predation on bacteria. The synthesis of poly P, a polymer of tens or hundreds of phosphate residues linked by high energy, ATP-like bonds, is catalyzed in most bacteria by poly P kinase (PPK1). The eukaryote D. discoideum possesses a homolog of PPK1. We report here that mutants of D. discoideum PPK1 (DdPPK1) have reduced levels of poly P and are deficient in development. Fruiting bodies are smaller and produce fewer spores, which appear to germinate like the wild type (WT). The DdPPK1 mutant formed smaller plaques on bacterial lawns compared with those of the WT. Predation by D. discoideum, assessed by uptake and digestion of Klebsiella aerogenes, showed that fewer bacteria were taken up by the DdPPK1 mutant compared with the WT and were killed less rapidly, indicating a role of poly P and/or DdPPK1 in phagocytosis. On Pseudomonas aeruginosa lawns, cleared plaques were observed with the bacterial PPK1 mutant but not with the WT P. aeruginosa. Thus, poly P is important in predation both for the predator and prey.
| S-EPMC549442 | biostudies-literature
Project description:Inorganic polyphosphate (polyP) deficiency in enteric bacterial pathogens reduces their ability to invade and establish systemic infections in different hosts. For instance, inactivation of the polyP kinase gene (ppk) encoding the enzyme responsible for polyP biosynthesis reduces invasiveness and intracellular survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in epithelial cells and macrophages in vitro. In addition, the virulence in vivo of a S. Typhimurium ?ppk mutant is significantly reduced in a murine infection model. In spite of these observations, the role played by polyP during the Salmonella-host interaction is not well understood. The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In fact, many intracellular pathogens can survive within D. discoideum cells using molecular mechanisms also required to survive within macrophages. Recently, we established that S. Typhimurium is able to survive intracellularly in D. discoideum and identified relevant genes linked to virulence that are crucial for this process. The aim of this study was to determine the effect of a polyP deficiency in S. Typhimurium during its interaction with D. discoideum. To do this, we evaluated the intracellular survival of wild-type and ?ppk strains of S. Typhimurium in D. discoideum and the ability of these strains to delay the social development of the amoeba. In contrast to the wild-type strain, the ?ppk mutant was unable to survive intracellularly in D. discoideum and enabled the social development of the amoeba. Both phenotypes were complemented using a plasmid carrying a copy of the ppk gene. Next, we simultaneously evaluated the proteomic response of both S. Typhimurium and D. discoideum during host-pathogen interaction via global proteomic profiling. The analysis of our results allowed the identification of novel molecular signatures that give insight into Salmonella-Dictyostelium interaction. Altogether, our results indicate that inorganic polyP is essential for S. Typhimurium virulence and survival in D. discoideum. In addition, we have validated the use of global proteomic analyses to simultaneously evaluate the host-pathogen interaction of S. Typhimurium and D. discoideum. Furthermore, our infection assays using these organisms can be exploited to screen for novel anti-virulence molecules targeting inorganic polyP biosynthesis.
| S-EPMC5797601 | biostudies-literature
Project description:Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.
| S-EPMC2168402 | biostudies-literature
Project description:Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity; this effect requires GrlD and Ppk1, but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, and to both decrease energy expenditures and retain nutrients.
| S-EPMC10056890 | biostudies-literature
Project description:Five or more orthophosphates bound together by high-energy phosphoanhydride bonds are highly ubiquitous inorganic molecules called polyphosphate. Polyphosphate acts as a signaling molecule eliciting a number of responses in eukaryotic cells, but the mechanisms mediating these effects are poorly understood. Proliferating Dictyostelium discoideum cells accumulate extracellular polyphosphate. At extracellular concentrations similar to those observed in stationary phase cells, polyphosphate inhibits proteasome activity and proliferation, and induces aggregation. Here we identify GrlD as a putative G protein-coupled receptor that mediates binding of extracellular polyphosphate to the cell surface. Cells lacking GrlD do not respond to polyphosphate-induced proteasome inhibition, aggregation, or proliferation inhibition. Polyphosphate also elicits differential effects on cell-substratum adhesion and cytoskeletal F-actin levels based on nutrient availability, and these effects were also mediated by GrlD. Starving cells also accumulate extracellular polyphosphate. Starved cells treated with exopolyphosphatase failed to aggregate effectively, suggesting that polyphosphate also acts as a signaling molecule during starvation-induced development of Dictyostelium. Together, these results suggest that a eukaryotic cell uses a G protein-coupled receptor to mediate the sensing and response to extracellular polyphosphate.
| S-EPMC6724513 | biostudies-literature
Project description:BackgroundDevelopment of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under both developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods.ResultsHere, we combine the superior depth and specificity of RNA-seq-based analysis of mRNA abundance with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large shifts. For each time point we treated the entire transcriptome as single phenotype, and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represented gradual changes in mRNA abundance, or molecular phenotype, and the gaps represented times during which many genes are differentially expressed rapidly, and thus the phenotype changes dramatically. Comparing developmental experiments revealed that gene expression in filter developed cells lagged behind those treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis suggested that the transition to multicellularity coincided with rapid accumulation of transcripts associated with DNA processes and mitosis. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. Our analysis also demonstrated a high level of synchrony among the developing structures throughout development.ConclusionsOur data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and among multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for studies of developmental gene expression.
| S-EPMC4403905 | biostudies-literature
Project description:A critical aspect of toxicity evaluation is developmental and reproductive toxicity (DART) testing. Traditionally, DART testing has been conducted in vivo in mammalian model systems. New legislation aimed at reducing animal use and the prohibitive costs associated with DART testing, together with a need to understand the genetic pathways underlying developmental toxicity means there is a growing demand for alternative model systems for toxicity evaluation. Here we explore the potential of the eukaryotic social amoeba Dictyostelium discoideum, which is already widely used as a simple model system for cell and developmental biology, as a potential nonanimal model for DART testing. We developed assays for high-throughput screening of toxicity during D. discoideum growth and development. This allowed the toxicity of a broad range of test compounds to be characterized, which revealed that D. discoideum can broadly predict mammalian toxicity. In addition, we show that this system can be used to perform functional genomic screens to compare the molecular modes of action of different compounds. For example, genome-wide screens for mutations that affect lithium and valproic acid toxicity allowed common and unique biological targets and molecular processes mediating their toxicity to be identified. These studies illustrate that D. discoideum could represent a predictive nonanimal model for DART testing due to its amenability to high-throughput approaches and molecular genetic tractability.
| S-EPMC8538044 | biostudies-literature
Project description:The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn't' play a major role in the IP₈ developmental increase.
| S-EPMC3887064 | biostudies-literature
Project description:In mammals, D-Ser is synthesized by serine racemase (SR) and degraded by D-amino acid oxidase (DAO). D-Ser acts as an endogenous ligand for N-methyl-D-aspartate (NMDA)- and ?2 glutamate receptors, and is involved in brain functions such as learning and memory. Although SR homologs are highly conserved in eukaryotes, little is known about the significance of D-Ser in non-mammals. In contrast to mammals, the slime mold Dictyostelium discoideum genome encodes SR, DAO, and additionally D-Ser specific degradation enzyme D-Ser dehydratase (DSD), but not NMDA- and ?2 glutamate receptors. Here, we studied the significances of D-Ser and DSD in D. discoideum. Enzymatic assays demonstrated that DSD is 460- and 1,700-fold more active than DAO and SR, respectively, in degrading D-Ser. Moreover, in dsd-null cells D-Ser degradation activity is completely abolished. In fact, while in wild-type D. discoideum intracellular D-Ser levels were considerably low, dsd-null cells accumulated D-Ser. These results indicated that DSD but not DAO is the primary enzyme responsible for D-Ser decomposition in D. discoideum. We found that dsd-null cells exhibit delay in development and arrest at the early culmination stage. The efficiency of spore formation was considerably reduced in the mutant cells. These phenotypes were further pronounced by exogenous D-Ser but rescued by plasmid-borne expression of dsd. qRT-PCR analysis demonstrated that mRNA expression of key genes in the cAMP signaling relay is perturbed in the dsd knockout. Our data indicate novel roles for D-Ser and/or DSD in the regulation of cAMP signaling in the development processes of D. discoideum.
| S-EPMC5928759 | biostudies-literature
Project description:The stringent response, involving the regulatory molecules inorganic polyphosphate (poly P) and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel exopolyphosphatase responsible for poly P hydrolysis. Using two different poly P-accumulating M. tuberculosis recombinant strains, we found that increased poly P content drives the organisms into early growth arrest, and contributes to tolerance to the cell wall-active agent isoniazid, increased resistance to stress conditions, and improved survival in macrophages. Transcriptomic and metabolomics analysis revealed metabolic downshift manifested by reduced expression of the transcriptional and translational machinery, and shift from utilization of glucose as a carbon source. In summary, regulation of the poly P balance is critical for persister formation in M. tuberculosis. The transcriptome of poly P accumulation strains, Rv1026 knock-down and ppk1 knock-in were compared to empty vector strains by RNA-seq.
2015-02-20 | E-GEOD-57868 | biostudies-arrayexpress